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our visits were part of our lab time, which unfortunately meant that we were interacting with the second graders at the end of their day. activities. all activities chosen except for the brain puzzle were adapted from brain - related activities found on dr.. we chose activities using the following criteria : they needed to be time - flexible, fun, and cheap. activities needed to be able to be included in a modular fashion, depending on how much time we had in the classroom and on the abilities of the students on that particular day. since we knew that we would be reaching almost 500 students, any disposables our biggest expense was the brain puzzle, because we wanted to make enough so that each student could have one and take it home. we constructed 500 brain puzzles using recycled manila folders, brain images, simple text about basic functions of the brain, plastic ziplock bags, tape and glue. we found a colorful labeled brain image on the web (many exist ; we used one from http://www.sharpbrains.com/blog/2008/06/05/your-brain-on-trading-101/) and saved costs by printing them out four to a page. we cut each brain into 710 pieces and placed the pieces into a ziplock bag which was then taped into the inside of the folder. we typically did the brain puzzle as the first activity, after introductions and the discussion about college. once the second graders had assembled the puzzle, the denison students briefly described and answered questions about the parts of the brain (fig. 1). depending on the time available and the focus of the second graders, this activity took between 10 and 20 minutes. afterwards, when the second graders did different activities relating to their senses or to movement, we related these experiences to the relevant parts of the brain. the goal of the mystery sock activity was to have students identify common objects using their sense of touch, which we then related back to the parietal lobe. our mystery socks consisted of plain white sports socks filled with items that would present some interesting tactile contrasts and that were objects that i had around the house : markers, glue sticks, whiffle balls, playdough, pennies, legos, small metal trucks and cars, and tissues. each denison team had the same assortment of eight socks. during the activity, each child tried to guess the contents of each sock by using their sense of touch alone. the hardest part of this activity was keeping the second graders from shouting out what they thought was in the sock. but by asking them to keep it a secret, we were able to keep the experience novel for everyone. students really enjoyed guessing once everyone had felt all of the socks. a good strategy for keeping track of which students had felt each sock was to have the students form lines in front of the denison students, who were holding the socks (fig. the goal of the jelly bean activity was for students to recognize the extent to which taste is affected by smell, and thus also the general concept of how the senses can work together. for our jellybean experiments, we used lifesaver jellybeans because they are inexpensive and have strong flavors and fragrances that most people enjoyed. typically, denison students ran this exercise by sorting the jellybeans by color and giving all of the second graders the same color jellybean. during the first round, the second graders were allowed to use all their senses to identify the flavor. during the second round they were asked to close their eyes, to avoid the color cue, and for the third round, they were asked to pinch their nose to minimize the flavor - amplifying effect of odor. the mystery noises activity involved producing noises which the second graders had to identify. in some cases, denison students were able to play electronic noises of ice cream trucks, cows mooing, popcorn popping, etc. on their laptops. these sounds were all found on www.youtube.com. when the internet was not available in the classrooms, denison students created sounds by ripping paper, clapping erasers, dropping a penny, closing a door, etc. the goal of this exercise was to show how signals can be passed from one neuron to another. students formed two or three lines with equal numbers of students and were instructed to tap the next person in line once they felt a tap themselves. a tagged player must hold the hand of the first player and together they have to chase the other players. as more and more players are tagged, they are added to the chain of neurons. the game ends when all of the players are part of the chain. student engagement. over the course of each activity, the denison student assigned to monitor second grader engagement observed each student in the classroom. this was facilitated by the fact that for activities 15 students were typically grouped at desk clusters or in rows. they noted each student s gender and decided if he / she was engaged or unengaged and recorded this in a data sheet with a + or a - mark. students were considered engaged when they watched the denison students leading the activity, followed directions, interacted with peers when instructed (as in the tag and message games), and worked independently as assigned (as in the brain puzzle), etc. students were considered unengaged when they were not looking at the students leading the activities or were talking out of turn, had their head down on their desk, or were otherwise not following directions. students who received mostly for an activity were given a 1 for that activity, students who received an approximately equal mix of and + were given a 2, and students who received mostly + were considered to have an engagement of 3 for that activity. since these scores are ordinal, we compared student engagement between boys and girls using the mann - whitney test with the improved normal approximation (zar, 1999). this test was calculated busing jmp 8 software (sas institute inc., 2008). effect sizes were calculated using cohen s d statistics, in which the difference between the means is divided by the mean standard deviation (cohen, 1988). denison students also filled out a weekly individual form reporting on how the activities went and what they learned. they also filled out an anonymous survey commenting on the service learning experience as a whole. our visits were part of our lab time, which unfortunately meant that we were interacting with the second graders at the end of their day. activities. all activities chosen except for the brain puzzle were adapted from brain - related activities found on dr.. we chose activities using the following criteria : they needed to be time - flexible, fun, and cheap. activities needed to be able to be included in a modular fashion, depending on how much time we had in the classroom and on the abilities of the students on that particular day. since we knew that we would be reaching almost 500 students, any disposables our biggest expense was the brain puzzle, because we wanted to make enough so that each student could have one and take it home. we constructed 500 brain puzzles using recycled manila folders, brain images, simple text about basic functions of the brain, plastic ziplock bags, tape and glue. we found a colorful labeled brain image on the web (many exist ; we used one from http://www.sharpbrains.com/blog/2008/06/05/your-brain-on-trading-101/) and saved costs by printing them out four to a page. we cut each brain into 710 pieces and placed the pieces into a ziplock bag which was then taped into the inside of the folder. we typically did the brain puzzle as the first activity, after introductions and the discussion about college. once the second graders had assembled the puzzle, the denison students briefly described and answered questions about the parts of the brain (fig. 1). depending on the time available and the focus of the second graders, this activity took between 10 and 20 minutes. afterwards, when the second graders did different activities relating to their senses or to movement, we related these experiences to the relevant parts of the brain. the goal of the mystery sock activity was to have students identify common objects using their sense of touch, which we then related back to the parietal lobe. our mystery socks consisted of plain white sports socks filled with items that would present some interesting tactile contrasts and that were objects that i had around the house : markers, glue sticks, whiffle balls, playdough, pennies, legos, small metal trucks and cars, and tissues. each denison team had the same assortment of eight socks. during the activity, each child tried to guess the contents of each sock by using their sense of touch alone. the hardest part of this activity was keeping the second graders from shouting out what they thought was in the sock. but by asking them to keep it a secret, we were able to keep the experience novel for everyone. students really enjoyed guessing once everyone had felt all of the socks. a good strategy for keeping track of which students had felt each sock was to have the students form lines in front of the denison students, who were holding the socks (fig. the goal of the jelly bean activity was for students to recognize the extent to which taste is affected by smell, and thus also the general concept of how the senses can work together. for our jellybean experiments, we used lifesaver jellybeans because they are inexpensive and have strong flavors and fragrances that most people enjoyed. typically, denison students ran this exercise by sorting the jellybeans by color and giving all of the second graders the same color jellybean. during the first round, the second graders were allowed to use all their senses to identify the flavor. during the second round they were asked to close their eyes, to avoid the color cue, and for the third round, they were asked to pinch their nose to minimize the flavor - amplifying effect of odor. the mystery noises activity involved producing noises which the second graders had to identify. in some cases, denison students were able to play electronic noises of ice cream trucks, cows mooing, popcorn popping, etc. on their laptops. these sounds were all found on www.youtube.com. when the internet was not available in the classrooms, denison students created sounds by ripping paper, clapping erasers, dropping a penny, closing a door, etc. the goal of this exercise was to show how signals can be passed from one neuron to another. students formed two or three lines with equal numbers of students and were instructed to tap the next person in line once they felt a tap themselves. a tagged player must hold the hand of the first player and together they have to chase the other players. as more and more players are tagged, they are added to the chain of neurons. the game ends when all of the players are part of the chain. student engagement. over the course of each activity, the denison student assigned to monitor second grader engagement observed each student in the classroom. this was facilitated by the fact that for activities 15 students were typically grouped at desk clusters or in rows. they noted each student s gender and decided if he / she was engaged or unengaged and recorded this in a data sheet with a + or a - mark. students were considered engaged when they watched the denison students leading the activity, followed directions, interacted with peers when instructed (as in the tag and message games), and worked independently as assigned (as in the brain puzzle), etc. students were considered unengaged when they were not looking at the students leading the activities or were talking out of turn, had their head down on their desk, or were otherwise not following directions. students who received mostly for an activity were given a 1 for that activity, students who received an approximately equal mix of and + were given a 2, and students who received mostly + were considered to have an engagement of 3 for that activity. since these scores are ordinal, we compared student engagement between boys and girls using the mann - whitney test with the improved normal approximation (zar, 1999). this test was calculated busing jmp 8 software (sas institute inc., 2008). effect sizes were calculated using cohen s d statistics, in which the difference between the means is divided by the mean standard deviation (cohen, 1988). denison students also filled out a weekly individual form reporting on how the activities went and what they learned. they also filled out an anonymous survey commenting on the service learning experience as a whole. we successfully visited all 24 second grades in newark, oh, and interacted with about 560 second graders. the activities presented in each classroom varied depending on teacher preparedness, time available and other constraints (such as achievement testing occurring elsewhere in the building). every visit included the brain puzzle and the jelly bean taste test, and most groups did the mystery socks, but fewer groups did the other activities. all activities except for message transmission had a score of at least 2.5 out of 3, with the mean engagement for all activities being 2.63. the most engaging activity, for both boys and girls, was the neuron chain tag (2.77 and 2.81 for boys and girls). a summary of the mean engagement scores of the six activities, analyzed by gender, is shown in figure 3. when student engagement was analyzed as a function of gender, effect sizes measured as cohen s d statistics for these activities were small to moderate (0.203 and 0.285). the other four activities (mystery socks, mystery noises, message, and neuron chain tag) showed no difference in engagement between male and female second graders, although the message activity might well show a difference with a larger sample size. the first hypothesis, that girls would be more engaged in the brain puzzle than boys was supported (p < 0.0226, d = 0.203 ; figure 3, table 1). the second hypothesis, that the sensory activities would be gender neutral, was supported by two out of the three sensory activities (mystery socks and mystery noises), but not by jelly beans (p < 0.0074, d = 0.285). the third hypothesis, that the more active games would engage boys more than girls, was not supported. message transmission and neuron chain tag - the favorite of both boys and girls - was gender neutral. it is worth considering the biases that could have crept in to the observations of the effect of gender on student engagement. although our class did not explicitly discuss the theoretical gender bias of our chosen activities, saying only that we wanted to have a variety of types of activities to accommodate different learning styles, these students may have formed predictions and this may have affected their scoring. i tried to minimize the likelihood of bias by creating a rubric for scoring student engagement that relied on simple, clear observations. i was initially surprised that girls were equally or more engaged than boys for all six activities. however, this could be due to the fact that engagement has to do with following instructions and looking at whoever is giving instructions. perhaps, if engagement had been structured to be about enthusiasm, jumping out of one s seat, asking questions, and interrupting out of eagerness, boys might have received higher scores. this experiment could be the subject of an outreach in another offering of this course. eighteen out of 23 students (78%) thought that the experience of teaching about college and about the brain enhanced their understanding of the content material, saying that they learned more about the brain and [its ] functions by teaching 2nd graders. typical responses from the 22% who did not feel that this experience helped them directly with course content included that the experience did nt enhance class subject matter but [they ] did learn more about kids and learning styles. twenty - one out of 23 students (91%) felt that the experience gave them a window into solving community problems, saying that this outreach exemplifies and supports denison s mission and that they felt proud to have taken action. the two who did not respond affirmatively felt that the college awareness part was certainly important, but did nt think that greater brain awareness could solve this community s problems. twenty out of 23 students (87%) felt that they had developed a greater sense of themselves as agents of change as a result of the experience. the three who did not respond affirmatively felt that earlier volunteering experiences had transformed them already. i feel like i may have left a lasting impression on students who may otherwise get little attention, and i became passionate again about volunteering. some students reflected that the experience made me realize that people in the surrounding community do nt have the great opportunities and constant encouragement that i do. they noted that simple things, like sitting at the table with the second graders, and giving them a lot of attention and encouragement, made a big difference. some comments included : this definitely enhanced my denison experience, the peak program [was ] extremely beneficial to both volunteers and participants, they wanted to stay longer to have more impact, and that the outreach made me examine myself as a teacher and as a student at the same time. i think that my students, as well as the second graders, were highly engaged. i think that the denison students benefitted from seeing male - female differences in learning styles and from analyzing and discussing the data. i think that they also benefitted personally - either finding a drive to volunteer, or perhaps realizing how fortunate they were. altogether, we devoted six out of our fourteen lab times to this outreach (one planning, one preparing and practice, four weeks in the classrooms). part of this extensive time commitment was due to the mission of peak and the program s desire to have every student in the targeted grade level uniformly involved in the outreach. since this class was a non - majors class, i felt that the benefits of this experience outweighed the costs (losing time for additional wetlabs or independent projects). i am not sure that i could rationalize using the same amount of time doing outreach for my upper level neurophysiology course. after this experience, however, i will be looking for ways that we can reach out to the community in a different way for a shorter period of time. supplementary material : script with lesson plans for each activity (1), student engagement forms (2), and lab supply list (3). | this article describes a neuroscience outreach program developed by college undergraduates and aimed at second graders. over a period of four weeks, twenty - five denison students enrolled in a non - majors course on gender and the brain visited twenty - four second grade classrooms to engage a total of 464 students. we had a mission to both promote college awareness and to specifically bring some brain science into the classroom. the desire to engage students with the brain was in part a wish to celebrate brain awareness week and in part a wish to follow a feminist tenet of bridging theory and practice via activism. the college students chose six activities : a brain puzzle, a sock content guessing game, a jelly bean olfaction and taste test, mystery noises, a message transmission game, and a version of tag. during our outreach with the second graders, my students monitored student engagement and compared engagement between male and female second graders. engagement was high for nearly all activities but girls were more engaged than boys during the brain puzzle and jelly bean activities. effect sizes measured as cohen s d statistics were small to large (0.2 to 0.93). the other four activities (mystery socks, mystery noises, message transmission and neuron chain tag) showed no difference in engagement between male and female second graders. our program benefited the denison students as well, introducing many to community involvement and awakening in them an interest in teaching or working with kids. |
over the last decade, chronic exposure to ambient air pollution has become increasingly recognized as an important risk factor underlying adverse pregnancy outcomes (apos) [19 ]. in parallel, the associations between socioeconomic status (ses) and apos are among the most robust findings in perinatal research [1012 ], which persist even in settings with universal access to health care [1316 ]. while interest in the intersection between health and the social environment is long standing [1719 ], renewed attention has been propelled by two independent progressions in quantitative research. the first is the popularization of multilevel statistical models and the ability to separate the individual - level effects from those of their encompassing social and physical environments [2026 ]. the second is the emerging research on the biological effects of psychosocial stress on health and its modification by environmental factors. there is now mounting evidence that stress can interact with chemical exposures to exacerbate the toxic effect and the physiological response to a greater extent than either insult (stress or chemical) acting alone [2731 ]. furthermore, the accumulation of low - level exposures to multiple chemicals via multiple sources and pathways shows evidence of dose addition and synergism [3234 ]. for example, synergism was observed between aqueous cigarette tar and other respirable particles (e.g., asbestos fibers, particulate matter, and diesel exhaust). recognition of these interactions has been incorporated into several conceptual models and study designs of cumulative risk of chemical and nonchemical exposures [3639 ] with models recently developed to identify these potentially double - exposed populations [40, 41 ]. two complimentary reviews of these models have been recently published [42, 43 ]. although the causes of apos are multifactorial, the placenta plays the main intermediary role between the mother 's physical and social environment and the fetus [4450 ]. importantly, a perturbed intrauterine environment inhibiting the fetal growth trajectory may also have long - term adult health implications as suggested by the developmental origins of disease hypothesis [5153 ]. therefore efforts to understand the underlying mechanisms of the physical and social environment that contribute to the disproportionate risk of apos across the socioeconomic spectrum are required in order to target preventative and restorative interventions. this review will examine how the shared pathoetiological effects of exposure to particulate air pollution and ses act on the fetal - placental unit leading to adverse pregnancy outcomes. this will be accomplished by building on conceptual pathway models of air pollution and ses etiologic mechanisms on apos [54, 55 ]. we review the role of the placenta in this context, describing its physiology and obstetrical pathologies followed by a description of particulate air pollution and its toxicokinetics in relation to placentation and how it can lead to apos. we highlight specific indicators of ses and their biological pathways that intersect with air pollution exposure and how this may contribute to increased susceptibility for apos. our aim is for this review to be a resource for researchers interested in environmental - perinatal epidemiology. understanding how correlated social and environmental exposures at times overlap to produce potentially synergistic and modifiable effects will help guide future research and intervention strategies with the aim to improve the overall health of the population [3640 ]. formed from two genetically distinct organisms, it is multifunctional and vital to fetal development yet situated outside the fetal body with a limited life span. notable characteristics unique to humans and the great apes include deep interstitial implantation and a highly invasive hemochorial phenotype thus allowing the direct interaction of maternal blood and fetal chorionic tissues. interestingly, this particular aspect of placental evolution has less to do with nutrient transfer efficiency than previously thought and more likely implicates the highly regulated maternal - fetal immunological relationship [5759 ]. the first trimester is a critical period in pregnancy involving implantation and initial placentation, two events highly susceptible to disturbance. the great obstetrical syndromes such as early / recurrent miscarriage, pregnancy induced hypertension and preeclampsia (pih / pe), fetal growth restriction (fgr), placental abruption, prelabour rupture of the fetal membranes (prom), and spontaneous preterm labour may share common etiological mechanisms arising from defective deep placentation (ddp) [61, 62 ]. together, these conditions may complicate between 17 and 29% of all pregnancies and are for the purpose of this review referred to collectively as apos. furthermore, these conditions may lead to epigenetic programming of adult disease susceptibility including obesity, diabetes, cardiovascular and reproductive diseases, all with their own substantial societal costs [52, 6466 ]. ddp refers to the shallow invasion of the placental bed into the maternal decidua and myometrium including incomplete remodeling of the uterine spiral arteries [62, 67 ]. the latter is a vital event during which under normal conditions the endothelial lining of the spiral artery walls is remodeled to accommodate the inundation of maternal blood flow starting in the second trimester. spiral arteries that fail to undergo this vascular remodeling are not only narrower in diameter but also remain responsive to vasoconstrictive compounds such as stress hormones. the etiological trigger(s) leading to ddp are thought to involve either early placental oxidative stress which triggers an inflammatory response or, vice versa, an atypical inflammatory maternal immune response to the semi - allogenic fetal - placental unit leading to placental oxidative stress and further inflammation [69, 70 ]. the difference between a normal and an affected pregnancy is a matter of degrees on a continuum with individual biological and behavioural variability nested within the social and physical environment [12, 2426, 68, 69, 7173 ]. air pollution is a general term used to describe the presence of agents (particulates, biologicals, and chemicals) in outdoor or indoor air that negatively impact human health. several common air pollutants have been associated with apos, including carbon monoxide (co), nitrogen dioxide (no2), sulfur dioxide (so2), ozone, particulate matter (pm), and polycyclic aromatic hydrocarbons (pahs) ; however, attention has focused on the latter two compounds showing strong molecular evidence of cytotoxicity, mutagenicity, dna damage, oxidative stress, and inflammation [55, 7479 ]. while the observed risks of apos in relation to air pollution tend to be modest, the population attributable risk can be quite large due to the pervasiveness of exposure to the general population. significant risks have been observed even in settings with relatively low ambient air pollution exposure [80, 81 ]. preterm birth (ptb) and fgr are major risk factors of perinatal mortality and serious infant morbidities contributing to increased health care and societal costs [8287 ]. particulate matter (pm) it is defined according to particle size into the inhalable coarse fraction (pm10, 2.510 m), the fine respirable fraction (pm2.5, 2.5 m), and the ultrafine fraction (ufp, 0.1 m). their ubiquity and recognized human health risks have deemed them as toxic [88, 89 ]. first, particle size dictates the location of deposition in the respiratory system [88, 90 ]. for example, pm10 is mainly derived from mechanical processes such as windblown soil, pollen, minerals and dust from roads, farms, and industrial operations. conversely, pm2.5 is a primary by - product of combustion and atmospheric reactions with precursor gases such as so2, nitrogen oxides, ammonia, and volatile organic compounds (vocs). pm2.5 can remain suspended in air for days to weeks and are consequently more prone to long - range transport. derived mainly from the earth 's crust, pm10 typically contains oxides of iron, calcium, silicon, and aluminum, whereas pm2.5 mixtures derived from anthropogenic combustion sources are mainly composed of sulphates, nitrates, ammonium, trace metals, elemental carbon, and organic hydrocarbons (e.g., pahs). chemical differences and relative proportions also differ within the pm10 and pm2.5 mixtures with regional (urban - to - rural) and interurban (urban - to - urban) differences as well as intraurban spatial variation [88, 9193 ]. therefore trimester and demographic differences in residential mobility and intraurban population differences are important study design issues to consider [94, 95 ]. finally, pm10, pm2.5, and ufps differ by their toxicological mechanisms such as their oxidative potential, which may reflect their differences in size, surface area, and/or their chemical constituent compositions, although they tend to be correlated [76, 92, 96, 97 ]. transition metals such as copper, nickel, lead, chromium, iron, vanadium, and cobalt among other metals are variably present in ambient air absorbed to pm2.5 [92, 93 ]. their direct oxidative action or redox potential to create reactive oxidative species (ros) is one possible mechanism as to how pm2.5 induces oxidative dna and protein damage [78, 97 ]. there is accumulating evidence that suggests ufps may be the fraction of pm responsible for many of the adverse health effects reported in air pollution studies [78, 79, 97, 98 ]. ufps are a small proportion by mass but make up a large proportion in particle number and have gone either unmeasured or misclassified as pm2.5 [88, 98 ]. their small size facilitates better tissue penetration deep into lung alveoli and into epithelial cells restricting their clearance via macrophage phagocytosis. animal studies have shown that ufps can translocate across the lung epithelium into blood circulation and accumulate in other organs, including the liver, spleen, kidneys, heart, brain, and reproductive organs. the high surface area of ufps favours the absorption of pahs and possibly transition metals which has shown to localize in the mitochondria inducing major structural damage. this could be a possible explanation to ufp 's exhibited higher oxidative potential compared to larger pm fractions of the same material. recent attention has been given to proinflammatory and endocrine - disrupting properties of diesel emissions, a major source of ufps in ambient air [31, 99101 ]. polycyclic aromatic hydrocarbons (pahs) are organic substances that constitute a class of over 100 individual chemical compounds made up of carbon and hydrogen atoms formed into rings. while toxicological data exist for individual pahs (benzo[a]pyrene being the most commonly used pah indicator), they almost always occur as complex mixtures (e.g., soot, tobacco smoke, creosote, and diesel exhaust). thus it is difficult and arguably futile to assess the toxicity of individual pah components only to be compounded by the likelihood of interactions [75, 104, 105 ]. combustion of organic matter and fossil fuels is the main source of atmospheric pahs with their distribution and magnitude concentrated along transportation corridors (road and rail) and land - use areas with heavy industrial activities. however, main stream and environmental tobacco smoke (ets) remain a leading source of pah exposure. pahs are generally nonvolatile (i.e., stable) and have low water solubility. as a consequence residency times in the atmosphere depend on weather conditions, pah molecular weight, and the emission source (e.g., stack versus tailpipe) with atmospheric deposition as the main source of pahs to soil, vegetation, and surface water. once in aquatic systems, pahs are often found absorbed to suspended particles or bound to sediments settled on the bottom where they persist or are slowly biodegraded by microorganisms. while pahs can bioaccumulate in some aquatic and terrestrial organisms, they tend to not biomagnify in food systems due to their metabolism in higher order species [102, 106 ]. however, it is the inefficient clearance and action of the highly reactive pah metabolites that are suspected to cause cytotoxicity, mutagenicity, dna damage, oxidative stress, and tumorgenesis [75, 106 ]. much of the work elucidating the mechanisms in which pm and pahs elicit adverse cellular effects have been conducted using cardiovascular disease (cvd) and lung cancer as models [7678, 97, 107109 ]. although seemingly different diseases, they share several similarities with ddp and apos with respect to associated risk factors. first, both apos and cvd related outcomes are associated with pm exposure levels which vary by ses [40, 110, 111 ] but are also associated with other socially patterned risk factors such as smoking, poor or inadequate diet, psychosocial stress, obesity, and diabetes [12, 112114 ]. cvd and apos also share many other risk factors such as the presence of systemic inflammation and preexisting hypertension. interestingly, pih / pe is a risk factor for maternal cvd later in life and also in the offspring if affected by iugr [115117 ]. cvd and disorders of ddp have similarly affected cellular tissues in their respective target systems (i.e., endothelial cells of the cardiovascular system and in the highly vascularised placenta) which are particularly susceptible to oxidative and inflammatory injury [97, 118 ]. high plasma homocysteine concentrations are positively associated with vasculopathy and infarction in the placental - uterine and coronary systems increasing the risk of spontaneous ptb and cvd events, respectively [119, 120 ]. fittingly, high density lipoprotein cholesterol may be protective against spontaneous ptb and cvd events [120, 121 ]. finally, pm and pah - induced mutagenicity, cytotoxicity, dna damage, and oxidative stress linked to lung cancer have also been observed in the fetal - placental unit [122, 123 ], and exposure early in pregnancy may contribute to the risk of congenital anomalies and early (subclinical) pregnancy loss [124127 ]. the social environment plays a significant role in maternal and perinatal health with indicators of low socioeconomic status (ses) consistently among the strongest predictors of adverse pregnancy outcomes [1012 ]. the causal pathways in which ses contribute to apos and ill health in general can be conceptualized in terms of downstream or mediating exposures, stresses, and behaviours acting on the individual through upstream society - level determinants such as poverty, poor education, income inequality, and social discrimination / marginalization over the lifespan. indicators of low ses associated with ptb and fgr include maternal anthropometry (prepregnancy bmi, height, and gestational weight gain), nutrition and micronutrient status, cigarette use, genital tract infections and inflammation, cocaine and other drug use, physically demanding work, quantity and quality of prenatal care, and psychosocial factors including anxiety, depression, and stress (e.g., lack of social, familial, and marital support, poverty or financial hardship, physical / verbal abuse, and neighbourhood crime) [12, 24, 26, 54 ]. for the purpose of this review, the focus here will be on three that engage with the oxidative stress and inflammation pathways to potentially interact with exposure to particulate air pollution. they include (1) a diet - micronutrient pathway [55, 128131 ], (2) cigarette smoke exposure [35, 132135 ], and (3) stress - mediated (allostatic) activation of the hpa - axis and corresponding glucocorticoid production [47, 72, 136138 ]. nutrition and diet can influence perinatal health in opposing directions. poor / under - nutrition such as high fat / calorie dense food and low micronutrient intake is more prevalent among women from low ses backgrounds which may partly explain higher rates of some apos [12, 139142 ]. conversely, adequate diet and micronutrient status provides resilience against oxidative stress and inflammation caused by various exposures including air pollution, allostatic stress, infection, or smoking [55, 118, 128, 129, 131, 143 ]. maternal exposure to mainstream or environmental cigarette smoke during pregnancy their exposure prevalence is associated with indicators of low ses as well as other socially patterned risk factors [147149 ] and remains one of the most modifiable risk factors with potential for beneficial intervention. other risk factors associated with low ses such as obesity, pre - existing and gestational diabetes, and hypertension [13, 113, 150 ] also engage the oxidative stress and inflammatory pathways and could therefore also potentially interact with pm exposure to increase susceptibility to adverse effects as evidenced in studies of cardiovascular health [114, 151, 152 ]. recent studies have observed increased risks of preeclampsia and gestational diabetes associated with measures of air pollution [153156 ] with one study showing positive effect modification by preexisting and gestational diabetes. evidence shows that chronic life stressors associated with low ses at multiple levels of organization (individual, household, and community) result in a cumulative biological toll on the body affecting multiple systems and increasing susceptibility to numerous ailments [21, 157160 ] including apos [15, 26, 161, 162 ]. the concept of allostasis and allostatic load / overload has been proposed to describe the individual stress response to an event as a necessary and adaptive process thereby removing the implicit negative connotation attached to the term stress. stress can be positive or tolerable when it improves function and performance and may have long - term adaptive benefits. however, this may depend on available coping resources such as one 's psychological resistance, resilience, and ability to recover. negative or toxic stress occurs when real or perceived environmental / social demands, or the anticipation of such, become too extreme or unpredictable thereby exceeding one 's (perceived) ability to cope (e.g., no sense of control, adverse childhood experiences, and other forms of trauma) [164, 165 ]. therefore, allostasis is the multisystem biological response that promotes adaptation using system mediators such as cortisol, (nor)epinephrine, vasopressin, renin, and glucagon, whereas allostatic load and overload is the cumulative toll (wear and tear) on biological systems after prolonged or poorly regulated (hyper / hypoactivated) allostatic responses [165, 166 ]. for example, the cardiovascular system is extremely sensitive to stress in terms of increased blood pressure ; however, metabolic disorders such as diabetes and obesity as well as immune function impairment are also linked to chronic stress. furthermore, lifestyle coping mechanisms as a response to chronic stress have the ability to either buffer or exasperate the effect (e.g., exercise, diet, sleep, and social interactions or lack thereof). therefore in light of the above, it is our belief that the fetal - placental unit is the site where the physical and social environments converge and interact to influence reproductive health which we describe further below. figure 1 illustrates the interconnectedness between particulate air pollution (pm / pah) and ses on how they may act discretely or in a combined manner to yield apos. using figure 1 as a guide, the following text will review the two major mechanisms (oxidative stress and inflammation) through which the physical and social environments are believed to negatively affect the fetal - placental unit and how they may combine / interact to lead to the multifactorial nature of apos. aptly known as the oxygen paradox, oxygen is both essential and toxic to the multicellular aerobic organisms whose very evolution was dependent on leveraging this anaerobic waste by - product into a higher energy producing advantage. observed in all mammals, a steep oxygen tension gradient from 20% in our atmosphere to 3 - 4% oxygen concentration in most internal tissues is the primary defense against oxidative damage. secondary and tertiary layers of protection include antioxidant defenses as well as damage removal, repair, and apoptotic response systems [168, 169 ]. these genetically adaptive responses are upregulated in the presence of reactive oxygen species (ros) generated as natural by - products of cellular aerobic metabolism and exposure to various toxins. it is produced by the mitochondria as a metabolic by - product but also from the metabolism of various growth factors, drugs, and toxins by oxidizing enzymes such as nadph - oxidase and cytochrome p450 (cyp450). superoxide is reduced by superoxide dismutase (sod) into hydrogen peroxide (h2o2) which is then further reduced into water by glutathione peroxidase (gpx) and catalase. under normal physiological conditions h2o2 acts as intracellular secondary messengers ; however, it 's accumulation along with superoxide can react with free iron ions or nitric oxide to form highly toxic hydroxyl (oh) or peroxynitrite (onoo) ions, respectively [70, 168 ]. free iron is a common metal found absorbed to pm, and the antioxidant heme oxygenase-1 (ho-1) facilitates its conjugation and removal through the increased availability of ferritin thereby preventing the formation of reactive hydroxyl molecules [92, 170172 ]. deficiencies in ho-1 have been associated with several apos such as recurrent miscarriage, fgr, and preeclampsia [171, 172 ]. common antioxidants include enzymatic (e.g., sod, gpx, catalase, and ho-1) and nonenzymatic compounds (e.g., vitamin c and e, glutathione, -carotene, and ubiquinone). genetic polymorphisms and/or micronutrient deficiencies in antioxidant enzymes precursors can impair antioxidant capacity, while chronic exposure to toxicants, psychosocial stress, bacteria, viruses, and other inducers of inflammation can foster prooxidant burden [70, 77, 118, 172 ]. oxidative stress is unavoidable ; however, under optimal conditions the presence of ros leads to homeostatic adaptation and are safely removed. failure to effectively manage oxidative stress can result in altered cellular function as excess ros degrade lipids, proteins, and dna potentially initiating pathological processes. refer to for an extensive review on the role of cellular ros in pregnancy outcomes. it is well recognized that the maternal immune system plays a central role throughout the entire pregnancy, from preimplantation to parturition, and is influenced by the inflammatory response of the mother to her environment as well as to her partner. alternative to previously hypothesized, the maternal immune system is not passive or suppressed during implantation and development of the semiallogeneic placenta and fetus. rather, it exerts executive influence on the establishment and progression of the pregnancy as an immune - mediated quality control mechanism to maximize maternal and offspring health [44, 173 ]. this is achieved by favouring pro- or anti - inflammatory environments at different times during pregnancy for different purposes. for instance, implantation, initial placentation, and parturition are characterized by a proinflammatory environment whereas an anti - inflammatory state prevails for most of midgestation. the favoured localized immunological response however is highly modified by the infectious, inflammatory, stress, nutritional, and metabolic status of the individual and thus can be influenced by environmental agents such as pm [175177 ] and/or available coping, social, and nutritional resources [44, 128, 164, 178 ]. therefore, inflammation is believed to be one pathway involved in both pm and ses - mediated apos. chronic and acute inflammation is a complex response process mediated by a real or perceived attack from foreign substances. the innate immune response is the rapid automatic response to externally originating (exogenous) substances such as pathogens, but also from internal (endogenous) danger signals including products of trauma, ischemia, necrosis, or oxidative stress. the response includes the release of proinflammatory signaling cytokine proteins such as interleukins il-1, il-6, and tumour necrosis factor (tnf-) which serve to recruit neutrophils to affected tissues. however, the recruited neutrophils release ros and hydrolytic proinflammatory enzymes (inducible nitric oxide synthase (inos), cyclooxygenase (cox-2), and prostaglandins (pg - e2)) which disturb normal cells in addition to affected tissues. this in turn leads to increased ros and oxidative stress [180, 181 ]. the placenta plays a role at the maternal - fetal interface as the main producer of endocrine steroid and protein hormones as well as the immunologic barrier between mother and fetus which positively interact for the success of the pregnancy [44, 173 ]. this is achieved through a nonlinear series of positive and negative feedback pathways with the stimulation or suppression of molecules with pro- and anti - immunosuppressant properties (interleukins, galectins, placental growth factor, and human chorionic gonadotropin (hcg)) [182184 ]. the production of these cytokines, chemokines, and other immune - regulatory agents mediates the coordination, migration, and function of several maternal immune cells (e.g., uterine natural killer cells (unk)) that participate in early pregnancy events such as endometrial receptivity of embryo implantation, tissue remodeling, immune tolerance, and vascular adaptation to invading placental trophoblast cells [44, 182184 ]. interference or aberrant production / secretion of these substances by various stressors including infection, toxins, and those acting through the hpa - axis may result in the impaired maternal immune response leading to the hallmark ddp syndrome complications described above (early pregnancy loss, pih / pe, prom, fgr, and premature labour, figure 2) [44, 61, 69, 134, 175, 185187 ]. due to immortal time bias, miscarriage is not easily measured in population or cohort studies without careful design methodologies [188, 189 ] ; however, associations between infertility and air pollution have been made [190, 191 ]. for example, obese mice showed increased ros synthesis and oxidation in oocytes with a reduced ability of zygotes to develop to the blastocyst stage providing evidence that impaired cellular antioxidant capacity can limit successful ovulation and fertilization. dividing mitotic cells are particularly sensitive to oxidative damage and are shown to enter a transient growth - arrested state as a protective mechanism until the stress has passed. thus, severe or chronic oxidative stress may hamper cell division or cause cellular necrosis reducing or terminating embryo viability [72, 169 ]. alternatively, an exaggerated inflammatory state via a viral, toxic, and/or allostatic load could lead to a maternal immune maladaptation to conception restricting trophoblast stem cell accumulation in the early embryo responsible for the production of hormones that enables successful implantation (figure 2) [44, 72 ]. oxidative stress is implicated in first trimester miscarriage from premature placental perfusion of maternal oxygenated blood and accompanying ros into the early embryonic environment. early embryo development occurs in a low oxygen state, and it is not until the tenth to twelfth week of gestation that maternal blood begins to gradually infiltrate the intervillous space of the yet fully developed placenta. the limited oxygen environment is thought to act as a protective mechanism against the deleterious and teratogenic effects of ros on early stem cells at a time of extensive cell division [64, 138 ]. this early hypoxic environment also plays a vital physiological role in placental cell type differentiation switching from proliferative villous cytotrophoblasts into invasive extravillous trophoblasts (evt) important in spiral artery remodeling. at the end of the first trimester, oxygen tension rises sharply which coincides with the infusion of oxygenated maternal blood into the placenta and triggers an apoptotic cascade that serves to establish the definitive discoid placenta. evt invasion is insufficient allowing for the premature onset of maternal intraplacental circulation and its consequential burst of ros on the conceptus [70, 192 ]. severe cases may result in pregnancy failure while more modest cases may initiate fetal - maternal adaption to impaired spiral artery remodeling leading to the ddp pathology and further complications later in pregnancy such as fgr and pih / pe (figure 2) [69, 70, 193 ]. while oxidative stress and inflammation are conditions of normal pregnancy, they are consistently elevated in cases of pih / pe and central in its pathology. pih / pe stems from a defect in early trophoblast invasion insufficient to fully convert the spiral arteries into low - resistance channels [68, 194 ]. the retention of smooth muscle cells remains active to circulating vasoconstricting agents such as stress hormones (e.g., glucocorticoids) and other stimulants. the diminished and intermittent perfusion of maternal blood into the intravillous space produces transient hypoxia resulting in a chronic ischaemia - reperfusion (i / r) type injury. this further provokes ros synthesis and excess shedding of placental microvesicles which have proinflammatory, antiangiogenic, and procoagulant activity initiating endothelial dysfunction [6870 ]. elevated circulating levels of placental debris and ros biomarkers in the placental tissues of preeclamptic women are well documented [68, 179, 194 ]. similarly, prom can be considered part of the ddp syndrome but may represent a phenotype resulting from a less severe ddp pathophysiology compared to preeclampsia [61, 62 ]. excess oxidative stress arising from multiple causes (infection, inflammation, smoking, and cocaine use) has been implicated in prom in addition to its role in ddp. both pih / pe and prom are leading causes of preterm birth, while pih / pe is a major risk factor for fgr (figure 2). deficiencies in ho-1 have been associated with preeclampsia as well as morphological changes in the placenta and elevations in maternal blood pressure. the bioactive ho-1 metabolites co and bilirubin may protect against preeclampsia through their vasodilatory properties and the suppression of the antiangiogenic factor sflt, respectively [171, 172 ]. fgr has many causes however often arises from placental insufficiency due to compromised supply of oxygen and nutrients to the fetus which may have both short- and long - term health consequences on the offspring [51, 82, 195 ]. fgr is strongly associated with early onset or more severe cases of preeclampsia, and there is a clear etiological link between fgr and ddp as it involves abnormal placentation and reduced uteroplacental blood flow (figure 2) [62, 70 ]. alternatively, perturbed calcium homeostasis can induce chronic low - level stress within the endoplasmic reticulum leading to suppressed protein synthesis and a reduced growth trajectory of the placenta. cadmium, an environmental toxin and highly present in cigarette smoke, is a major antagonist of cellular calcium activities (transport, uptake, and binding) as well as in the transfer of other nutrients and zinc homeostasis within the placenta [134, 185, 196 ]. furthermore, cadmium is a known endocrine disruptor shown to impair hormone synthesis in the placenta including progesterone and leptin, important hormones in early pregnancy [49, 175, 186 ]. both smoking and air pollution exposure were associated with lower birth weights along with low blood progesterone levels and high placental cadmium concentrations compared to a non - exposed control group. inflammation is proposed as one potential mechanism leading to spontaneous preterm labour, both with intact membranes or prom. the classification of patients who deliver preterm can be categorized into two non - mutually exclusive clusters : those who present with inflammatory lesions (e.g., acute chorioamnionitis and funisitis) and those with vascular lesions who tend to have longer gestational periods. the consequence of uteroplacental ischemia as a result of such lesions will depend on the severity, the timing, and duration of the insult. while a complete blockage of uterine arteries will lead to fetal death, less severe ischemia will result in different clinical phenotypes as a result of adaptive mechanisms for fetal survival. this may include fetal growth restriction if chronic underperfusion of oxygen and nutrients persists, the onset of maternal hypertension to sustain or increase uterine blood flow, and/or the initiation of preterm labour as a maternal / fetal adaptation to continued growth restriction in utero (figure 2) [61, 197 ]. cardiovascular lesions indicating thrombosis and atherosis are shown to be indirectly caused by exposure to pm2.5 and ufps via inflammatory and/or oxidative injury. exposure to pm2.5 and its constituents, including pahs and metals, induce oxidative stress and inflammation in many biological systems through various means (figure 3) [48, 7779, 97, 176, 177, 198 ]. one method is the direct generation of ros from free radicals and oxidants on particle surfaces including soluble transition metals such as iron, copper, chromium, and vanadium. as mentioned above, free iron can react with available superoxide or hydrogen peroxide to form highly reactive hydroxyl radicals [70, 77 ]. pahs and other organic molecules absorbed to pm2.5 and ufps may account for a large proportion of their oxidative potential due to their ability to enter the cell and disrupt the mitochondria. altered function of mitochondria may produce excess quantities of nadph - oxidase which in turn generates large amounts of cellular superoxide, a process already in overdrive throughout pregnancy but particularly in the first trimester [70, 77 ]. interpolated ambient pm10 exposure was shown to be negatively associated with the number of placental mitochondrial dna, a molecular marker of mitochondrial disruption and inflammation. this association was reversed with increasing distance from major roads, a proxy for traffic - related air pollution. alternatively, pm / pah mediated oxidative stress can be induced by the activation of the inflammation system. immunotoxic compounds can promote the release of proinflammatory cytokines, tnf-, and cox-2, which in turn act in a positive feedback loop to generate more ros and oxidative stress. for example, modelled pm10 and pm2.5 exposure has been positively associated with elevated c - reactive protein (crp) levels, a biomarker of systemic inflammation, in both maternal first trimester blood and fetal cord blood in a dose - dependent manner [176, 200 ]. crp is produced in the liver and part of the acute - phase response released during inflammatory reactions from cytokines produced in the lungs. raised crp is a risk factor for cardiovascular disease as a marker of unstable atheromatous plagues leading to thrombosis and ischemic events. exposure to diesel exhaust in healthy human volunteers resulted in pulmonary inflammation in addition to systemic inflammation, prothrombotic changes, and other cardiovascular effects consequent of proinflammatory events [99, 201 ]. this hyper proinflammatory state, along with oxidative stress, is hypothesized to contribute to several apos [69, 70, 174, 181, 202 ]. indirectly, the cellular detoxification of pahs can induce oxidative stress and cytotoxicity by forming potent ros metabolite by - products. specifically, pahs and other organic xenobiotics (notably pcbs and dioxins) are detoxified by the cytochrome p-450 (cyp) superfamily of phase i and phase ii metabolizing enzymes. the expression of these enzymes is highly modulated by genetic polymorphisms, steroid / sex hormones such as glucocorticoids, insulin, estrogens, and progesterone, and micronutrient / dietary deficiencies [74, 75, 128, 203, 204 ]. furthermore, hypoxia, infection, and inflammation are shown, in general, to downregulate cyp enzymes which may affect the clearance and bioavailability of growth factors, hormones, drugs, and toxins [203, 205 ]. cyp1a1 is the only isoform also significantly expressed in the placenta throughout pregnancy responsible for metabolizing steroid / sex hormones, growth factors, and fatty acids in addition to toxins. these exogenous and endogenous substances act as ligands to activate the aryl hydrocarbon receptor (ahr), a transcription factor that mediates the biotransformation of such ligands (pahs, estradiol, etc.) into more polar and bioavailable metabolites by upregulating cyp enzymes. however, certain metabolites of pahs (e.g., o - quinones, arene oxide, and diol epoxide) bind to dna, rna, and protein macromolecules to form toxic adducts that disrupt dna replication and are considered mutagenic [72, 75 ]. such dna adducts have been found in newborn cord - blood positively correlated with maternal exposure to pahs. pahs have also shown to significantly decrease the accumulation of trophoblast stem cells in the early placenta thereby limiting their differentiation into other cell types vital for hormone synthesis and ongoing placental development, a process that could contribute to ddp. direct prenatal exposure to airborne pahs has been associated with fgr with an increased exposure - related risk in the first trimester [206, 207 ]. secondary (phase ii) metabolizing enzymes are required to further detoxify reactive pah - metabolites in which their inefficient clearance results in prolonged exposure leading to sustained cytotoxicity and mutagenicity. phase ii enzymes include glutathione s - transferases (gsts), udp - glucuronosyltransferases (ugts), nad(p)h - dependent quinone oxidoreductase-1 (nqo1), and aldehyde dehydrogenase-3 (aldh3) [75, 205 ]. adequate diet and micronutrient status provides resilience against oxidative stress and inflammation caused by various exposures including air pollution, allostatic stress, infection, and smoking (figure 4) [55, 118, 128, 129, 131, 143 ]. many micronutrients such as essential trace metals are vital cofactors in several antioxidant enzyme systems. similarly, selenium and its incorporation into the amino acid selenocysteine are required for the functionality of all selenoenzymes, including gpx and gst. thus, selenium is essential in several aspects of human health, particularly conditions involving oxidative stress and inflammation such as cvd, immune function, cancer, and reproduction, but also thyroid regulation and brain diseases [208, 209 ]. ros may have direct effects on oocyte quality and appears to be modulated by dietary antioxidant supplements. women who are obese tend to have higher rates of infertility that correlate with increased levels of oxidative stress biomarkers in their blood as excess glucose availability leads to higher mitochondrial ros synthesis [70, 118 ]. selenium deficiency and corresponding reduced gpx activity has been documented in cases of recurrent miscarriage and spontaneous abortions [210212 ] and has also been associated with preeclampsia and preterm birth [213, 214 ]. however, given the supposed role of oxidative stress in preeclampsia, treatment with certain antioxidants (notably vitamins c and e) has not produced reliable preventative results in experimental trials. one hypothesis is that inappropriate antioxidant regiment and/or administration too late in gestation are responsible and new therapeutic candidates include melatonin and selenium. interestingly, national programs in finland and new zealand fortifying food with selenium have been associated with a significant reduction in the rate of preeclampsia. furthermore, fatty acids and low density lipid (ldl) cholesterols necessary for the placental synthesis of oestrogens and progesterone are particularly vulnerable to oxidative injury. regulation of placental nutrient transport is controlled by several different mechanism, including imprinted genes, placental signaling pathways, various cytokines, and hormones such as insulin, leptin, glucocorticoids, and oestrogens (for review see). the major placental transfer mechanisms include simple diffusion of lipophilic substances (e.g., oxygen, co2, fatty acids, steroids, fat soluble vitamins, and anesthetic gases), restricted diffusion of hydrophilic substances, facilitated diffusion via a membrane bound carrier (e.g., glucose and other carbohydrates), and active transport which requires energy (e.g., amino acids, iron, calcium, and other divalent cations) [45, 217 ]. placental physiology, including spiral artery remodeling and placental villous surface area are major determinants dictating placental transport capacity, and the degree of placental developmental disruption correlates with the severity of obstetrical complications associated with ddp [51, 62 ]. nutrition and diet can influence perinatal health in opposing directions (i.e., it can be an antagonist or agonist). poor / undernutrition such as high fat / calorie dense food and low micronutrient intake is more prevalent among women from low ses backgrounds which may partly explain higher rates of some apos [12, 139142 ]. on the other hand, good nutrition and supplemental vitamin intake is capable of reducing the toxicity of everyday environmental stressors as well as preventing certain apos and congenital anomalies as shown with the successful reduction of neural tube defects with folic acid [128, 143, 218 ]. nutritional and/or genetically induced deficiencies in folate and vitamins b6 and b12 can disrupt the homocysteine - to - methionine pathway resulting in hyperhomocysteinemia (hhc), a known risk factor of cardiovascular morbidities (thrombosis, lesions, and infarcts) and markers of oxidative stress [54, 119, 219, 220 ]. hhc may similarly affect the highly vascularized placenta and has been associated with decidual vasculopathy and preterm birth [54, 120 ]. omega-3 fatty acids abundant from eating salmon were shown to improve markers of oxidative stress, which may impart neurodevelopmental resilience against stressors [222, 223 ]. dietary phytophenols from fruits, vegetables, herbs, and spices have shown to have antioxidant and anti - inflammatory properties capable of reducing infection - induced inflammatory and contractile pathways in human gestational tissues. significant differences in pregnancy outcomes between dominicans and african americans both exposed to similar levels of pahs in new york city neighbourhoods were thought to be due to healthful dietary / cultural practices in the dominican immigrant population. maternal smoking during pregnancy and exposure to ets remain to be two modifiable risk factors with the greatest potential for beneficial interventions (figure 4). their association with numerous apos including congenital anomalies is well documented [127, 144146 ], as have their associated prevalence with indicators of low ses and other socially patterned risk factors [147149 ]. the mechanisms involved leading to apos have been well reviewed [132, 134 ] ; however, it is notable that the two main toxins present in tobacco smoke can also be absorbed to pm2.5 (pahs more so than cadmium). cadmium (cd) exposure readily interferes with the active transport of essential minerals to the fetus, particularly zinc and calcium [46, 135, 196, 226228 ]. cadmium and lead (pb) exposure has also been shown to reduce glycogen concentrations thereby potentially limiting available glucose to the fetus. cadmium has shown to disrupt placental leptin synthesis, a hormone with several vital functions including placental angiogenesis, immunomodulation, amino acid and fatty acid transport, as well as fetal pancreatic development important in the regulation of insulin - like growth factors and fetal body fat accumulation [49, 51 ]. finally, synergistic effects in the generation of oxidative hydroxyl radicals have been observed between tobacco smoke and both ambient pm2.5 and diesel exhaust particles specifically. interestingly, the counterintuitive association between smoking and lower risk of preeclampsia was recently shown to vary according to the timing and intensity of smoking. it is possible that the increased exposure to co from smoking in late gestation acts as a vasodilator and at the same time inhibits the release of sflt-1, a hallmark antiangiogenic factor implicated in the endothelial dysfunction present in preeclampsia [115, 230 ]. reviewed elsewhere, the brain is the primary target and mediating organ through which ses - related stress pathways are translated to other body systems via the hypothalamic - pituitary - adrenal (hpa) axis. the hpa - axis is actively involved in several biological systems, including the cardiovascular, metabolic, immunological, and endocrinal effects in both mother and fetus to promote allostatic adaptation [165, 231 ]. here, the neuroendocrine hormones of the hpa axis, corticotrophin releasing hormone (crh), adrenocorticotropic hormone (acth), and glucocorticoids (gc), respectively, coordinate the biological response via feedback loops. the human placenta is also capable of releasing crh and other neuropeptides which interact with the hpa axis to regulate the maternal stress response as well as other normal pregnancy functions. proper levels of in utero glucocorticoids are essential for successful embryo implantation, fetal organ maturation, and the initiation of labour with glucocorticoid levels gradually increasing over the course of gestation. normally, levels of maternal cortisol rise sharply in the third trimester causing the release of placental crh in a positive adrenal - placental feedback loop. placental crh stimulates fetal cortisol secretion which in turn suppresses placental progesterone and activates the release of prostaglandins and oxytocin to promote uterine contractions [47, 232 ]. however, early and increased levels of fetal glucocorticoids can impair growth and predispose to adult - onset diseases [136, 233, 234 ]. the placental enzyme 11-hydroxysteroid dehydrogenase type 2 (11-hsd2) protects the fetus from excess endogenous glucocorticoids by converting active cortisol into inactive cortisone. 11-hsd2 is hormonally regulated making it susceptible to endocrine disruption from chemical and nonchemical stressors such as maternal anxiety, inflammation, infection, cadmium exposure, and low caloric intake [136, 138, 224, 235, 236 ]. placental hypoxia associated with pih / pe has been shown to suppress 11-hsd2 activity which may be an adaptive response to counteract compromised fetal growth by allowing more cortisol to reach the fetus for organ development. low concentrations / activities of 11-hsd2 and high levels of cortisol have been associated with ptb and fgr [136, 237, 238 ], two outcomes also associated with poor maternal psychosocial / mental health [233, 234, 239 ]. factors affecting 11-hsd2 activity that are associated with low ses include allostatic overload leading to the excess production of glucocorticoids that can overwhelm the fetal protective mechanism (figure 4) [136, 231, 240 ]. indirectly, allostatic load is capable of disrupting the metabolic system leading to impaired glucose tolerance, insulin resistance, diabetes, and/or obesity, all of which are risk factors for various apos [138, 165, 231 ]. general maternal undernutrition and/or a low dietary protein intake has been shown to impair placental glucose transport and inhibit 11-hsd2 activity in pregnant rats leading to fgr, indicating a possible mechanism through poor diet [224, 241 ]. additionally, cadmium has also shown to inhibit 11-hsd2 activity in both human and rodent placentas, and prenatal cadmium exposure has been shown to increase fetal corticosterone concentrations in rats which resulted in reduced birth weights. this suggests a possible mechanism from active or passive tobacco smoke exposure or ambient pm2.5 exposure [135, 242, 243 ]. collectively, it is possible for the cumulative exposures of pm2.5, smoking, ets, poor dietary intake, and other ses - related factors to interact through the same 11-hsd2 mechanism to increase the risk of impaired fetal growth (figure 4). the ubiquitous exposure to particulate air pollution and its constituents (e.g., pahs and metals) is but one class of environmental contaminants that can act through oxidative stress, inflammation, and/or endocrine disruption to promote developmental toxicity and adverse perinatal health [177, 244, 245 ]. summarized in figure 2, a perturbed early in utero environment can lead to defective deep placentation resulting in a cascade of fetal - placental adaptive mechanisms contributing to a range of pregnancy complications and adverse outcomes. here the underlying biological, social, and physical risk factors likely intersect to produce excessive or atypical oxidative stress, inflammatory response, and biological antagonism to either initiate the defective deep placentation pathology and/or contribute to the severity of its phenotype. socioeconomic disparities are known to confound the environmental exposure effects ; however, they may also act as potential effect modifiers given their overlapping etiological mechanisms with pm2.5 exposure. while the traditional biomedical paradigm that views populations as a collection of independent individuals has yielded useful information regarding risk factors, elucidating the intersecting pathways involved in apos will require placing individual biologic and behavioural determinants within the social and spatial context [22, 246 ]. it is now well recognized that ses operates at multiple levels of organization, and neighbourhood or community - level factors can work to either ameliorate or exacerbate certain risk factors [15, 2426 ]. the healthy migrant paradox exemplifies these effects in which home country, education, and neighbourhood qualities combine to modify the expected perinatal outcomes often observed with low income households [161, 247 ]. the ses risk factors that overlap or interact with the pm - mediated mechanisms include smoking, nutrition, and psychosocial stress acting through the hpa - axis and allostatic load. given this knowledge, interventions aimed at ameliorating these factors may be the best way to counteract the negative influences of low ses and air pollution exposure on fetal development. maternal smoking continues to be one of the most modifiable risk factors to lower the risk of apos [134, 147 ]. furthermore, maternal smoking also tends to interact negatively with nutrient intake and status [133, 248 ]. smokers in general have poorer nutritional profiles than nonsmokers with both behavioural and biological factors independently accounting for the differences in micronutrients such as folate and essential vitamins and minerals [133, 248250 ]. while smokers tend to have lower dietary nutrient intakes, they also have an accelerated requirement for micronutrients due to increased inflammatory cell turnover caused by the oxidative stress of smoking, an effect more pronounced among heavy and long - time smokers. these interacting effects of smoking and nutrition are further compounded by their association with other indicators of low ses such as low education and income contributing to allostatic load [139, 251 ]. nutrient intake may be ameliorative after an insult has occurred as shown in rat models of fetal alcohol syndrome where an omega-3 fatty acid enriched diet reversed the cellular effects of prenatal ethanol exposure on the fetal brain. therefore with respect to policy interventions, nutrition in the form of improved food security and micronutrient intake may serve to counteract the negative influences of both low ses and air pollution exposure [252257 ]. the complex mixture of particulate air pollution, especially pm2.5 and ufps which includes absorbed pahs and various metals, also emerges as an important target for risk reduction and management. the deserved scrutiny stems from their ubiquity in the environment, the myriad of emission sources, and their established association with apos [88, 98, 258 ]. the pervasiveness of pm2.5 and ufps in the environment means that a high proportion of people are exposed resulting in a high etiological fraction. therefore, even a modest reduction in exposure will have a large population effect with reduction of the societal costs of apos. notably, their sources are primarily local, such as vehicle emissions and industrial land - use. this makes them modifiable risk factors that can be addressed at the municipal and provincial / state level with better urban planning to reduce vehicle traffic, increasing access to green - space and enforcing air quality regulations [260263 ]. not unlike the accumulation of evidence on smoking and health outcomes or that of air pollution on cardiovascular and pulmonary health, the epidemiological and toxicological research over the past two decades has established a consistent dose - response association with high biological specificity, temporality, and plausibility [3, 55, 177 ]. taken in concert, these characteristics and further corroborating research should lend strength for evidence - based policy for intervention strategies targeting high risk areas in order to reduce the environmental burden of disease attributed to particulate air pollution [98, 265, 266 ]. adverse pregnancy outcomes such as fetal growth restriction and preterm birth are a public health priority of global importance. we have brought together the multidisciplinary literature on the current state of evidence linking the physical and social environment to specific adverse pregnancy outcomes. the evidence suggests that various exposures, whether socially or environmentally determined, may be interpreted by the fetoplacental unit in similar ways resulting in a common pathological foundation for adverse outcomes, namely, deficient deep placentation. given this background, well planned future epidemiology studies using multilevel models exploring various biological effects of the social and physical environment will have the potential to provide the evidence to establish crucial windows of fetal vulnerability with an aim to identify and mitigate modifiable risk factors. | exposure to particulate air pollution and socioeconomic risk factors are shown to be independently associated with adverse pregnancy outcomes ; however, their confounding relationship is an epidemiological challenge that requires understanding of their shared etiologic pathways affecting fetal - placental development. the purpose of this paper is to explore the etiological mechanisms associated with exposure to particulate air pollution in contributing to adverse pregnancy outcomes and how these mechanisms intersect with those related to socioeconomic status. here we review the role of oxidative stress, inflammation and endocrine modification in the pathoetiology of deficient deep placentation and detail how the physical and social environments can act alone and collectively to mediate the established pathology linked to a spectrum of adverse pregnancy outcomes. we review the experimental and epidemiological literature showing that diet / nutrition, smoking, and psychosocial stress share similar pathways with that of particulate air pollution exposure to potentially exasperate the negative effects of either insult alone. therefore, socially patterned risk factors often treated as nuisance parameters should be explored as potential effect modifiers that may operate at multiple levels of social geography. the degree to which deleterious exposures can be ameliorated or exacerbated via community - level social and environmental characteristics needs further exploration. |
the cochlear implant (ci) transforms acoustic signals from the environment into electric impulses, which are then used to stimulate intact fibers of the auditory nerve. with this treatment, individuals with profound hearing loss (hl) current technology and speech processing strategies allow many ci recipients to achieve impressive accuracy in open - set speech recognition, and the ci is arguably the most effective neural prosthesis ever developed [13 ]. however, the success of the outcome depends both on duration of deafness prior to implantation [4, 5 ] and on the onset of deafness before (prelingually) [47 ] or after (postlingually) critical stages in the acquisition of language. in many cases, the greatest gains of performance occur in the first three months of use [911 ]. the dramatic improvements following implantation not only demonstrate the efficiency of the ci technology, but also point to the role of cortical plasticity as a means to reactivate brain function. plasticity is a term used to describe the reorganization of the central nervous system by means of synaptic changes and rewiring of neural circuits. in cases of cochlear implantation, neural plasticity associated with deprivation of auditory input and adaptation to the absence of stimuli is of particular interest. reduced input to the brain from impaired auditory pathways results in significant changes in the central auditory system and is accompanied by a recruitment of deprived cortices in response to input from the intact senses [1317 ]. when auditory input to the brain is reintroduced, this novel auditory experience may itself induce additional plasticity. the sensory reafferentation provided by the ci thus offers a unique opportunity to study the effects of preceding deafness on functional brain organization. in normal - hearing (nh) adults, language processing is associated with extensive frontal activation in the left cerebral hemisphere, including the anterior (brodmann 's areas (ba) 45 and 47) and posterior (ba 44 and 45) parts of the left inferior frontal gyrus (lifg), the latter often referred to as broca 's area [19, 20 ]. traditionally, this area is mainly assigned an expressive language function, but several studies show a relationship between the perception of language and left frontal activity, both when stimuli are presented aurally [2124 ] and visually [2428 ]. neuroimaging experiments comparing auditory responses of ci users and normal - hearing control participants, while listening to speech or complex nonspeech, generally reveal bilateral activity in the primary and secondary auditory cortices, including both superior and middle temporal gyri [12, 2934 ]. one consistent outcome of these studies is the more dominant right temporal activity of ci users listening to speech, that is, the observation of more bilateral activity than would be expected on the basis of the classical presumption of left - lateralized activity of language processing in normal - hearing. however, in these studies, activation of other classic language regions such as broca 's area was not a consistent finding. naito and colleagues found broca 's area to be activated only when the ci participants silently repeated sentences [31, 36 ]. compared brain activity in experienced ci users according to their levels of speech comprehension performance. they found that, unlike ci users with low speech comprehension, single words and speech yielded raised activity in the left inferior prefrontal cortex (lipc) in ci users with excellent speech perception. some observed activations outside the classic language areas, including anterior cingulate, parietal regions, and left hippocampus, have been attributed to nonspecific attentional mechanisms and memory in ci users [31, 32 ]. furthermore, some studies have reported convincing evidence of visual activity in response to auditory stimuli or auditory activity in response to visual stimuli in ci users. although much debate about the identity of the brain systems that are changed and the mechanisms that mediate these changes exists, the general belief is that this cross - modal reorganization is associated with the strong visual speech - reading skills developed by ci users during the period of deafness, which are maintained or even improved after cochlear implantation, despite progressive recovery of auditory function [5, 11, 33, 3841 ]. the possible reasons for these mixed results may include differences in experimental paradigms, small sample sizes, heterogeneous populations, variance in statistical thresholds, and a lack of longitudinal control of plasticity. with the present study, we tested the cortical mechanisms underlying the restoration of hearing and speech perception in the first six - month period following implant switch - on. we expected to see inactive neuronal pathways reactivated in ci recipients, within three to six months of switch - on, and engagement of cortical areas resembling those of normal - hearing control participants. furthermore, in previous findings, notwithstanding, we expected to see broca 's area involved in speech perception. finally, we expected to see a difference in the progress of adaptation and the involvement of cortical areas between ci users with postlingual hearing loss and ci users with prelingual hearing loss. over the course of two years, patients who were approved for implantation were contacted by mail and invited to take part in the research project, including positron emission tomography (pet) and speech perception measures. from a total of 41 patients, 15 accepted and were included in the study (6 women, 9 men, mage = 51.8, age, range : 2173 years). four participants had a prelingual onset of hl, indicated by their estimated age at onset of deafness and main use of signed language as communicative strategy. the remaining 11 participants had a postlingual or progressive onset of hl, as indicated by their main use of residual hearing, supported by lip - reading. in accordance with local practice, all ci participants followed standard aural / oral therapy for six months in parallel with the study. the therapy program includes weekly one - hour individually adapted sessions and trains speech perception and articulation. table 1 lists the demographic and clinical data for the 15 participants. to obtain a normal - hearing reference, we recruited a group of nh adults (4 women, 2 men, mage = 54.29 years, age range : 4764 years) for a single pet / test session. all nh participants met the criteria for normal - hearing by passing a full audiometric test. the study was conducted at the pet center, aarhus university hospital, denmark, in accordance with the declaration of helsinki, and approved by the research ethics committee of the central denmark region. nh participants underwent pet once, while ci participants were tested consecutively at three points of time : (1) within 14 days after switch - on of the implant (baseline, bl), (2) after three - months (midpoint, mp), and (3) after six months (endpoint, ep). for purposes of analysis, two subgroups were identified as (1) the postlingual (post) hl subgroup (n = 11) and (2) the prelingual (pre) hl subgroup (n = 4). the hagerman test is an open - set test which presents sentences organized in lists of ten. the sentences have identical name - verb - number - adjective - noun structures, which the participant is required to repeat. normally, the test is presented in background noise. however, considering the participants ' inexperience with the implant, we removed the background noise. the participants were given one training list with feedback and two trial lists without feedback (max. sound was played back on a laptop computer through an active loudspeaker (fostex 6301b, fostex company, japan) placed in front of the participant. the stimuli were presented at 65 db sound pressure level (spl), and ci users were instructed to use their preferred ci settings during the entire test session and to adjust their processors to a comfortable loudness level. furthermore, participants who used a hearing aid were instructed to turn it off and leave it plugged in. the ci participants performed the hagerman tests in separate sessions at the same milestones as selected for pet data acquisition (bl, mp, and ep). the nh group performed a single hagerman test along with their pet scan session. to identify effects of time, we performed a repeated measures analysis of variance. due to nonnormal distributions in the data, this was done using a nonparametric friedman 's anova in the post and pre subgroups separately. post hoc tests and between - group analyses were performed by mann - whitney nonparametric tests and bonferroni corrected at alpha 0.016. the hag data were analyzed in spss and plotted with sigmaplot for windows 11.0 (systat software inc.). a high - resolution t1-weighted mr scan was acquired prior to pet scanning. in the case of ci participants, all participants were examined in 2 conditions : (1) multitalker babble (bab) from multiple simultaneous speakers with a complexity close to that of speech and perceived by the listeners as speech - like but devoid of meaning and (2) running speech (rs), narrating the history of a familiar geographical locality at the rate of 142 words per minute, generated in danish by a standard female voice. the stimuli were played back on a laptop computer in the freeware sound editor software audacity (http://audacity.sourceforge.net/) and delivered directly from the computer 's headphone jack to the external input port of the implant speech processor. bimodally aided participants removed their hearing aid and were fitted with an earplug in the nonimplanted ear during the tomography. these measures were taken to preclude background noise and possible cross - talk from the contralateral ear. the nh participants listened to the stimuli binaurally through a pair of headphones (akg, k 271). all stimuli were presented at the most comfortable level. to define this level, participants were exposed to the two stimuli once before the tomography. in the tomograph, prior to bolus injection, participants had no information about the nature of the next stimulus, but they were instructed to listen attentively in all cases. after each of the four scans, participants were required to describe what they had heard and, if possible, review the content of the narration. pet. positron emission tomography (pet) is a molecular imaging method that yields brain activity, by means of detecting changes in regional cerebral blood flow (rcbf). this is done by computing and comparing the spatial distributions of the uptake of a blood flow tracer. pet measurements are generally limited with respect to spatial and temporal resolution and the invasiveness of the procedure, which requires injection of oxygen-15-labelled water. in this case, however, anatomical and temporal specificity could not have been improved by using functional magnetic resonance imaging (fmri), as the auditory implants are not mri compatible. in addition, pet is an almost noiseless imaging modality, which is useful for both ci participants and for the study of speech. finally, because only the head of the participant is positioned in the tomograph, compared with the whole body imposition of fmri, it is possible to communicate visually with the participant during tomography. we measured raised or reduced cerebral activity as the change of the brain uptake of h2 o oxygen-15-labeled water, which matches the distribution of cerebral blood flow (cbf), using an ecat exact hr 47 tomograph (siemens / cti). emission scans were initiated at 60,000 true counts per second after repeated intravenous bolus injections of doses of tracer with an activity of 500 mbq (13.5 mci). the tomography took place in a darkened room with participants ' eyes closed. the babble and running speech conditions the uptake lasted 90 seconds (single frame) at intervals of 10 min. after correction for scatter and measured attenuation, each pet frame was reconstructed with filtered back projection and smoothed with a postreconstruction 10 mm gaussian filter resulting in a resolution of 11 mm full - width - at - half - maximum (fwhm). rules of regulation mean that participants who volunteer for scientific experiments may receive a total maximum radiation of 6 millisieverts (msv) within one year. here, the total radiation dose administered over the three times of scanning was approximately 5.58 msv. due to these restrictions, no preoperative baseline scans could be acquired. participants ' mr images were co - registered to an mr template averaged across 85 individual mr scans in talairach space, using a combination of linear and nonlinear transformations. each summed pet emission recording was linearly coregistered to the corresponding mr image using automated algorithms. to smooth the pet images for individual anatomical differences and variation in gyral anatomy, images were blurred with a gaussian filter resulting in final 14 mm at full - width half - maximum (fwhm) isotropic resolution. all images were processed using statistical parametric mapping 8 (spm8 ; wellcome neuroimaging department, uk, (http://www.fil.ion.ucl.ac.uk/spm/)). local maxima of activation clusters were identified using the montreal neurological institute (mni) coordinate system and then cross - referenced with a standard anatomical brain coordinate atlas. differences in global activity were controlled using proportional normalization (gray matter average per volume). significance threshold for task main effects was set to p < 0.05, family wise error (fwe) corrected for multiple comparisons. we tested the effect of side of implant and type of implant in a separate preanalysis. as we found neither main effects nor interactions with functional data involving these variables, we concluded that these factors had no significant effect on the results. the first analysis identified the main effects of time, speech / babble contrast, and history of hearing loss (post hl versus pre hl) and possible interactions between these effects in the whole brain, across ci participants. this analysis was performed as a single spm matrix in a factorial 3-way design with time, contrast, and subgroup as factors. to define a region - of - interest (roi) analysis 2. the second analysis identified possible main effects of contrast, time, and interactions between these factors in the inferior frontal gyrus (ifg), more specifically broca 's area (ba 44/45). this analysis was performed as two 2-way factorial analyses of the post hl and the pre hl subgroups separately. to define a region - of - interest (roi), we created a mask based on bilateral inferior frontal gyri (broca 's region), including the putative brodmann regions 44, 45, and 47 using the wfu pick - atlas. the third analysis investigated main effects of contrast and group (ci versus nh) at the ci baseline and possible interactions between these effects. this analysis was performed as a single spm matrix in a factorial 2-way design with condition and group as factors. to define a roi we found a main effect of speech / babble contrast across ci participants, regardless of subgroup, in bilateral superior temporal gyri (f(1,78) = 60.14). a t - test confirmed that the effect was driven by higher activity during running speech (table 2 ; figure 1). there was no significant main effect of time or group, nor any interaction between the effects. the roi analysis revealed significant interaction between the effects of contrast and group in ba 21/22 in the left superior temporal gyrus (f(1,78) = 20.42) (table 2 ; figure 2). a plot of contrast estimates showed a difference between running speech and babble that was larger in the postlingual subgroup than in the prelingual subgroup (figure 2). in the bilateral ifg roi analysis, we found a main effect of speech / babble contrast in ba 47 in the postlingual subgroup. a t - test confirmed that the effect was driven by higher activity during running speech. furthermore, we found a main effect of time in left ifg (broca 's area ba 45 ; f(2,60) = 14.19 ; p = 0.006, fwe corrected) (figure 3), with no significant interaction between contrast and time. the prelingual subgroup had no main effects in the bilateral ifg roi analysis (table 3). we found a main effect of speech / babble contrast across the ci and nh groups bilaterally in superior temporal gyri, in the left middle temporal gyrus, and in the right inferior parietal lobule. t - tests showed that the superior temporal gyri bilaterally and the left middle temporal gyrus were more active during running speech, while the right inferior parietal lobule was more active during babble. a t - test showed that this effect was due to higher activity of this area in the nh group than in the ci group. no interaction was found between the effect of contrast and the effect of group in whole - brain analysis (table 4). the roi analysis based on the main effect of contrast yielded a main effect of ci versus nh in secondary auditory cortex including ba 22 in the right superior temporal gyrus. a t - test showed that this effect was due to higher activity of this area in the nh group than in the ci group. furthermore, in the roi analysis, we found an interaction between the effect of speech / babble contrast and the effect of group in the right inferior parietal lobule (table 4). we found a significant effect of time in the post group ((2,2) = 19.62, p < 0.001), but not in the pre group. post hoc tests showed that the effect was driven by the 51.3 percentage points endpoint gain (mann - whitney u = 12.5, p = 0.002). furthermore, the post group scored significantly higher than the pre group at all three points of measurement (bl : mann - whitney u = 0.000, p = 0.004 ; mp : mann - whitney u = 0.000, p = 0.004 ; ep : mann - whitney u = 0.000, p = 0.004). ceiling performance (100% correct) was observed in all nh participants (figure 4). in this study, we aimed to examine the plastic changes which underlie the recovery of hearing after cochlear implantation. our results showed that across all ci recipients and all points of measurement, the bilateral middle and superior temporal gyri (including, more specifically, brodmann areas 21 and 22) were significantly more active when participants listened to running speech than when they listened to multi - talker babble. thus, on average, the auditory brain regions, known to be involved in the processing of complex auditory stimuli [4951 ] displayed a clear distinction between speech - like noise and speech in recently implanted ci recipients. this confirms that both hemispheres are involved in the speech perception process, even during monaural auditory stimulation [29, 36, 52, 53 ]. furthermore, the results showed a difference in the way ci recipients with postlingual hearing loss and recipients with prelingual hearing loss distinguish between speech and babble. the ci users with postlingual hearing loss displayed a greater activation during speech than during babble in ba 21 and 22 in the left superior temporal gyrus, indicating differential processing of the stimuli by the two subgroups. we speculate that the postlingually deaf listeners disengage attention when they are presented with the incomprehensible babble stimulus. in contrast, the prelingually deaf ci listeners may be equally attentive to the two stimuli, regardless of their nature, as reflected in undifferentiated activity. the postlingual subgroup not only possessed a moderate level of speech perception at baseline (within 14 days after switch - on of the implant), but also made significant gains in performance, the majority of which occurred in the first three month period. in contrast, the prelingual subgroup had no baseline speech perception and only modest, if any, progress during the study period. this finding is consistent with expectation and implies an association between behavioral performance and brain activity related to the history of hearing loss. in prelingual deafness, the neuronal connections of the auditory pathways (e.g., measured as cortical auditory evoked potentials) may not be established in the appropriate time window of opportunity [5456 ]. the subsequent electric stimulation at some time in adulthood may produce some hearing sensation, but the discrimination of sounds and time intervals remain defective [57, 58 ]. the findings are compatible with naito., who suggested that the reduced speech activation in prelingually deaf implant users could be explained by insufficient development of neuronal networks or their degeneration due to prolonged deafness. follow - up studies in the present population may provide interesting insight into the degree to which speech perception progresses in the prelingual subgroup in the long term. we found a main effect of time exclusively in broca 's area, and only in the postlingual subgroup. this is in line with recent studies showing that, in ci users, the activation of broca 's area during speech processing is negatively correlated with the duration of deafness and positively correlated with the progress in the restoration of speech comprehension [7, 8, 37, 59 ]. this indicates that the changes in the auditory recovery process are most profoundly manifested in this specific area, which is associated with speech perception and production. this suggests that the area becomes increasingly activated, regardless of whether the stimulus makes semantic sense or not, or is active in the distinction between sense and nonsense. however, the absence of difference between speech and babble in broca 's area may also be explained by an increasingly high activity at rest in ci patients, as reported by strelnikov.. our whole - brain analysis revealed a significant activation of bilateral superior temporal gyri and left middle temporal gyrus during speech across ci participants at baseline and nh individuals., who, in addition to bilateral superior temporal gyri, found significant speech activation in the right middle temporal gyrus, the left posterior inferior frontal gyrus (broca 's area), and the left hippocampus in both normal participants and ci users. these further findings could be explained by the use of silence as contrast relative to the use of babble in the present study. interestingly, even though the nh participants received unilateral stimulation (6 right ; 6 left), the naito study found no significant difference in any brain area in either nh participants or ci users between right ear stimulation and left ear stimulation. the significant involvement of the right parietal lobule in the ci participants during babble suggests that, at this initial stage of the ci adaptation, ci listeners, unlike nh individuals, need to pay attention to the speech - like noise to determine its possible character. the observation that during speech stimulation the nh participants involved the caudate nucleus more than the ci participants may be explained by a reduction of the effort needed by the nh participants to deal with the well - known task of receiving a message. the caudate nucleus is a part of the striatum, which subserves among other tasks the learning of slowly modulated skills or habits. to the normal - hearing listener, the reception of auditory information is an every - day experience similar to following a known route ; for example, see wallentin. for a similar argument. in contrast, to ci listeners, auditory stimuli are nonhabitual in the strongest sense of the word, thus relying on other sources of processing. interestingly, naito. made a similar finding and speculated that though the caudate nucleus has been associated with various tasks ranging from sensorimotor tasks to pure thinking, the result may provide further evidence that the area has cognitive function and shows increased activity along with the increased activity in cortical language areas. in contrast to the significantly higher right - lateralized activation observed in nh individuals in the present study, giraud. showed a left - lateralized activation of temporal and frontal regions in nh controls. however, direct comparison between the two studies is difficult as there are several differences in study design and implant experience of the participants. found increased activity in right cerebellar cortex when running speech was comprehended relative to babble, but only in ci listeners with high speech comprehension. the authors speculated that this could be due to cognitive work of cerebellum subserving verbal working memory or a contribution of the right cerebellar hemisphere to precise representation of temporal information for phonetic processing. however, this finding was not replicated in the present study, which may reflect differences in duration of implant use and a mixture of speech comprehension levels. giraud and colleagues consistently demonstrated activation of areas ba 17/18 in the visual cortex when ci users responded to meaningful sounds. the authors argued that the process was associated with improvement of lip - reading proficiency, which is supported by findings in a behavioral study by rouger. a similar cross - modal interaction between vision and hearing was not replicated in the current study. the giraud study involved repetition of words and syllables and naming of environmental sounds, contrasted with noise bursts, as opposed to the current study, which involved passive listening to a story contrasted with speech - like noise. furthermore, sound was presented in free field, whereas in the current study, the auditory stimuli bypassed the microphones of the speech processor and were fed directly to the auxiliary input. finally, the strict conservative statistical methods used here preclude reporting of results that are not statistically significant when corrected for multiple comparisons. in scandinavia as in most european countries, cochlear implantation is administered by the public health care system and offered for free to all patients who meet the clinical implantation criteria (< 40% open - set word - recognition scores). this includes patients with a prelingual hearing loss, despite recognition that these patients may have very limited linguistic benefit. as a result of this policy, the group of ci users in general is very heterogeneous, which was also the case in this study. while the difference in group size was far from optimal and may have confounded the results, we maintain that it is of high importance to also study cortical activity in prelingually deaf patients following cochlear implantation. in a previous study involving music training, we found that while little gain was achieved in speech perception, prelingual deafness did not preclude acquisition of some aspects of music perception. this implies that plastic changes take place also in the long - term deaf brain and that specific training measures could help these patients in achieving improved implant outcome [58, 63 ]. our normal - hearing control group had a less optimal size, which may have made direct comparisons between groups less valid than desired. however, ethical restrictions limit the number of healthy participants in studies involving pet. as stated, the degree of deafness (i.e., residual hearing) varied across subjects and may have influenced the adaptation to the implant. unfortunately, a correlation analysis between preoperative speech understanding and pet and behavioral results was not possible since such data were not available for all participants. the present pet study tested brain activation patterns in a group of recently implanted adult ci recipients and a group of normal - hearing controls, who listened to speech and nonspeech stimuli. ci listeners with postlingual hearing loss showed differential activation of left superior temporal gyrus during speech and speech - like stimuli, unlike ci listeners with prelingual hearing loss. this group difference was also reflected in a behavioral advantage for patients with postlingual hearing loss. furthermore, broca 's area was activated as an effect of time, but only in ci listeners with postlingual hearing loss. comparison of the ci listeners and the normal - hearing controls revealed significantly higher activation of the caudate nucleus in the normal - hearing listeners. the study demonstrates that processing of the information provided by the cochlear implant is highly related to the history of hearing loss. patients whose hearing loss occurred after the acquisition of language involve brain areas associated with speech comprehension, which is not the case for patients whose hearing loss occurred before the acquisition of language. finally, the findings confirm the key role of broca 's area in restoration of speech perception, but only in individuals in whom broca 's area has been active prior to the loss of hearing. | the most dramatic progress in the restoration of hearing takes place in the first months after cochlear implantation. to map the brain activity underlying this process, we used positron emission tomography at three time points : within 14 days, three months, and six months after switch - on. fifteen recently implanted adult implant recipients listened to running speech or speech - like noise in four sequential pet sessions at each milestone. ci listeners with postlingual hearing loss showed differential activation of left superior temporal gyrus during speech and speech - like stimuli, unlike ci listeners with prelingual hearing loss. furthermore, broca 's area was activated as an effect of time, but only in ci listeners with postlingual hearing loss. the study demonstrates that adaptation to the cochlear implant is highly related to the history of hearing loss. speech processing in patients whose hearing loss occurred after the acquisition of language involves brain areas associated with speech comprehension, which is not the case for patients whose hearing loss occurred before the acquisition of language. finally, the findings confirm the key role of broca 's area in restoration of speech perception, but only in individuals in whom broca 's area has been active prior to the loss of hearing. |
endoscopic resection has now been accepted in south korea as the definitive treatment for cases of early gastric cancer (egc) that indicate it, mainly because endoscopic techniques and accessories have improved, and because the widespread national cancer screening program has increased detection of egc. furthermore, the number of egc cases that indicate endoscopic submucosal dissection (esd) and for which this procedure is considered has risen, because many technical difficulties have been overcome, and because esd has become the treatment of choice for egc that indicate it. moreover, it has been necessary to expand the use of esd, because the general population is aging, and quality of life after treatment for egc is an important consideration. however, the increased use of esd to treat egc should not be allowed to jeopardize patient survival. therefore, esd should only be used to treat egc in cases where (1) the risk of lymph node or distant metastasis is negligible, (2) complete resection is feasible, (3) the risk of complications is minimal, and (4) the chances of survival are comparable to those of surgical resection. nonetheless, as more cases of egc are considered as indicating esd, the rate of incomplete resection has increased with the expansion of indication of esd for egc. of course, incomplete resection does not necessarily indicate residual tumor ; nonetheless, additional treatment should be considered in patients with who are at high risk of residual tumor, whereas patients at low risk of residual tumor can simply be followed up closely. the term complete resection means that no tumor cells remain after treatment this can be revealed by both endoscopy and histopathology. however, as microscopic synchronous tumor can not be found without total mapping of the stomach, tumor - negative lateral and vertical resection margins are usually interpreted as indicating complete resection. microscopic synchronous tumor is reported to exist in 7.4% of cases despite complete resection. in cases of piecemeal resection, completeness in addition, in cases of irregular tumor arrangement or insufficient resection margin (less than 2 mm), residual tumor may not be detected. false - negative tumor margins can also occur when the specimen is mapped in an incorrect direction or with a wide interval. the specimen should be flattened and fixed immediately after resection, because the tissue may otherwise contract, leading to a false - positive result. similarly, false - positive tumor margins can be caused by the cautery effect, whereby microscopic tumor cells around the resection margin are ablated. the depth of tumor invasion, as well as the distance from the vertical resection margin, can be measured using pathologic mapping. when the tumor invades more than 500 m into the submucosal layer, the risk of lymph node or distant metastasis increases, irrespective of whether resection is complete. furthermore, lymphovascular tumor invasion confers an increased risk of lymph node or distant metastasis. put another way, the risk of residual tumor, or of lymph node / distant metastasis, increases with deeper submucosal or lymphovascular tumor invasion, irrespective of whether resection is complete. conversely, it may be that no residual tumor is found during follow - up without additional treatment in cases of incomplete resection, because false - positive tumor margins often occur. in addition, it is possible that no lymph node / distant metastasis has occurred in cases of deeper submucosal or lymphovascular tumor invasion. a resection is defined as curative when there is (1) no residual tumor, (2) no lymph node / distant metastasis, and (3) no additional treatment during long - term follow - up ; this definition is conferred irrespective of whether resection is complete, and without regard for either depth of submucosal invasion or presence of lymphovascular invasion. tumor - positive resection margin is a risk factor for residual tumor. in two retrospective studies, residual tumor was found in 29% of patients with tumor - positive lateral resection margin. additional risk factors for residual tumor were : piecemeal resection, diffuse type histology, and tumor - positive vertical or vertical and lateral resection margin. however, residual tumor was found in only 5.8% of patients with mucosal cancer and tumor - positive lateral - only resection margin, implying that curative resection had been achieved in most such patients. conversely, residual tumor was found in 20% of patients with submucosal tumor invasion, even though resection was complete ; therefore, submucosal tumor invasion is a strong risk factor for residual tumor, irrespective of whether resection is complete. it has been reported that the risk factors for residual tumor are : large tumor size, extent of tumor invasion at the lateral resection margin, and undifferentiated histology [7 - 11 ]. therefore, in patients who display these signs, as well as those that show tumor - positive resection margin, additional treatment should be considered. it is well known that tumor size, depth of tumor invasion, and undifferentiated histology are risk factors for lymph node metastasis [2,12 - 15 ]. for this reason, curative resection can not be confirmed in cases with a high risk of lymph node metastasis, even if resection is complete. to illustrate, in one retrospective study, the rate of complete resection was significantly lower, and rates of submucosal and lymphatic tumor invasion were significantly higher, in patients with undifferentiated histology than in patients with differentiated histology, and residual tumor was found in 40%, and lymph node metastasis in 13%, of patients who had undergone additional surgical resection. according to the expanded criteria for esd, when there is a high risk of lymph node metastasis, additional surgical resection is needed, irrespective of whether resection is complete. clinicians should decide on treatment strategy after incomplete resection of egc based on the tumor differentiation, tumor size, depth of tumor invasion, tumor involvement at resection margin, and lymphovascular tumor invasion. when mucosal cancer is less than 2 cm and there is microscopic tumor involvement at the lateral resection margin, but no definite gross residual tumor or lymphovascular tumor invasion, the patient should be closely followed up without immediate additional treatment, because the cautery effect may have caused a false - positive result. however, in cases of extensive tumor involvement at the lateral resection margin, large tumor size, or undifferentiated histology, additional treatment should be considered, because of the high risk of residual tumor. immediate additional esd should be used when tumor involvement at the lateral resection margin has been confirmed using histopathological mapping ; however, the disadvantages of such an approach are that the location of the residual tumor may be ambiguous, and that there may be no residual tumor (false - positive result). additional surgical resection should be considered in cases with a high risk of lymph node metastasis, irrespective of whether resection is complete ; that is, when there is large tumor size, submucosal or lymphovascular tumor invasion, or undifferentiated histology. additional esd can be performed to manage any residual tumor found during follow - up ; however, this may be technically difficult because of submucosal fibrosis (fig. 1). when curative resection is expected to be difficult using additional esd, surgical resection must be used. alternatively, argon plasma coagulation (apc) can be used to ablate the tumor when esd or surgical resection is contraindicated because of old age or severe comorbidity, or when the case involves residual adenoma rather than cancer (fig. however, as apc may not totally ablate the residual tumor, serial follow - up should be performed, and repetitive apcs may be required. as tumor - positive vertical resection margin indicates extensive submucosal tumor invasion and a high risk of lymph node metastasis, additional surgical resection is mandatory in such cases. moreover, additional surgical resection should be considered in cases that go beyond the expanded criteria, irrespective whether resection is complete because such cases involve a high risk of lymph node or distant metastasis. specifically, in a retrospective study, the tumor had recurred during follow - up in 10.1% of such patients after esd beyond the expanded criteria. the criteria for esd to treat egc have been expanded as endoscopic technical progress has been made ; for this reason, the rate of incomplete resection has also increased. when planning treatment strategy, clinicians should consider the quality of life and long - term survival of patients, as well as the risk of residual tumor and lymph node metastasis. | endoscopic resection of early gastric cancer is defined as incomplete when tumor cells are found at the resection margin upon histopathological examination. however, a tumor - positive resection margin does not always indicate residual tumor ; it can also be caused by tissue contraction during fixation, by the cautery effect during endoscopic resection, or by incorrect histopathological mapping. cases of highly suspicious residual tumor require additional endoscopic or surgical resection. for inoperable patients, argon plasma coagulation can be used as an alternative endoscopic treatment. immediately after the incomplete resection or residual tumor has been confirmed by the pathologist, clinicians should also decide upon any additional treatment to be carried out during the follow - up period. |
traditional methods of anchorage preparation often rely on patients ' cooperation and thus may be unpredictable. to ensure attainment of ideal treatment goals, temporary anchorage devices (tads) tads are devices temporarily fixed to bone for the purpose of enhancing orthodontic anchorage and which are subsequently removed after use. a commonly used tad would be the miniscrew implant, which is a fixation device placed for anchorage control using mechanical stability without the intention of osseointegration. miniscrew implants are often chosen among other tads due to its ease of insertion and removal, relative affordability, and numerous applications in various anatomical locations. in the national dental centre of singapore (ndcs), miniscrew implants were first introduced in the year 2004 but there is currently no available datum on their success rate in ndcs. success rates seem to vary amongst operators and its use is not widespread due to the purported high dislodgement rate and the need for surgical placement. in the orthodontic literature, there is also no clear information on whether patient - related, location - related, or miniscrew implant - related factors influence the success of miniscrews in ndcs. meta - analyses [3, 4 ] conducted have shown that a myriad of factors seem to affect their failure rates, but most variables still need additional evidence to support any possible associations. this is due to the extensive types and brands of miniscrew implants used and the heterogeneity of the included studies which may affect the success rates reported. thus, the aim of this retrospective study is to find out the success rate of miniscrew implants in ndcs pertaining to our local population, and whether they are a reliable form of tad. secondary objectives of this research will include finding out if patient - related factors, location - related factors, and miniscrew implant - related factors have any impact on success rates. records of patients who received miniscrew implants as part of their orthodontic treatment plan during the period of january 2010 to june 2012 were retrospectively examined. details of these patients were obtained from the surgical logbooks maintained in the day surgery department in ndcs. patients with the following data on the electronic dental records of ndcs were included : comprehensive demographic information including dental and skeletal relationships, dates of miniscrew placement, miniscrew loading, and miniscrew removal or dislodgement, type, length, and diameter of miniscrew, location of the miniscrew.smokers and patients with systemic medical conditions or those on long - term medications were excluded. comprehensive demographic information including dental and skeletal relationships, dates of miniscrew placement, miniscrew loading, and miniscrew removal or dislodgement, type, length, and diameter of miniscrew, location of the miniscrew. to see if there is any association with clinical success of miniscrew implants, 11 variables were collected for analysis. the 11 variables were divided into 3 categories : patient - related, miniscrew implant location - related, or miniscrew implant design - related factors as shown in table 1. patient - related factors include the age and gender of the patient, the dental malocclusion according to the british standards institute incisor classification, and the skeletal (sagittal and vertical) relationship based on the orthodontist 's clinical diagnosis and documentation. location - related factors of the miniscrew include the side of placement (right, left, or at the midline) and the jaw involved (maxilla or mandible). the miniscrew position in the oral cavity (anterior region, posterior region, retromolar, palate) was also examined. the posterior region refers to the buccal dentoalveolus distal to the canines, the tuberosity area and the infrazygomatic crest area. miniscrew implant - related factors include the type (vectortas or absoanchor) of miniscrew, its length (6 mm, 7 mm, 8 mm, 10 mm, 12 mm), and its diameter (1.3 mm, 1.4 mm, 2.0 mm). the miniscrew implant placement surgery was done by randomly assigned periodontists or oral and maxillofacial surgeons working in ndcs. they were told to brush the surgical site gently to maintain good oral hygiene and a bottle of 0.2% chlorhexidine mouth rinse was prescribed to be used twice daily for a week. this study examines early and late successes of the miniscrews at 2 time points : on the day of orthodontic loading and 12 months after insertion of the miniscrew implant. the outcome examined at the first time point (t1) will be the miniscrew implant 's initial stability, prior to orthodontic loading. success of the miniscrew implant at that juncture is defined by absence of infection of the surrounding soft tissues or any reason warranting its immediate removal or replacement prior to loading. failure of the miniscrew implant is defined as dislodgement of the miniscrew implant prior to loading or a miniscrew that have become excessively mobile such that orthodontic anchorage objectives can not be met. likewise, if the miniscrew implant has caused irreversible biological damage to adjacent structures as recorded by the clinician and was thus unusable, it was also considered a failure. the outcome at the second time point (t2) was assessed 12 months after the miniscrew 's insertion date or after its use as skeletal anchorage has ceased, whichever came first. success of the miniscrew implant at this juncture is defined by no dislodgement from the date of initial loading to the 12-month mark after the date of insertion or when intentional removal is carried out prior to the 12-month mark. it will mean that the miniscrew has sustained orthodontic loading forces throughout that time period and has served its skeletal anchorage function. similarly, failure of the miniscrew will be defined as dislodgement from the surgical site after orthodontic loading, any time before the 12-month period. the research protocol was approved by the singhealth institutional review board with cirb reference 2012/1057/d. descriptive statistics were initially performed to calculate the overall success rate of the miniscrew implants, as well as their specific success rates with regard to the 11 variables studied. the hosmer - lemeshow test was used to test for goodness of fit for the logistic regression model and results showed a good fit (at t1, p = 0.70 ; at t2, p = 0.11). the overall success rate was 94.7% at t1 (95% ci 92.1%97.3%) and 83.3% at t2 (95% ci 78.7%87.9%). the detailed information on success rates at t1 and t2 is shown in tables 2 and 3. out of the 214 successful miniscrew implants at t2, 37 of them were removed intentionally prior to the 12-month mark. these 37 miniscrews had a successful loading duration ranging from 2 to 12 months, and this is presented in figure 1. mean loading time for failed miniscrews at t2 was 3.5 months, ranging from 1 to 10 months and this is shown in figure 2. in the univariate analyses, length of miniscrew was significantly associated with success at t1 (p = 0.001). in the multivariate analysis of success rate at t1, only length of miniscrew implant was still found to be significantly associated (p = 0.002) with miniscrew implant success after being adjusted for age, gender, vertical skeletal malocclusion, recipient jaw, and type of miniscrew implant. due to multicollinearity, some variables in the univariate analyses were not included in the multivariate analysis. in the univariate analyses, sagittal skeletal malocclusion (p = 0.025) and vertical skeletal malocclusion (p = 0.028) multivariate analysis of the success of miniscrew implants at t2 found vertical skeletal malocclusion (p = 0.043) and length of miniscrew (p = 0.030) to be significantly associated with success rate. of the patient - related factors, there were no statistically significant differences between the variables at t1. but using univariate analyses at t2, there were associations between sagittal skeletal malocclusion and miniscrew implant success and also between vertical skeletal malocclusion and miniscrew implant success. miniscrew implants placed in patients with class iii malocclusion had a lower chance of success compared with those placed in patients with class i malocclusion (p = 0.01, or = 0.26, 95% ci 0.080.79). miniscrew implants in average angle patients had a higher chance of success compared with those placed in high angle patients (p = 0.025, or = 3.18, 95% ci 1.138.98). after adjusting for age, gender, sagittal skeletal malocclusion, dental malocclusion, recipient jaw, type of miniscrew implant, and length of miniscrew, vertical skeletal malocclusion was still found to be significantly associated (p = 0.043) with miniscrew implant success. miniscrew implants in average angle patients had a higher chance of success compared with those placed in high mandibular plane angle patients (p = 0.013, or = 4.22, 95% ci 1.3513.16). none of the location - related factors was significantly associated with success at t1 and t2. although at t1, for side of placement, there seem to be higher success rates for miniscrew implants placed in the midline (100%) compared to the left (94.9%) or ride side (94.5%). this was also reflected at t2 ; midline miniscrew implants had a 100% success rate compared to the left (85.4%) or right (80.8%). for recipient jaw, success rate of miniscrew implants in the mandible is higher at both t1 and t2 compared to the maxilla. but this is also not significant. similarly, the different sites of placement had no significant difference in success rates, although the retromolar area showed the highest success at t1 (100%) and t2 (94.4%). of the miniscrew implant - related factors, only length of miniscrew implant was significantly associated with success in the multivariate analyses at t1 (p = 0.002) and at t2 (p = 0.030). those with length 8 mm and 1012 mm had a higher chance of success at t1 compared to those with length 6 - 7 mm, respectively (8 mm : or = 11.88, 95% ci 2.7351.71, p = 0.001 ; 1012 mm : or = 10.50, 95% ci 1.1893.51, p = 0.035). at t2, those with length 1012 mm were found to have a higher chance of success compared with those with 6 - 7 mm (or = 17.95, 95% ci 1.83176.01, p = 0.013). the success rate of miniscrew implants in our study was 94.7% at t1 and 83.3% at t2. success rate at t1 is comparable to the success rate by lim. who reported a 93.1% success rate when they assessed initial stability of the miniscrews 1 week after placement. similarly, success rate at t2 is comparable to the rates in other retrospective studies of asian patients, (83.8%89.9%) [69 ]. this is in spite of the various miniscrew implant systems used, the varying operators and surgical techniques, and diverse management protocols reported by the different centres. the mean loading time for failed miniscrews in this study was 3.5 months, ranging from 1 to 10 months. most of the failures (30 out of 39) occurred within the first 5 months after loading. this is in accord with the findings which estimated that the highest failure rate occurred during the first 50150 days following loading. although a success rate of 83.3% is reasonable, there is still a 1 in 5 chance of failure using miniscrew implants for orthodontic anchorage.. demonstrated that palatal implants and miniplates showed a better survival rate compared to miniscrews. it will be interesting to find out how the success rate of other skeletal anchorage systems is compared against miniscrew implants in ndcs, and whether they can provide an improved and significantly more reliable form of tad for orthodontic use. this will be elucidated in a future study. due to the retrospective nature of this study, datum was sometimes lacking and not every variable mentioned in the literature was investigated and confounding factors may be present. the miniscrew implant placement surgery was done by randomly assigned periodontists or oral and maxillofacial surgeons working in ndcs. other than standard postoperative care instructions given to the patient, surgical techniques and surgical experience of the clinician may vary and affect the results of our study. operator 's surgical experience in miniscrew placement has been investigated in the literature, but this variable was excluded as we felt it was difficult to classify clinicians into groups according to years of experience or number of miniscrews inserted. this is because some clinicians do not work full time in ndcs, and it will be inaccurate to place a clinician in the inexperienced group who may have had prior experience in other centres before operating in ndcs. unlike a study in laboratory settings, insertion torque, loading forces, and direction of insertion thus, no data on the above variables could be obtained from the patient charts and treatment note records. also, it is clinically hard to record accurately a constant magnitude of force due to the rapid force level decay of orthodontic elastomeric chains, which are most commonly used in ndcs for orthodontic loading. the effect of delayed, early, or immediate loading on success rates was also not investigated as the individual patient 's orthodontic appointment varies after insertion of the miniscrew implant and there are no standard loading protocols followed by the orthodontists. types of tooth movement involved were investigated by other studies on success rates but this was not investigated as miniscrew implants are sometimes used for a combination of movements (e.g., both intrusion and distalization), thus making it difficult for any meaningful comparison of success rates to be made between any particular tooth movement. using univariate analysis at t2, sagittal skeletal malocclusion was associated with success rate. miniscrew implants placed in patients with class iii malocclusion had lower success compared with class i malocclusion. however, according to studies by antoszewska. and miyawaki., there is no obvious physiological reason why dentoalveolar abnormality or malocclusion type should affect success rate. hence, our initial finding may just be due to chance. using a multivariate analysis of success at t2, vertical skeletal malocclusion this was agreed upon by antoszewska. who found that, out of all the patient - related factors, only the vertical dimension seemed to play a role in determining success rates. our results showed that average mandibular plane angle patients had a significantly higher success rate compared to high mandibular plane angle patients. this corresponds with the study by miyawaki. who reported that the average mandibular plane angle group had significantly higher success rates compared to the high mandibular plane angle group. it was found that density of cortical bone was higher in subjects with small frankfort - mandibular plane angles and gonial angles. accordingly, high mandibular angle patients may have less dense cortical bone and this might affect success rates of miniscrew implants placed. this is supported by results of a meta - analysis which showed a positive association between the primary stability of miniscrew implants and cortical bone thickness of the surgical site. none of the location - related factors was significantly associated with success at both t1 and t2. for side of placement, at both t1 and t2, success rates for miniscrew implants placed in the midline were the highest, followed by the left then the right side but this did not reach statistical significance. park. and wu. reported that the left side had significantly higher success rates than the right side. in this study, placement of miniscrew implants on the left side does has a slightly higher success rate compared to the right side at both t1 and t2. this may be because most surgeons are right - handed, making it easier to insert miniscrews on the patient 's left side. also, there may be better hygiene maintenance on the left side in right - handed patients, who are most prevalent in the population. miniscrews located in the midline had the highest success rate in our study and these were all located in the palate. this is similar to the results of a study by lim. which showed a 100% success rate in the mid - palatal area. reasons for a high success rate in the mid - palatal region might be due to the abundance of compact bone and thin gingival tissue in the area, optimizing miniscrew implant insertion. the success rate of miniscrew implants in the mandible is higher compared to the maxilla at both t1 and t2 but this is not significant. this concurred with results from studies by miyawaki. and lim. who found no statistically significant association with success rates in the maxilla or mandible. the slightly higher success rate in the mandible may be attributed to thicker cortical bone in the mandible which is ideal for miniscrew implant stability. the different sites of placement had no significant difference in success rates in our study, and this supports the results by chen. who showed that placement site (maxilla or mandible, left or right side, anterior or posterior) presented no statistically significant association with success rates. this is in contrast to the study by tseng. who found that the only statistically significant factor affecting miniscrew success rates was location. success rates were the highest in the anterior tooth - bearing region of the maxilla, followed by the posterior tooth - bearing region of the maxilla, and success declines correspondingly in the anterior dentoalveolus of the mandible, posterior dentoalveolus of the mandible, and lastly the ramus. chen. also observed that the differences in success rates were significant in the different sites : success rate was best in maxillary anterior dentoalveolus followed by maxillary posterior dentoalveolus and then lastly in the mandibular posterior dentoalveolus. in this study, the success rates of miniscrews were compared at the anterior or posterior dentoalveolus separately from those inserted in the maxillary or the mandibular basal bone. since both maxillary and mandibular anterior miniscrews are grouped into one general category and vice versa for the posterior miniscrews, this may have decreased the statistical significance of the results. of the miniscrew implant - related factors, only length of miniscrew implant was significantly associated with success at both t1 and t2. lengths of 1012 mm had the highest success rate, followed by 8 mm and then the 6 - 7 mm lengths. this is probably due to the fact that longer miniscrews have the highest contact surface area for mechanical retention. this is in accord with the findings of chen. who found that length of microimplant is a significant risk factor. success rate for the longer microimplant (8 mm) used in their study was significantly higher than the shorter microimplant (6 mm). similarly, tseng. found that as success rate increases with length, it was the highest for miniscrews with lengths 12 mm and 14 mm. diameter of miniscrew implant had no statistically significant association with success in our study though it shows increasing success with increasing diameters. type of miniscrew implant showed no significant association with success although higher success rates were reported for the vectortas miniscrews compared to absoanchor microimplant at both t1 and t2. in ndcs, the more popular absoanchor microimplants used are the small head (sh1312) series, which has a diameter of 1.3 mm only and the lengths used in our study sample range from 6 to 10 mm, depending on the site of placement. in contrast, the vectortas miniscrews used in this study sample have a diameter of at least 1.4 mm or 2.0 mm, and lengths that range from 612 mm. due to the larger diameter and longer length of the vectortas miniscrews, success rates may be similarly increased. however, since there are no prior studies evaluating the success rates of the two types of miniscrew implants, no comparisons can be made. patient - related factors like vertical skeletal malocclusion were found to influence success : average mandibular plane angle patients have a higher chance of success compared to high mandibular angle patients probably due to the less dense cortical bone of the latter. miniscrew implant location - related factors have no significant effect on success but careful site selection must still be done to avoid encroaching on vital structures and to optimize orthodontic mechanics. of the miniscrew implant - related factors, thus, as long as surrounding anatomy permits, a longer miniscrew implant for better mechanical retention is recommended for higher success rate. | objective. to find out the success rate of miniscrew implants in the national dental centre of singapore (ndcs) and the impact of patient - related, location - related, and miniscrew implant - related factors. materials and methods. two hundred and eighty - five orthodontic miniscrew implants were examined from ndcs patient records. eleven variables were analysed to see if there is any association with success. outcome was measured twice, immediately after surgery prior to orthodontic loading (t1) and 12 months after surgery (t2). the outcome at t2 was assessed 12 months after the miniscrew 's insertion date or after its use as a temporary anchorage device has ceased. results. overall success rate was 94.7% at t1 and 83.3% at t2. multivariate analysis revealed only the length of miniscrew implant to be significantly associated with success at both t1 (p = 0.002) and t2 (p = 0.030). miniscrew implants with lengths of 1012 mm had the highest success rate (98.0%) compared to other lengths, and this is statistically significant (p = 0.035). at t2, lengths of 1012 mm had significantly (p = 0.013) higher success rates (93.5%) compared to 6 - 7 mm (76.7%) and 8 mm (82.1%) miniscrew implants. conclusion. multivariate statistical analyses of 11 variables demonstrate that length of miniscrew implant is significant in determining success. |
a 60-year - old man presented to our institute with a swelling in the superior medial aspect of his right orbit. it has been noticed for the last 1 year and has been gradually increasing in size. there was no history of defective vision, pain, or sudden increase in size. on examination, a soft to firm globular mass in the superomedial aspect of the orbit displaced the right eye downward and outward. it was not compressible or reducible, and the deeper margins were not palpable [fig extraocular movements in the right eye showed restriction in elevation, levo - elevation, and adduction. clinical photograph showing right eye eccentric proptosis with a mass in the superomedial aspect of the eye on magnetic resonance imaging, a 39 mm 26 mm lobulated well - encapsulated lesion was seen in the superomedial aspect of the right orbit. it was hyperintense in t2 and flair sequence and isointense in t1 imaging [fig. 2 ]. the mass displaced the medial rectus, encircling the superior rectus, and was extra- and intra - conal. an orbitotomy was performed after obtaining consent the mass was excised. t2 and flair sequence hyperintense lobulated well - encapsulated lesion was seen in the superomedial aspect of the right orbit involving both intra- and extra - conal space histopathological examination revealed areas of hemorrhage and clusters of short spindle cells in eosinophilic fibrillary background [fig. 3 ]. cells have elongated nucleus and scanty eosinophilic cytoplasm along with foci of myxoid stroma and irregular vascular channels, thick- and thin - walled lined by flat endothelial cells (a) glial tissue in a fibrocollagenous background (h and e, 10). (b) short spindle cells in an eosinophilic fibrillary background along with foci of myxoid stroma and irregular vascular channels (h and e, 40) postoperatively, the patient had improved extraocular motility, no diplopia, and the proptosis showed regression. at 1 year follow - up, glial heterotopia is abnormally located collection of normal glial tissue distant to the central nervous system. the most common location is around the nasal cavity, and it can occur in sites such as ethmoid sinus, middle ear, pharyngeal area, parapharyngeal space, pterygopalatine fossa, submandibular region, scalp, head, neck, and lung. they are generally present at birth but can manifest at any time in life. of the previously reported orbital glial heterotopia, the age of presentation has varied between 15 days and 20 years. our patient presented in the sixth decade, and the glial tissue was present in the orbit without any bony defect. there is only one other case reported in literature in a 59-year - old man who presented with optic disc swelling, and the orbital mass was found to be ectopic glial tissue. patients may present with varying symptoms of proptosis, diplopia, ptosis, or lid swelling due to mass effect. the most widely accepted theory in the pathogenesis of heterotopic glial tissue is that there is embryological herniation of glial tissue through a bony defect which subsequently may close, leaving no communication with the cranial cavity. on histopathology, heterotopic glial tissue found in the orbit is composed of astrocytes, gemistocytic astrocytes, fibrovascular connective tissue, lacrimal gland, cerebellar tissue, primitive retina, and skeletal muscle in varying proportions. there can be associated laminated calcific bodies or calcospherules ; it can be cystic filled with cerebrospinal fluid or clear fluid. some authors opine that this could be dysembryogenic in origin, and some opine that it could have got admixed with the developing muscle. surgery may be difficult and bony defects have to be looked for, which may then need multi - specialty approach. the authors certify that they have obtained all appropriate patient consent forms. in the form the patient(s) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal. the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity can not be guaranteed. the authors certify that they have obtained all appropriate patient consent forms. in the form the patient(s) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal. the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity can not be guaranteed. | heterotopic glial tissue is very rare in the orbit. our case was an adult, which is unique since most cases reported in literature involve children. we describe a case of a 60-year - old man who presented with an orbital mass, which histopathologically revealed heterotopic glial tissue. |
the transmission of light energy drastically increases following cataract extraction in the aphakic or pseudophakic eye. conventional intraocular lenses (iols) tend to block only ultraviolet light wavelengths (400 nm), but unlike the natural crystalline lens, they do not block light in the short - wave visible region of the spectrum commonly referred to as the blue - light spectrum. blue - light - filtering (blf) iols were originally designed to reduce this increase in actinic light exposure. a large body of data dating back to ham 's original studies in 1976 suggests that visible blue light is associated with special risks (e.g., photooxidative damage) [4, 5 ] when the blue - light blocking normally provided by anterior structures of the eye is lost. without natural blue - light - blocking mechanisms, the blue light that reaches the retina is sufficiently energetic to initiate oxidative damage, and the retina / retina pigment epithelium complex contains high amounts of photosensitizers (e.g., lipofuscin) with an action spectrum that matches the waveband of approximately 400 to 500 nm (hence, the description of the blue - light hazard). another ramification of filtering light in the visible spectrum is that it alters the incoming stimulus and changes visual function, as has been demonstrated by studies using psychophysical methods to measure the optical density of naturally occurring intraocular blfs like the anterior lens or macular pigment. such filtering has a practical advantage in improving vision in photopic conditions in a variety of species, including humans [8, 9 ]. for example, wooten and hammond originally argued that blf could influence visual range (how far one can see outdoors) by selectively attenuating the deleterious effects of atmospheric blue haze (see the empirical validation of the original modeling by hammond and colleagues). manufacturers of spectacle lenses or sunglasses designed for this purpose face the challenge of reducing glare without reducing visibility. this is often accomplished by using strategies that mimic biological mechanisms for adapting to variations in light intensity. for example, photochromic lenses becoming darker in proportion to light intensity are analogous to how the visual system adjusts sensitivity. polarizing lenses absorb horizontally oriented glare and pass vertical orientations that tend to not produce glare. filtering shorter wavelengths tends to also spare visibility since blue light is off the peak of the photopic spectrum. hence, blfs reduce retinal exposure to the wavelengths that tend to be the most actinic, photophobic, and susceptible to atmospheric (i.e., rayleigh) scatter while minimally absorbing the medium- to long - wave light that mediates object perception. one assumption inherent in many blf iol studies is that the filtering itself is one of the primary factors that produce the visual improvements observed with blue - light - absorbing iols. the current study was designed to examine the isolated effects of blue - light filtering. this was done by measuring photostress recovery and glare disability thresholds in pseudophakic patients implanted with clear (i.e., non - blf) iols. a clip - on spectacle lens with filtering characteristics matched to a commonly used blf iol (the acrysof natural iol ; alcon laboratories, inc.) was then compared to a matched clear lens in a within - patient, same - eye crossover design (see transmission spectra [1, 14 ]). this was a prospective, randomized, patient - masked crossover study conducted at 6 clinical sites in the united states from september 2014 to january 2014 (this trial is registered with clinicaltrials.gov nct01938989). the participating clinicians (listed in the acknowledgments) preselected candidates who fit several criteria. patients were required to be 21 years of age, be in good ocular health (based on a clinical interview), and be able to adequately participate in the psychophysical testing. patients with ocular pathology, degeneration, or media opacity that could have affected study assessments were excluded. slit - lamp examinations were used to confirm the presence of clear ocular media and the absence of clinically significant posterior capsule opacification. patients were excluded from participation if they had any conditions that could be exacerbated, triggered, or worsened by exposure to high - intensity light. patients were randomized to the order of use of blf and non - blf (clear) clip - on glasses, which were worn over patients ' habitual correction. equivalence of the transmission spectrum of the blf glasses with the natural chromophore used in a commercially available iol (acrysof natural ; alcon laboratories, usa) was confirmed before use in the study. the blf and non - blf clip - on glasses at each study site were reused for all patients evaluated at that site. patients were masked to the identity of the test and control clip - on glasses. the primary efficacy measure was photostress recovery time with the blf versus non - blf clip - on glasses. supportive efficacy measures included glare disability threshold, monocular corrected visual acuity as assessed using the 100% contrast etdrs chart under photopic lighting, and pupil size. the study protocol was approved by an independent ethics committee and institutional review board (aspire irb ; santee, ca, usa) for all participating clinical sites. patients were informed about the aims and methods of the study, and all patients signed a statement of informed consent. the apparatus used to measure glare disability and photostress recovery time was a 2-channel maxwellian view system and is shown in figure 1. the apparatus used white leds (lxml - pwn2, 4100 k ; phillips lumileds lighting company, san jose, ca, usa) that provided a relatively broad spectrum of energy (major peaks at 445 and 570 nm) as the glare source. the intensity of this glare source was electronically controlled (via pulse - width modulation) with customized computer software. one channel presented a target which was a 1-diameter disk (peak wavelength, 570 nm) containing a contrast grating stimulus (4 cycles per degree). the luminance of the bars within the grating was 0.1 candela / m. this target stimulus was used for both the glare disability and photostress measurements. to aid patients in maintaining the same position during the procedure, an eyepiece with a soft rubber eye cup prior to testing, patients were aligned to the optical system (this was facilitated by the structure of the eye cup). the blf- and non - blf - absorbing filters that we tested were incorporated into the eyepiece so that they were not visible to the patient. careful adjustments were made such that the image was in focus and in the plane of the patient 's pupil. when testing photostress recovery, the 1 target stimulus was shuttered at 500 ms on and off and a second channel provided a photobleaching light of high intensity (corneal irradiance, 5 log trolands). the photobleaching light was presented for 5 seconds. the patients were informed before the photostressor appeared and were instructed to keep their eyes open for the duration of the exposure. at the end of the 5-second exposure, timing began to determine the length of time required until the target stimulus became visible again. at the point of reemergence, the participant pressed a button and the timing stopped. there were 3 repetitions separated by a waiting period of 2 minutes for each patient. when testing glare disability, the target stimulus was presented at 2 seconds on and 1 second off. a second channel provided an annulus with an 11-degree inner diameter and 12-degree outer diameter. before each trial, the annulus was set at a level well below that which would cause the target stimulus to be veiled. the intensity of the annulus was then adjusted by the experimenter until the patient indicated verbally that the target stimulus was no longer visible. this procedure was repeated for a total of 5 measurements (recorded as time in seconds), and patients were instructed to maintain their criterion threshold across trials. five measurements were planned to be recorded per eye, unless a patient 's values had more than ~5% variability among measurements, in which case up to 4 additional measurements were conducted. careful calibrations were conducted in this study to ensure that the stimuli did not vary across sites. prior to each experimental sitting, a dedicated radiometer was used to ensure that total light output remained constant (s370 optometer with a pin-10 photohead, udt instruments, hawthorne, ca, usa). photometric calibrations were done using a telescopic spectral radiometer (model pr650, photoresearch inc. chatsworth, ca, usa) with the stimuli projected onto a white reflectance standard calibrated to the instrument. spatial alignment of the channels was checked every session by increasing the intensity of the light source and checking the precise location of the projected image against a fixed plate on the optical table. endpoints were analyzed in the efficacy analysis data set, which consisted of patients who provided data on 1 of the efficacy endpoints. the arithmetic mean photostress recovery time with blf versus non - blf glasses was compared using a 1-sided paired t - test. the difference between arithmetic means, 1-sided 95% ci, and p values were calculated. the arithmetic mean of glare disability threshold (x) for each patient was transformed by solving for y in the equation y = 4.89 + 5.43x. glare disability threshold data were compared for blf versus non - blf glasses using paired t - tests. corrected visual acuity, pupil size, and demographic / baseline characteristic data were summarized descriptively. assuming a log - transformed photostress recovery time sd of 0.35, a minimum sample size of 153 patients was determined to provide 80% power to detect a 20% difference in photostress recovery time. one hundred fifty - four of 156 enrolled patients completed the study (97.5%). one patient was invalidated because they violated inclusion / exclusion criteria (implantation with a blf iol). nine other patients had incomplete data sets due to physical limitations or inability to maintain alignment with the optical system. most patients were white (94.9%) and there were more women (58.3%) than men (41.7%). mean photostress recovery time was significantly lower when patients were wearing blf compared to non - blf clip - on glasses (p = 0.0001 ; 95% ci, 2.08 to 0.66 seconds ; table 2). the mean sd difference between blf and non - blf glasses was 1.4 4.3 seconds. glare disability thresholds were significantly higher (i.e., patients could tolerate more light before losing sight of the central grating target) with blf versus non - blf glasses (p = 0.00014 ; 95% ci, 0.060.18). the difference between blf and non - blf glasses was 0.12 0.38 log units. mean sd acuity was 0.051 0.105 logmar with the blf clip - on glasses (range : 0.30 to 0.34 logmar) and 0.049 0.099 logmar with the non - blf clip - on glasses (range : 0.3 to 0.38 logmar). mean sd pupil size was 3.54 0.80 mm and 3.52 0.79 mm with the blf and non - blf clip - on glasses, respectively. this study was aimed at assessing the role of additional blue filtration on vision when assessed under intense light conditions. patients with pseudophakia with clear implants had a short - wave filter placed in front of their eyes which had absorption characteristics matching the young natural crystalline lens [1, 14 ]. this filtering lens was placed behind optical baffling and an eye cup so that patients would remain blinded to the comparison with a lens transparent to visible light. consistent with past studies (see table 3) that have compared clear and blf iols, we found a visual advantage of blfs when testing glare disability and photostress recovery. blue - light filtration increased tolerance to veiling white light and significantly lowered glare disability thresholds. for example, the amount of energy (i.e., light intensity) required to veil the central target in the glare disability assessment was about 23% higher when using the blf lens compared to the clear lens (this percentage is based on a linear translation of the logged values). these changes could translate to meaningful differences in everyday life. as a practical example, if someone is driving 60 mph and is blinded by the sun or bright headlights, a 10-second photostress recovery time translates to about 880 feet traveled before normal visual function is recovered. regaining visual function 28% more quickly means seeing about 246 feet sooner (about two thirds the length of a football field). the mechanism for how blue - light filtering reduces photostress is straightforward : it simply reduces the intensity of the exposure and, hence, decreases recovery time. photostress is caused by short intense light exposure (which is particularly damaging when dark adapted due to higher levels of photosensitizing photopigment). such exposure causes adaptive change and photopigment isomerization which results in temporary loss of vision. endogenous blf mechanisms such as the natural crystalline lens or macular pigment and artificial mechanisms such as blf iols can absorb the incoming light and the forward scatter reducing the resultant loss of vision. this filtering protection, which operates over short exposures to intense light, does not lower overall sensitivity since the visual system adjusts sensitivity to offset stable changes in illumination [39, 40 ]. it has been argued [41, 42 ], for instance, that intraocular filters (macular pigment or blf iols) do not reduce glare disability. the basis for this argument is ecological ; to wit, the authors note that the empirical studies that have found that blf iols or macular pigment reduce glare disability have done so because they are biased by using bluer glare than target illumination, guaranteeing that glare light is preferentially reduced by yellow chromophores. headlights and sunlight cause glare and illuminate targets. that 's why clinical glare tests use glare and target illumination with similar spectra simulating real - world conditions. the question is whether similar spectra really simulate real - world conditions ; we would argue that they do not. unless a target (an object in the patient 's line of sight) reflects all wavelengths equally (unlikely unless it is a mirrored surface or a perfect white), it would never have the same spectrum as a glare source such as the sun. perhaps a more reasonable framing would be whether it is likely that a glare source (like the sun) would have more short - wave energy when compared to a probable target (hence, favoring a yellow intraocular filter). this is likely for several reasons.the most common source of glare is the white light of the sun, which has a strong short - wave component.glare sources often come in from the side or above, whereas targets are, by definition, objects that are within our line of sight. hence, even under the highly unlikely circumstance that the glare source and target share the exact same spectra, the composition would not be the same at the plane of the retina. light reflected from an object within our line of sight passes through the atmosphere and short - wave energy scatters out of the light path (rayleigh scatter does the opposite for objects in the surround).target stimuli are often composed of medium - wave light. one argument for the evolution of spectral sensitivity that peaks in the medium - wave region of the visible spectrum is that such a spectrum matches objects that are commonly perceived in the environment. many of the major pigments throughout nature are represented in the medium - and - longer - wave region. for example, chlorophyll is green and most of the carotenoids are yellow, orange, and red (few pigments one sees in a natural landscape are blue).the light stimulus for s - cones (i.e., blue - light - sensing cones), is largely filtered by yellow intraocular filters, which are relatively sparse, contribute little to the luminosity function, and mostly mediate color perception. spatial vision is largely mediated by medium- and long - wave cones (this spectra tuned to match ecological condition). the most common source of glare is the white light of the sun, which has a strong short - wave component. glare sources often come in from the side or above, whereas targets are, by definition, objects that are within our line of sight. hence, even under the highly unlikely circumstance that the glare source and target share the exact same spectra, the composition would not be the same at the plane of the retina. light reflected from an object within our line of sight passes through the atmosphere and short - wave energy scatters out of the light path (rayleigh scatter does the opposite for objects in the surround). one argument for the evolution of spectral sensitivity that peaks in the medium - wave region of the visible spectrum is that such a spectrum matches objects that are commonly perceived in the environment. many of the major pigments throughout nature are represented in the medium - and - longer - wave region. for example, chlorophyll is green and most of the carotenoids are yellow, orange, and red (few pigments one sees in a natural landscape are blue). the light stimulus for s - cones (i.e., blue - light - sensing cones), is largely filtered by yellow intraocular filters, which are relatively sparse, contribute little to the luminosity function, and mostly mediate color perception. spatial vision is largely mediated by medium- and long - wave cones (this spectra tuned to match ecological condition). based on these reasons, it could be argued that many of the available clinical glare tests that match the spectra of the glare source and target are not ecologically valid (rather, they are designed to diagnose clinical conditions). the best way to measure glare disability is to use a glare source that matches a source that is commonly encountered, such as the sun, and to use a target that is strongly at the peak of the photopic spectral sensitivity curve, thereby matching the spectral content of most objects one would view. several empirical studies have examined the effects of macular pigment and blf iols on visual function under glare conditions (see table 3). most of the blf iol studies have used a case - control design, with the exception of hammond. (2010), which compared glare disability and photostress recovery using a contralateral design where visual function was tested in 1 eye implanted with a blf iol compared to the other eye with a clear iol. that study found significant visual benefit in the eye with the blue - light - filtering iol. hammond and colleagues [17, 18 ] and gray and colleagues [15, 16 ] used stimuli or test circumstances that closely match real - world scenarios ; in the study by hammond, visual stimuli were matched to daytime sunlight, and gray tested patients using a driving simulator. in these studies, there was a clear benefit of the blf iols with regard to photostress recovery and glare disability thresholds, similar to the findings of the current study.. reported significant improvements using a blf iol when contrast sensitivity was measured under glare conditions, whereas k. hayashi and h. hayashi, muftuoglu., and neumaier - ammerer. found no difference in glare disability threshold or contrast sensitivity function between clear and blf iols. however, these studies used light sources without a significant blue - light component and/or glare devices without strong discriminative ability. the use of clip - on glasses worn over pseudophakic patients ' habitual correction is a potential limitation of this study. additionally, a single light source was used and psychophysical testing was not performed under varied light conditions (e.g., mesopic and scotopic). in this study, photostress recovery time and glare disability thresholds were significantly reduced by blue - light filtration, whereas visual acuity was not compromised. our findings are consistent with the visual benefit one might predict from simply returning the eye closer to its natural state, in this case, the young natural crystalline lens. yellow (i.e., blue - light filtering) chromophores added to an iol may help ameliorate complications of photostress and glare disability. | to evaluate the effects of filtering short wavelength light on visual performance under intense light conditions among pseudophakic patients previously implanted with a clear intraocular lens (iol). this was a patient - masked, randomized crossover study conducted at 6 clinical sites in the united states between september 2013 and january 2014. one hundred fifty - four bilaterally pseudophakic patients were recruited. photostress recovery time and glare disability thresholds were measured with clip - on blue - light - filtering and placebo (clear ; no blue - light filtration) glasses worn over patients ' habitual correction. photostress recovery time was quantified as the time necessary to regain sight of a grating target after intense light exposure. glare disability threshold was assessed as the intensity of a white - light annulus necessary to obscure a central target. the order of filter used and test eye were randomized across patients. photostress recovery time and glare disability thresholds were significantly improved (both p < 0.0001) when patients used blue - light - filtering glasses compared with clear, nonfiltering glasses. compared with a nonfiltering placebo, adding a clip - on blue - absorbing filter to the glasses of pseudophakic patients implanted with clear iols significantly increased their ability to cope with glare and to recover normal viewing after an intensive photostress. this result implies that iol designs with blue - light - filtering characteristics may be beneficial under intense light conditions. |
dandy - walker malformation is characterized by partial or complete agenesis of the cerebellar vermis, cystic dilation of the fourth ventricle, and an enlarged posterior fossa combined with superior displacement of the cerebellar hemisphere. disrupted cerebellar development is associated with neuromotor and cognitive developmental problems, and patients with dandy - walker malformation frequently present with symptoms of severe cerebellar ataxia, truncal hypotonia, and various developmental delays. it was previously reported that motor control impairments in patients with dandy - walker malformation were related to microstructural abnormalities in the connections from the cerebellum to the brainstem or cerebrum. however, it is difficult to evaluate cerebellar peduncle lesions as they are not wholly isolated from adjacent structures on conventional mri. the recent development of diffusion tensor imaging allows evaluation of the integrity and orientation of isolated neural tracts. diffusion anisotropy has been used to evaluate the extent of fiber damage in diseases that affect the neural tracts. diffusion tensor tractography, a three - dimensionally visualized version of diffusion tensor imaging, can be used to visualize neural tracts in three - dimensions. in the current study, we report a patient with severe ataxia due to dandy - walker malformation, who showed functional recovery associated with changes in the cerebellar peduncles. a 20-month - old female patient and her parents visited our department of pediatrics for severe ataxia and developmental delay. the patient was born at 40 weeks of gestation by normal vaginal delivery with a birth weight of 2 980 g. no specific perinatal history or family history of neurological disease or developmental delay was found. the patient showed severe developmental delay and could not sustain a sitting posture by herself with hands on the bottom due to severe ataxia, although she could turn her body over and creep on her belly. on neurological examination, she presented a marked psychomotor delay with severe truncal ataxia and mild axial hypotonia. conventional mri of the brain was taken and showed hypoplasia of the cerebellar vermis and enlargement of the fourth ventricle, indicating dandy - walker malformation. she had also undergone diffusion tensor mri to estimate the status of the subcortical neural structure. the patient received comprehensive rehabilitative therapy for approximately 10 months, and showed considerable improvement in the ataxia and trunk control. at the commencement of rehabilitation the functional ambulation category was used to assess the functional level : 0, non - ambulatory ; 1, needs continuous support from one person ; 2, needs intermittent support from one person ; 3, needs only verbal supervision ; 4, help is required on stairs and uneven surfaces ; 5, can walk independently anywhere. the initial functional ambulation category score before rehabilitative therapy was 0. for measurement of ataxia, berg 's balance scale was also used by estimating the performance of functional tasks. the ability to maintain balance while performing a chain of 14 tasks (sit to stand, stand to sit, stand and sit unsupported, transfer from bed to chair, stand with eyes closed, stand with feet together, reach forward, pick up an object from the floor, single leg stance and tandem standing, turn and look over each shoulder, turn 360, and stepping) was examined. a 5-point scale ranging from 0 to 4 however, at 10 months after rehabilitation therapy, she was able to stand alone with assistance and to walk independently with a walker on an even floor. the functional ambulation category scores and berg 's balance scale scores were re - measured at 10 months after therapy, along with mri and diffusion tensor imaging (table 1). the follow - up result of conventional mri showed no definite interval changes, compared with that of initial conventional mri (figure 1). changes in bbs and fac in patient and control subjects t2-weighted mri of the patient. mri of the sagittal midline section at initial (a) and 10-month follow - up (b) demonstrating cystic enlargement of the fourth ventricle and hypoplasia of the cerebellar vermis. there were no definite interval changes between initial and follow - up on conventional mri. a : anterior ; p : posterior. diffusion tensor imaging was also performed on six healthy, age - matched children (two males and four females, total : mean age 24.3 months, range 1933 months ; two males : mean age 24 months, range 1929 months ; four females : mean age 24.5 months, range 1933 months). control subjects were volunteers whose parents applied for this study. all participants in the control group had no specific pre / perinatal medical history or developmental delay. the developmental state and neurological examination of control subjects were evaluated by a pediatric neurologist. the kolmogorov - smirnov test was used to determine the normalities of variable distributions (p > 0.05). pearson 's correlation analysis was also conducted to estimate the influence of postnatal months on the fractional anisotropy and apparent diffusion coefficient values in each cerebellar peduncle. we found no correlations between various diffusion tensor imaging parameter and postnatal months in the superior cerebellar peduncle, middle cerebellar peduncle, or inferior cerebellar peduncle (correlation coefficient r = 0.14, 0.17, and 0.01, respectively). diffusion tensor imaging data were acquired using a 1.5 t philips gyroscan intera system (hoffman - laroche, mijdrecht, netherlands) equipped with a synergy - l sensitivity encoding (sense) head coil using a single - shot, spin - echo planar imaging pulse sequence. for each of the 32 non - collinear and non - coplanar diffusion sensitizing gradients, the 67 contiguous slices were acquired parallel to the anterior commissure- posterior commissure line. the imaging parameters were : matrix, 128 128 ; field of view, 221 221 mm ; echo time, 76 ms ; repetition time 10 726 ms ; sensitivity encoding factor, 2 ; echo planar imaging factor = 59 and b = 1 000 mm / s ; number of excitations, 1 ; and a slice thickness of 2.3 mm. diffusion tensor imaging datasets were preprocessed using the oxford center for functional magnetic resonance imaging of brain software library. eddy current - induced image distortions and motion artifacts were removed using affine multi - scale two - dimensional registration. three cerebellar peduncles (superior cerebellar peduncle, middle cerebellar peduncle, inferior cerebellar peduncle) were evaluated using diffusion tensor imaging -studio software (cmrm, johns hopkins medical institute, baltimore, md, usa). fiber tracking was based on the fiber assignment continuous tracking algorithm, a brute - force reconstruction approach, and a multiple regions of interest approach. the cerebellar peduncles were identified by choosing the fibers that passed through both regions of interests on the color map. region of interest 1 was assigned to the junction of the superior cerebellar peduncle between the upper pons and cerebellum on the coronal view, and region of interest 2 to the area between the lateral wall of the fourth ventricle and the inferior cerebellar peduncle at the fourth ventricle level on the axial view. region of interest 1 represented the ventral junctional area of the middle cerebellar peduncle between the pons and cerebellum, and the caudal junctional area of the middle cerebellar peduncle on the coronal view. for the inferior cerebellar peduncle, region of interest 1 represented the restiform body (blue), and region of interest 2 represented the caudal part (green) to the superior cerebellar peduncle on the axial view at the upper pons level. fiber tracking was started at the center of the seed voxel with an fractional anisotropy value greater than 0.2 and ended at the voxel with a fiber assignment lower than 0.2 and a tract turning - angle lower than 60. the fractional anisotropy values and apparent diffusion coefficient of each of the cerebellar peduncles were estimated and defined abnormal as a lesion with fractional anisotropy and apparent diffusion coefficient values deviating more or less than two standard deviations below those of normal control values. all three pairs of cerebellar peduncles were well detected in all control subjects as a known anatomical pathway. no significant differences were observed between diffusion tensor imaging parameters of the right and left superior cerebellar peduncle, middle cerebellar peduncle, or inferior cerebellar peduncle in control subjects. the results of the initial diffusion tensor tractography in the patient revealed that the superior cerebellar peduncle and middle cerebellar peduncle were well detected, but that the inferior cerebellar peduncle was not detected in either hemisphere. no significant differences in fractional anisotropy and apparent diffusion coefficient values were found between the right and left superior cerebellar peduncle and the middle cerebellar peduncles in the patient. initial diffusion tensor imaging results showed that the fractional anisotropy values of the superior cerebellar peduncle and middle cerebellar peduncle decreased by two standard deviations below that of normal control values, and that the apparent diffusion coefficient values increased by two standard deviations. in the follow - up evaluation, both fractional anisotropy and apparent diffusion coefficient values of the superior cerebellar peduncle were within two standard deviations of control subjects. by contrast, the middle cerebellar peduncle showed increased fractional anisotropy and decreased apparent diffusion coefficient values, but remained below two standard deviations of normal control values. however, at the 10-month follow - up, diffusion tensor tractography revealed both inferior cerebellar peduncles, although the fractional anisotropy had decreased by two standard deviations below normal control values, and the apparent diffusion coefficient had increased by two standard deviations (figure 2 and table 2). the red tracts indicate right side tracts, and the yellow tracts indicate left side tracts. the blue arrows indicate the inferior cerebellar peduncles, which were not detected at initial diffusion tensor tractography, but appeared at the 10-month follow - up diffusion tensor tractography. roi : region of interest ; scp : superior cerebellar peduncle ; mcp : middle cerebellar peduncle ; icp : inferior cerebellar peduncle ; r : right ; p : posterior ; a : anterior. diffusion tensor imaging data were acquired using a 1.5 t philips gyroscan intera system (hoffman - laroche, mijdrecht, netherlands) equipped with a synergy - l sensitivity encoding (sense) head coil using a single - shot, spin - echo planar imaging pulse sequence. for each of the 32 non - collinear and non - coplanar diffusion sensitizing gradients, the 67 contiguous slices were acquired parallel to the anterior commissure- posterior commissure line. the imaging parameters were : matrix, 128 128 ; field of view, 221 221 mm ; echo time, 76 ms ; repetition time 10 726 ms ; sensitivity encoding factor, 2 ; echo planar imaging factor = 59 and b = 1 000 mm / s ; number of excitations, 1 ; and a slice thickness of 2.3 mm. diffusion tensor imaging datasets were preprocessed using the oxford center for functional magnetic resonance imaging of brain software library. eddy current - induced image distortions and motion artifacts were removed using affine multi - scale two - dimensional registration. three cerebellar peduncles (superior cerebellar peduncle, middle cerebellar peduncle, inferior cerebellar peduncle) were evaluated using diffusion tensor imaging -studio software (cmrm, johns hopkins medical institute, baltimore, md, usa). fiber tracking was based on the fiber assignment continuous tracking algorithm, a brute - force reconstruction approach, and a multiple regions of interest approach. the cerebellar peduncles were identified by choosing the fibers that passed through both regions of interests on the color map. region of interest 1 was assigned to the junction of the superior cerebellar peduncle between the upper pons and cerebellum on the coronal view, and region of interest 2 to the area between the lateral wall of the fourth ventricle and the inferior cerebellar peduncle at the fourth ventricle level on the axial view. region of interest 1 represented the ventral junctional area of the middle cerebellar peduncle between the pons and cerebellum, and the caudal junctional area of the middle cerebellar peduncle on the coronal view. for the inferior cerebellar peduncle, region of interest 1 represented the restiform body (blue), and region of interest 2 represented the caudal part (green) to the superior cerebellar peduncle on the axial view at the upper pons level. fiber tracking was started at the center of the seed voxel with an fractional anisotropy value greater than 0.2 and ended at the voxel with a fiber assignment lower than 0.2 and a tract turning - angle lower than 60. the fractional anisotropy values and apparent diffusion coefficient of each of the cerebellar peduncles were estimated and defined abnormal as a lesion with fractional anisotropy and apparent diffusion coefficient values deviating more or less than two standard deviations below those of normal control values. all three pairs of cerebellar peduncles were well detected in all control subjects as a known anatomical pathway. no significant differences were observed between diffusion tensor imaging parameters of the right and left superior cerebellar peduncle, middle cerebellar peduncle, or inferior cerebellar peduncle in control subjects. the results of the initial diffusion tensor tractography in the patient revealed that the superior cerebellar peduncle and middle cerebellar peduncle were well detected, but that the inferior cerebellar peduncle was not detected in either hemisphere. no significant differences in fractional anisotropy and apparent diffusion coefficient values were found between the right and left superior cerebellar peduncle and the middle cerebellar peduncles in the patient. as such initial diffusion tensor imaging results showed that the fractional anisotropy values of the superior cerebellar peduncle and middle cerebellar peduncle decreased by two standard deviations below that of normal control values, and that the apparent diffusion coefficient values increased by two standard deviations. in the follow - up evaluation, both fractional anisotropy and apparent diffusion coefficient values of the superior cerebellar peduncle were within two standard deviations of control subjects. by contrast, the middle cerebellar peduncle showed increased fractional anisotropy and decreased apparent diffusion coefficient values, but remained below two standard deviations of normal control values. however, at the 10-month follow - up, diffusion tensor tractography revealed both inferior cerebellar peduncles, although the fractional anisotropy had decreased by two standard deviations below normal control values, and the apparent diffusion coefficient had increased by two standard deviations (figure 2 and table 2). the red tracts indicate right side tracts, and the yellow tracts indicate left side tracts. the blue arrows indicate the inferior cerebellar peduncles, which were not detected at initial diffusion tensor tractography, but appeared at the 10-month follow - up diffusion tensor tractography. roi : region of interest ; scp : superior cerebellar peduncle ; mcp : middle cerebellar peduncle ; icp : inferior cerebellar peduncle ; r : right ; p : posterior ; a : anterior. diffusion tensor imaging data were acquired using a 1.5 t philips gyroscan intera system (hoffman - laroche, mijdrecht, netherlands) equipped with a synergy - l sensitivity encoding (sense) head coil using a single - shot, spin - echo planar imaging pulse sequence. for each of the 32 non - collinear and non - coplanar diffusion sensitizing gradients, the 67 contiguous slices were acquired parallel to the anterior commissure- posterior commissure line. the imaging parameters were : matrix, 128 128 ; field of view, 221 221 mm ; echo time, 76 ms ; repetition time 10 726 ms ; sensitivity encoding factor, 2 ; echo planar imaging factor = 59 and b = 1 000 mm / s ; number of excitations, 1 ; and a slice thickness of 2.3 mm. diffusion tensor imaging datasets were preprocessed using the oxford center for functional magnetic resonance imaging of brain software library. eddy current - induced image distortions and motion artifacts were removed using affine multi - scale two - dimensional registration. three cerebellar peduncles (superior cerebellar peduncle, middle cerebellar peduncle, inferior cerebellar peduncle) were evaluated using diffusion tensor imaging -studio software (cmrm, johns hopkins medical institute, baltimore, md, usa). fiber tracking was based on the fiber assignment continuous tracking algorithm, a brute - force reconstruction approach, and a multiple regions of interest approach. the cerebellar peduncles were identified by choosing the fibers that passed through both regions of interests on the color map. region of interest 1 was assigned to the junction of the superior cerebellar peduncle between the upper pons and cerebellum on the coronal view, and region of interest 2 to the area between the lateral wall of the fourth ventricle and the inferior cerebellar peduncle at the fourth ventricle level on the axial view. region of interest 1 represented the ventral junctional area of the middle cerebellar peduncle between the pons and cerebellum, and the caudal junctional area of the middle cerebellar peduncle on the coronal view. for the inferior cerebellar peduncle, region of interest 1 represented the restiform body (blue), and region of interest 2 represented the caudal part (green) to the superior cerebellar peduncle on the axial view at the upper pons level. fiber tracking was started at the center of the seed voxel with an fractional anisotropy value greater than 0.2 and ended at the voxel with a fiber assignment lower than 0.2 and a tract turning - angle lower than 60. the fractional anisotropy values and apparent diffusion coefficient of each of the cerebellar peduncles were estimated and defined abnormal as a lesion with fractional anisotropy and apparent diffusion coefficient values deviating more or less than two standard deviations below those of normal control values. all three pairs of cerebellar peduncles were well detected in all control subjects as a known anatomical pathway. no significant differences were observed between diffusion tensor imaging parameters of the right and left superior cerebellar peduncle, middle cerebellar peduncle, or inferior cerebellar peduncle in control subjects. the results of the initial diffusion tensor tractography in the patient revealed that the superior cerebellar peduncle and middle cerebellar peduncle were well detected, but that the inferior cerebellar peduncle was not detected in either hemisphere. no significant differences in fractional anisotropy and apparent diffusion coefficient values were found between the right and left superior cerebellar peduncle and the middle cerebellar peduncles in the patient. as such initial diffusion tensor imaging results showed that the fractional anisotropy values of the superior cerebellar peduncle and middle cerebellar peduncle decreased by two standard deviations below that of normal control values, and that the apparent diffusion coefficient values increased by two standard deviations. in the follow - up evaluation, both fractional anisotropy and apparent diffusion coefficient values of the superior cerebellar peduncle were within two standard deviations of control subjects. by contrast, the middle cerebellar peduncle showed increased fractional anisotropy and decreased apparent diffusion coefficient values, but remained below two standard deviations of normal control values. however, at the 10-month follow - up, diffusion tensor tractography revealed both inferior cerebellar peduncles, although the fractional anisotropy had decreased by two standard deviations below normal control values, and the apparent diffusion coefficient had increased by two standard deviations (figure 2 and table 2). the red tracts indicate right side tracts, and the yellow tracts indicate left side tracts. the blue arrows indicate the inferior cerebellar peduncles, which were not detected at initial diffusion tensor tractography, but appeared at the 10-month follow - up diffusion tensor tractography. roi : region of interest ; scp : superior cerebellar peduncle ; mcp : middle cerebellar peduncle ; icp : inferior cerebellar peduncle ; r : right ; p : posterior ; a : anterior. herein, we report a patient who showed functional improvement from a cerebellar peduncle lesion due to dandy - walker malformation and corresponding changes in the diffusion tensor imaging findings. the patient could not sit independently at the initial evaluation, but could walk with a walker after 10 months. on initial diffusion tensor tractography, the inferior cerebellar peduncle was not detected, the fractional anisotropy of the superior cerebellar peduncle and middle cerebellar peduncle were decreased by two standard deviations below that of normal subjects, and the apparent diffusion coefficient was increased by two standard deviations over normal control values. however, after 10 months follow - up, the inferior cerebellar peduncle was detected, and diffusion parameters improved, with an increase in fractional anisotropy and decrease in apparent diffusion coefficient to within two standard deviations of the normal subjects. a decrease in fractional anisotropy an increase in apparent diffusion coefficient values is caused by hindered water motion due to axonal damage. therefore, the decreased fractional anisotropy and increased apparent diffusion coefficient values of the cerebellar peduncles in our patient at initial examination suggest that the neural tract was affected, while the increased fractional anisotropy and decreased apparent diffusion coefficient values at follow - up indicate improvement from the lesions. moreover, the inferior cerebellar peduncle was not detected at the initial evaluation, but was well detected at the follow - up diffusion tensor tractography. these diffusion tensor imaging and diffusion tensor tractography results coincided with clinical improvement of the patient, which was not detected by conventional mri, suggesting that diffusion tensor imaging is useful for assessing cerebellar peduncles in cases of suspected cerebellar peduncle injury. several studies have identified normal cerebellar peduncles using diffusion tensor imaging, and a recent study demonstrated that patients with cerebellar peduncle injuries develop diffuse axonal injury or pontine infarcts. these results agreed with our findings that the recovery of cerebellar peduncle injury was associated with clinical improvement of ataxia. to the best of our knowledge, this is the first diffusion tensor imaging study examining recovery of injured cerebellar peduncles in a patient with dandy - walker malformation., we report on a patient who showed clinical improvement of ataxia according to the recovery of disrupted cerebellar peduncles using diffusion tensor imaging. diffusion tensor imaging can be performed along with conventional mri for patients with dandy - walker malformation. | we report a patient with severe ataxia due to dandy - walker malformation, who showed functional recovery over 10 months corresponding to a change in a cerebellar peduncle lesion. a 20-month - old female patient who was diagnosed with dandy - walker syndrome and six age- and sex - matched healthy control subjects were enrolled. the superior cerebellar peduncle, the middle cerebellar peduncle, and the inferior cerebellar peduncle were evaluated using fractional anisotropy and the apparent diffusion coefficient. the patients functional ambulation category was 0 at the initial visit, but improved to 2 at the follow - up evaluation, and berg 's balance scale score also improved from 0 to 7. initial diffusion tensor tractography revealed that the inferior cerebellar peduncle was not detected, that the fractional anisotropy of the superior cerebellar peduncle and middle cerebellar peduncle decreased by two standard deviations below, and that the apparent diffusion coefficient increased by two standard deviations over normal control values. however, on follow - up diffusion tensor tractography, both inferior cerebellar peduncles could be detected, and the fractional anisotropy of superior cerebellar peduncle increased to within two standard deviations of normal controls. the functional improvement in this patient appeared to correspond to changes in these cerebellar peduncles. we believe that evaluating cerebellar peduncles using diffusion tensor imaging is useful in cases when a cerebellar peduncle lesion is suspected. |
the cerebral epiphysis (pineal gland) is broadly involved in the synchronization of bodily functions(s) with the environment and serves as a regulator of regulators. it is known that, in addition to its synthesis of melatonin (mel) from serotonin, this organ secretes various proteins and peptides [1, 2 ]. the epiphysis additionally has a rich supply of adrenergic nerve fibers that greatly influence its secretory activity. the cerebral epiphysis, through its production of mel and its effect on serotonin, affects many neuroendocrine functions. however, many researchers regard mel as the sole mediator of epiphyseal functions. recently, a number of studies reported that epiphyseal proteins have the ability to regulate various physiological functions in numerous animals and thus hypothesized that these proteins effectively acted as epiphyseal hormones [37 ]. in spite of these studies, the functional role of epiphyseal hormones and proteins in antioxidant defense system of vital organs remains poorly understood. oxidative damage to vital organs, particularly to the liver and kidney, becomes very important in humans and animals when the antioxidant defense system is either absent or functioning inefficiently. inasmuch as the liver and kidney are metabolically highly active in xenobiotic metabolism and excretion, they have, compared to other organs, a greater load of free radical activity and thus are more prone to oxidative damage [8, 9 ]. as a consequence, reactive oxygen species (ros) tissue injuries are found more commonly in these organs, as are their sequelae of toxic damage, disease, and the ultimate death of the biological systems in which they occur [10, 11 ]. in healthy animals, there is a balance between the production of various ros and antioxidant defenses. it has been noted that the antioxidant defense systems of the organs of many species are inadequately equipped to take up an excessive load of free radicals and ros, and that when this does take place it is associated with increased oxidative damage in the affected organs [1012 ]. having to contend with oxidative stress brought about by uncontrolled oxidation of important molecules in foods and body tissues is thus a significant biological challenge faced by most living organisms [13, 14 ]. antioxidant therapies, which are based on upregulation of body antioxidant defense system, are now a commonly employed strategy for combating molecular damage in various tissues. variations of this approach, which make use of exogenously administered antioxidant agents, could potentially provide an important and inexpensive alternative treatment for diseases related to oxidative stress. hence, the present study was based on the perceived value of investigating several molecules for their potential benefit in bolstering the antioxidant defense systems of the liver and kidney. melatonin (n - acetyl-5-methoxytryptamine), which has been long known as the pineal 's major secretory product, has potent antioxidant properties [1517 ]. earlier, we reported on the antioxidant action of buffalo (bubalus bubalis) epiphyseal proteins (beps) under fluoride and arsenic - induced oxidative stress in blood, brain, and kidney [26, 1820 ]. however, the effect of epiphyseal hormone and proteins on liver and kidney antioxidant defense system has not previously been studied. thus in view of findings reported by ourselves and others, we hypothesized that pineal bep and mel might enhance levels of antioxidant defense activity (enzymatic and nonenzymatic) in the liver and kidney and could therefore be of benefit for animals undergoing oxidative stress. all the procedures, conducted on the experimental animals were duly approved by the institutional animal ethics committee (iaec) of indian veterinary research institute (ivri) for the purpose of control and supervision of experiments on animals. all chemicals used in the study were of analytical grade from himedia, loba chemie (mumbai, india), srl chemicals, india. buffalo (bubalus bubalis) epiphyseal proteins were supplied by the neurophysiology laboratory, division of physiology and climatology, ivri (izatnagar, india). the present study was carried out on eighteen sexually mature and healthy female wistar rats of 130142 g body weights, procured from the laboratory animal resource (lar) section of the ivri. rats were housed in polypropylene cages in a light / dark (ld) cycle of 12 h, in a pathogen - free, temperature- and humidity - controlled environment (set at 21 2c and relative humidity at 50 10%, resp.). after an acclimatization period of 1 week, they were weighed and randomly assigned to various groups with approximately equal initial group mean body weights. following allocation, the animals were marked with picric acid solution for individual identification. all the animals had free access to the standard laboratory animal diet and water, which were replenished on daily basis. the experimental design for the present study, including various groups, doses, route of administration, and duration of treatment is presented in table 1. beps were used as the experimental agent in as much as its safety and utility were confirmed in a study that we have previously published. appropriate dosages of bep and mel were optimized from experience in our previous work and thereafter they were dissolved in a suitable vehicle before administration at exactly 16.00 hrs. daily observations were taken for the behavioral changes and mortality, if any, throughout the experimental period. the samples were collected at the end of the experiment (day 28). the liver and kidney were collected, cleaned, rinsed in chilled saline, blotted, weighed, and stored at 20c. frozen liver and kidney tissues samples were partially thawed, and 200 mg of sample was weighed and taken in 2 ml of ice - cold saline. another 200 mg of the samples were weighed separately and taken in 2 ml of 0.02 m edta for gsh estimation. organ homogenates were prepared using an ika homogenizer (germany), under ice - cold conditions and collected, and then centrifuged for 10 min at 3000 rpm. thereafter, cell - free supernatant was collected and transferred to precooled microfuge tubes in duplicate and stored at below 20c. these supernatants were used for estimation of total proteins (organs), lipid peroxidation (lpo), and enzyme activity namely, catalase (cat), superoxide dismutase (sod), glutathione peroxidase (gpx), and glutathione reductase (gr) as well as non - enzymatic namely, reduced glutathione (gsh) antioxidant defense level. estimations of different antioxidant defense - related biochemical parameters in hepatic and renal tissues were carried out using a double beam uv - vis spectrophotometer (uv 5704 ss, ecil, india). lipid peroxidation (lpo)renal and hepatic tissues lpo was determined in terms of malondialdehyde (mda) production by the method of rehman. renal and hepatic tissues lpo was determined in terms of malondialdehyde (mda) production by the method of rehman. reduced glutathione (gsh)the concentration of gsh in renal and hepatic tissues was estimated by evaluating free - sh groups, using the 5, 5-dithiobis-2-nitrobenzoic acid (dtnb) method as described by sedlak and lindsay. the concentration of gsh in renal and hepatic tissues was estimated by evaluating free - sh groups, using the 5, 5-dithiobis-2-nitrobenzoic acid (dtnb) method as described by sedlak and lindsay. catalase (cat)activities of catalase enzymes were estimated as described by bergmayer and were expressed as nm h2o2 utilized per minute per milligram protein. activities of catalase enzymes were estimated as described by bergmayer and were expressed as nm h2o2 utilized per minute per milligram protein. superoxide dismutase (sod)superoxide dismutase activities were estimated using the method described by madesh and balasubramanian and are expressed as sod units [one unit of sod is the amount (g) of protein required to inhibit the mtt reduction by 50% ]. superoxide dismutase activities were estimated using the method described by madesh and balasubramanian and are expressed as sod units [one unit of sod is the amount (g) of protein required to inhibit the mtt reduction by 50% ]. glutathione peroxidase (gpx)glutathione peroxidase activities were determined by the method of paglia and valantine. the enzyme activity is expressed as u / mg of protein, and one unit of enzyme activity is defined as 1 nm of substrate (nadph) utilized / min / mg protein at 25c. the enzyme activity is expressed as u / mg of protein, and one unit of enzyme activity is defined as 1 nm of substrate (nadph) utilized / min / mg protein at 25c. glutathione reductase (gr)the enzyme activities were assayed by the method of goldberg and spooner, and activity is expressed as nm nadph oxidized to nadp / min / mg protein. the enzyme activities were assayed by the method of goldberg and spooner, and activity is expressed as nm nadph oxidized to nadp / min / mg protein. protein assayprotein contents in liver and kidneys homogenates were determined and calculated by the method of lowry. protein contents in liver and kidneys homogenates were determined and calculated by the method of lowry. statistical analysisdifferences between groups were statistically analyzed by one - way anova, and the differences between the means of groups were separated by the least significant difference (lsd) test. values differing by p <.05 are regarded as significant. a computer program (spss 10.01, spss inc. differences between groups were statistically analyzed by one - way anova, and the differences between the means of groups were separated by the least significant difference (lsd) test. values differing by p <.05 are regarded as significant. a computer program (spss 10.01, spss inc. all the animals were healthy, and no mortality was observed during the entire period of the experiment. the activities of hepatic and renal glutathione peroxidase (gpx), superoxide dismutase (sod), and the levels of lipid peroxidation (lpo) and glutathione (gsh) were measured to assess the level of antioxidant defense in female rats. bep significantly increased (p <.05) hepatic lpo over the control and melatonin - treated animals (table 2). however, no effects were observed on cat activity in hepatic of bep- and mel - treated groups. interestingly, gpx and sod activity in hepatic tissues was significantly increased (p <.05) in both bep- and mel - administered groups. no effect of mel was recorded on hepatic gsh and gr level, however, their levels were significantly higher (p <.05) in bep - treated animals. similarly, the renal lpo level was significantly (p <.05) greater in bep administered animals (table 3). on the other hand, renal cat, gsh, gr, and gpx levels were markedly enhanced in mel and bep - supplemented groups as compared to control animals (table 3) however, effects on cat and gsh were greater in bep - treated animals than in those which were given mel (table 3). markers of oxidative stress damage can be found in many disease states including renal damage, chronic heart disease, and liver disease [28, 29 ]. many diseases and increased toxicity are often associated with oxidative stress in different vital organs and are characterized by the reductions in the activity of specific enzymes activity, including catalase, sod, gpx, and gr. we observed marked increases in renal cat, gsh, gr, and gpx in mel- and bep - supplemented animals. interestingly, gpx and sod activities in hepatic tissues were also significantly increased (p <.05) in bep- and mel - administered groups. however, only hepatic gsh and gr levels were significantly higher (p <.05) in bep - treated animals. these pharmacological effects of bep and mel may be relevant to their potentiation of antioxidant defense activity in renal and hepatic tissues [1719 ]. present study findings do not support the hypothesis of increased endogenous antioxidant activity in response to the deleterious / toxic effect of bep in the rat 's liver and kidney. since, liver and kidney protective responses to any deleterious agents are immediate and transient, and so these endogenous protective / beneficial effects can not be for long periods (beyond 2-weeks), as in our study. therefore, findings of the present study support the antioxidant properties of bep. also, bep increased not only the catalase (kidney), sod (liver), gpx, and gr enzymes but also gsh level. gpx removes h2o2 by coupling its reduction to h2o2 with oxidation of reduced glutathione, gsh. it is the most important enzyme for extraperoxisomal inactivation of h2o2, especially in the liver and kidney. since the liver is a major source of gsh, metabolism of xenobiotics in the liver, which can drastically deplete liver gsh, may also result in gsh depletion in other tissues. in numerous reactions, gpx, gr, and gsh act as free radical scavenging molecules and therefore the finding in our study that these enzymatic and nonenzymatic antioxidant defense systems are upregulated in liver and kidney of animals that exhibit oxidative stress underscore the critical importance of bep and mel as potentiators of antioxidant activity. melatonin, the chief secretory product of the cerebral epiphysis, is a direct free radical scavenger and indirect antioxidant. in addition to its direct free radical scavenging activity, mel also enhances the synthesis of sod, gsh, cat, gr, and gpx. in the present study, it was shown that bep from the cerebral epiphysis possesses antioxidant properties exceeding in some cases the effects of the mel. the superior effects of beps might be due to their direct antioxidant effects, but also because beps have been implicated in the stimulation of mel production. in recent studies, of relevance here is that mel has a very short half - life and is metabolized and excreted within a few minutes. however, the presence of bep may represent a constant source of stimulation for mel synthesis. this might also be the reason that certain enzymes have greater antioxidative effects when compared to mel action alone. besides increasing the activity of the antioxidative enzymes, it is known that melatonin also increases their expression. this observation suggests that the indoleamine may have a physiological role in promoting endogenous antioxidative defense activity. these findings support the hypothesis that bep and mel modulate antioxidant defense of liver and kidney, and also demonstrate that these agents are generally equivalent in the potency of their of antioxidant activities. these experimental findings establish that buffalo epiphyseal proteins and melatonin are effective antioxidants and that they may play a protective role against hepatic and renal damage induced by oxidative stress. the findings suggest that epiphyseal proteins and melatonin may have therapeutic potential as antioxidants drugs for the management of oxidative stress. s. r. pandi - perumal is a stockholder and the president and chief executive officer of somnogen inc., a new york corporation. he declared no competing interests that might be perceived to influence the content of this paper. all remaining authors declare that they have no proprietary, financial, professional, nor any other personal interest of any kind in any product or services and/or company that could be construed or considered to be a potential conflict of interest that might have influenced the views expressed in this paper. | the cerebral epiphysis (pineal gland) secrets melatonin and number of other proteins and peptides. it was thus hypothesized that antioxidant properties of epiphyseal proteins and melatonin could potentially benefit from exogenous therapies. in view of the therapeutic potential of these proteins, the present experiment was conducted to investigate the effect of buffalo epiphyseal proteins (bep, at 100 g / kg bw, i.p.) and melatonin (mel, at 10 mg / kg bw, i.p) on changes in hepatic and renal antioxidant enzymes of adult female wistar rats. buffalo epiphyseal proteins significantly (p <.05) increased hepatic lipid peroxidation (lpo), superoxide dismutase (sod), glutathione reductase (gr), glutathione peroxidase (gpx), reduced glutathione (gsh), and renal lpo, catalase (cat), gr, gsh, gpx levels as compared to control animals. similarly, mel treatment significantly (p <.05) up - regulated hepatic sod and gpx activity, whereas cat, gr, gpx, and gsh levels in renal tissues were increased while sod and lpo remained unaffected. buffalo epiphyseal protein treatment produced greater effects on hepatic gpx and renal cat and gsh levels than did mel. these findings support the conclusion that buffalo epiphyseal proteins and melatonin activate a number of antioxidant mechanisms in hepatic and renal tissues. |
a beverage was produced by fermentation of an extract from 50 kinds of fruits and vegetables (see additional file 1). the extract was obtained using sucrose - osmotic pressure in a cedar barrel for seven days and was fermented by lactic acid bacteria (leuconostoc spp.) and yeast (zygosaccharomyces spp. and pichia spp.) for 180 days. the fermented beverage showed scavenging activity against 1,1'-diphenyl-2-picrylhydrazyl (dpph) radicals, and significantly reduced the ethanol - induced damage of gastric mucosa in rats. analysis by high performance anion exchange chromatography (hpaec) showed that this beverage contained high levels of saccharides, estimated between 550 and 590 g / l ; mainly glucose and fructose, and a small amount of undetermined oligosaccharides. recently, it was reported that different positions of glycosidic linkage of oligosaccharide isomers affected physiological properties as well as physical properties [3 - 5 ]. development of hplc analysis with high sensitivity and separation ability enables the detection and isolation of oligosaccharides in the fermented beverage. we have previously examined the preparation of saccharides of the fructopyranoside series from the fermented beverage of plant extracts, such as o--d - fructopyranosyl-(2->6)-d - glucopyranose, o--d - fructopyranosyl-(2->6)-o--d - glucopyranosyl-(1->3)-d - glucopyranose and o--d - fructopyranosyl-(2->6)-o-[-d - glucopyranosyl-(1->3)]-d - glucopyranose. the characteristics of o--d - fructopyranosyl-(2->6)-d - glucopyranose were non - cariogenicity and low digestibility, and the unfavorable bacteria that produce mutagenic substances did not use the saccharide. recently, we have studied isolation and identification of novel non - reducing trisaccharides, such as o--d - glucopyranosyl-(1->1)-o--d - fructofuranosyl-(21)--d - glucopyranoside and o--d - galactopyranosyl-(1->1)-o--d - fructofuranosyl-(21)- -d - glucopyranoside from the beverage, and those saccharides were confirmed to be produced by fermentation. in this paper, we have confirmed structures of the novel trisaccharides (fig. 1) : o--d - fructofuranosyl-(2->1)-o-[-d - glucopyranosyl-(1->3)]--d - glucopyranoside (named " 3--d - glucopyranosyl, -isosucrose "), o--d - glucopyranosyl-(1->2)-o-[-d - glucopyranosyl-(1->4)]-d - glucopyranose (4--d - glucopyranosyl sophorose) and o--d - fructofuranosyl-(2->6)-o--d - glucopyranosyl-(1->3)-d - glucopyranose (6--d - fructofuranosyl laminaribiose), isolated from the fermented beverage using methylation analysis, maldi - tof - ms and nmr measurements. structures of o--d - fructofuranosyl-(2->1)-o-[-d - glucopyranosyl-(1->3)]--d - glucopyranoside (1), o--d - glucopyranosyl-(1->2)- o-[-d - glucopyranosyl-(1->4)]-d - glucopyranose (2) and o--d - fructofuranosyl-(2->6)- o--d - glucopyranosyl-(1->3)-d - glucopyranose (3). saccharides 1, 2 and 3 were isolated from the fermented beverage of plant extracts using carbon - celite column chromatography, and were shown to be homogeneous using anion exchange hplc [tr, sucrose (relative retention time ; retention time of sucrose = 1.0) : 1.89, 2.23 and 2.40 respectively ]. the retention time of saccharides 1, 2 and 3 did not correspond to that of any authentic saccharides [glucose (0.62), fructose (0.68), sucrose (1.00), maltose (1.43), trehalose (0.58), laminaribiose (1.33), raffinose (1.23), 1-kestose (1.47), 6-kestose (1.75), neokestose (1.90), maltotriose (2.59), panose (1.87), nystose (2.06), fructosylnystose (3.81), o--d - fructopyranosyl-(2->6)-d - glucopyranose (0.83), o--d - fructopyranosyl-(2->6)-o--d - glucopyranosyl-(1->3)-d - glucopyranose (1.74), o--d - fructopyranosyl-(2->6)-o-[-d - glucopyranosyl-(1->3)]-d - glucopyranose (1.72), o--d - glucopyranosyl-(1->1)-o--d - fructofuranosyl-(21)- -d - glucopyranoside (1.24), o--d - galactopyranosyl-(1->1)-o--d - fructofuranosyl-(21)--d - glucopyranoside (0.84), 2(2--d - glucopyranosyl)isokestose (1.57), 2(2--d - glucopyranosyl)2isokestose (1.79), 2(2--d - glucopyranosyl)3isokestose (2.09), 2(2--d - glucopyranosyl)nystose (2.17), 2(2--d - glucopyranosyl)2nystose (2.63), o--d - glucopyranosyl-(1->2)-o--d - xylopyranosyl-(1->2)--d - fructofuranoside (1.51), o--d - glucopyranosyl-(1->2)-o--d - glucopyranosyl-(1->2)-o--d - xylopyranosyl-(1->2)--d - fructofuranoside (1.80). the degree of polymerization of saccharides 1, 2 and 3 was established as 3 by measurements of [m+na ] ions (m / z : 527) using tof - ms (see fig. 2), and analysis of the molar ratios of d - glucose to d - fructose in the acid hydrolysates. acid hydrolysates of saccharides 1 and 3 were liberated to glucose and fructose, and saccharide 2 was liberated to glucose. from the gc analysis, relative retention times of the methanolysate of the permethylated saccharides were investigated [tr (relative retention time ; retention time of methyl 2, 3, 4, 6-tetra - o - methyl--d - glucoside = 1.0 ; retention time, 9.60 min) ]. the methanolysate of permethylated saccharide 1 exhibited six peaks (see additional file 2) corresponding to methyl 2,3,4,6-tetra - o - methyl - d - glucoside (tr, 0.94 and 1.48), methyl 2,4,6-tri - o - methyl - d - glucoside (tr, 3.27 and 4.81) and methyl 1,3,4,6- tetra - o - methyl - d - fructoside (tr, 1.06 and 1.32). the methanolysate of permethylated saccharide 3 also exhibited six peaks (see additional file 2) corresponding to methyl 2,3,4-tri - o - methyl - d - glucoside (tr, 2.58 and 3.59), methyl 2,4,6-tri - o - methyl - d - glucoside (tr, 3.22 and 4.73), and methyl 1,3,4,6-tetra - o - methyl - d - fructoside (tr, 1.07 and 1.29). on the other hand, the methanolysate of permethylated saccharide 2 exhibited two peaks (see additional file 2) corresponding to methyl 2,3,4,6-tetra - o - methyl - d - glucoside (tr, 0.97 and 1.47). gc - ms analysis on the retention times and fragmentation patterns of the methyl glucosides showed the two peaks (10.08 min and 10.21 min) from the methanolysate of permethylated saccharide 2 to be methyl 3,6-di - o - methyl - d - glucoside. from these findings above, saccharides 1, 2 and 3 were proved to be, o - d - fructofuranosyl-(2->1)-o-[d - glucopyranosyl-(1->3)]-d - glucopyranoside, o - d - glucopyranosyl-(1->2)-o-[d - glucopyranosyl-(1->4)]-d - glucose and o - d - fructofuranosyl-(2->6)-o - d - glucopyranosyl-(1->3)-d - glucose, respectively. maldi - tof - ms spectra of saccharides 1, 2, and 3. 1 : saccharide 1, 2 : saccharide 2, 3 : saccharide 3. the structural confirmations of saccharides 1, 2 and 3 according to h and c nmr analyses and the subsequent complete assignment of h and c nmr signals of the three saccharides were carried out using 2d - nmr techniques. the hsqc - tocsy spectrum revealed theh and c signals of each glc, glc ' and fru. the cosy spectrum assigned the spin systems of these residues ; from h-1 to h-3 and h-1 ' to h-3 ' (fig. the j (h-1/h-2) value was 7.9 hz. these results indicated the glc 1 ->3 ' glc linkage, namely the laminaribiose moiety. these results indicated the glc ' 1 ->2 fru linkage, and all h and c nmr signals were assigned as shown in additional file 3. part of cosy (a), hsqc (b) and hmbc (c) spectra of saccharide 1. due to strong coupling between h-4 ' and h-5 ', these couplings could not be analyzed in first order. the nmr spectra of saccharide 2 showed that it was an anomeric mixture at the glc. these results indicated the glc " 1 ->4 glc linkage, namely the cellobiose moiety. the j (h-1'/h-2 ') value was 7.6 hz. these results indicated the glc ' 1 ->2 glc linkage, and all h and c nmr signals were assigned as shown in additional file 3. part of hsqc (a) and hmbc (b) spectra of saccharide 2. the nmr spectra of saccharide 3 were analyzed in the same manner as those of saccharide 2. the hsqc - tocsy spectrum revealed theh and c signals of each glc, glc ' and fru. the j (h-1/h-2) value was 7.9 hz. these results indicated the glc 1 ->3 glc ' linkage, namely the laminaribiose moiety. these results indicated the glc 6 1)-o-[-d - glucopyranosyl-(1->3)]--d - glucopyranoside (named " 3--d - glucopyranosyl, -isosucrose "), o--d - glucopyranosyl-(1->2)-o-[-d - glucopyranosyl-(1->4)]-d - glucopyranose (4--d - glucopyranosyl sophorose) and o--d - fructofuranosyl-(2->6)-o--d - glucopyranosyl-(1->3)-d - glucopyranose (6--d - fructofuranosyl laminaribiose). almost all of the monosaccharides were removed from the fermented and unfermented beverages of plant extracts by the batch method with charcoal. the saccharides 1, 2, and 3 were observed in the fermented beverage, but were not present in the unfermented one. therefore, saccharides 1, 2, and 3 were confirmed to have been produced during fermentation of the beverage of plant extracts (fig. b : the beverage of plant extract was fermented for 180 days. the beverage (100 ml) fermented for 0 or 180 days was mixed with charcoal (10 g), stirred for 3 h. and filtered. the charcoal was extracted with 30% ethanol (500 ml) three times. the ethanol extracts were combined, concentrated to dryness and solubilized with one ml of distilled water. we have previously found that the fermented beverage contained the novel saccharide, o--d - fructopyranosyl-(2->6)-d - glucopyranose, which is produced by fermentation. the saccharide was selectively used by beneficial bacteria, bifidobacterium adolescentis and b. longum, but was not used by unfavorable bacteria, clostridium perfringens, escherichia coli and enterococcus faecalis that produce mutagenic substances. it is interesting to study the biological functions of other oligosaccharides existing in the beverage. in this report, three novel oligosaccharides have been found from this beverage, and isolated from the beverage using carbon - celite column chromatography and preparative high performance liquid chromatography. structural confirmation of the saccharides was provided by methylation analysis, maldi - tof - ms and nmr measurements. these saccharides were identified as new trisaccharides : o--d - fructofuranosyl-(2->1)-o-[-d - glucopyranosyl-(1->3)]--d - glucopyranoside (named " 3--d - glucopyranosyl, -isosucrose "), o--d - glucopyranosyl-(1->2)-o-[-d - glucopyranosyl-(1->4)]-d - glucopyranose (4--d - glucopyranosyl sophorose) and o--d - fructofuranosyl-(2->6)-o--d - glucopyranosyl-(1->3)-d - glucopyranose (6--d - fructofuranosyl laminaribiose). these saccharides for preparation of the initial juice, 50 kinds of fruits and vegetables were used to produce the final extract as shown in a previous paper. the 50 fruits and vegetables were cut, sliced or diced into small pieces, mixed and put in cedar barrels. afterwards, an equivalent weight of sucrose was added to the samples, mixed well to allow high contact between the samples and sucrose, and then the barrels were left for one week at room temperature. the fermented beverage was obtained by incubation of the juice at 37c in the dark by natural fermentation using yeast (zygosaccharomyces spp. and pichia spp.) and lactic acid bacteria (leuconostoc spp.). after 7 days, the fermented beverage was kept in a closed enameled tank at 37c for 180 days for additional maturation and ageing, finally obtaining a brown and slightly sticky liquid. the oligosaccharides were analyzed using a dionex bio lc series apparatus equipped with an hplc carbohydrate column (carbo pack pa1, inert styrene divinyl benzene polymer) and pulsed amperometric detection (pad). the mobile phase consisted of eluent a (150 mm naoh) and eluent b (500 mm sodium acetate in 150 mm naoh) with a sodium acetate gradient as follows : 01 min, 25 mm ; 12 min, 2550 mm ; 220 min, 50200 mm ; 2022 min, 500 mm ; 2230 min, 25 mm ; using a flow rate through the column of 1.0 ml / min. the applied pad potentials for e1 (500 ms), e2 (100 ms), and e3 (50 ms) were 0.1, 0.6, and -0.6 v respectively, and the output range was 1 c. the fermented beverage of plant extracts (1000 g) was loaded onto a carbon - celite [1:1 ; charcoal (wako pure chemical industries, ltd ; osaka, japan) and celite-535 (nacalai tesque inc, osaka, japan) ] column (4.5 35 cm), and was successively eluted with water (14 l), 5% ethanol (30 l) and 30% ethanol (10 l). almost all of the glucose and fructose were eluted with water (4 l), and then saccharides 1, 2 and 3 were eluted with 30% ethanol (12 l). the 30% ethanol fraction containing saccharides 1, 2 and 3 was concentrated in vacuo and freeze - dried to give 894 mg of sample. subsequently, the 30% ethanol fraction was successfully repeatedly purified using an hplc system (tosoh, tokyo, japan) equipped with an amide-80 column (7.8 mm 30 cm, tosoh, tokyo, japan) at 80c, and eluted with 80% acetonitrile at 2.0 ml / min, and using refractive index detection. furthermore, the saccharides were purified by hplc with the ods-100 v column (4.6 mm 25 cm, tosoh, tokyo, japan) at room temperature, and eluted with water at 0.5 ml / min. purified saccharides 1 (2.5 mg), 2 (2.2 mg) and 3 (2.0 mg) were obtained as white powders. the permethylated saccharides were methanolyzed by heating with 1.5% methanolic hydrochloric acid at 96c for 10 or 180 min. the reaction mixture was treated with amberlite ira-410 (oh) to remove hydrochloric acid, and evaporated in vacuo to dryness. the resulting methanolysate was dissolved in a small volume of methanol and analyzed using gas liquid chromatography. for the analysis of the methanolysate, glc was carried out using a shimadzu gc-8a gas chromatograph equipped with a glass column (2.6 mm 2 m) packed with 15% butane 1,4-diol succinate polyester on acid - washed celite at 175c. gc - ms analysis was performed using a jms - ax500 mass spectrometer (jeol, japan) using a db-17ht capillary column (30 m 0.25 mm i.d., j & w scientific, usa). injection temperature was 200c. the column temperature was kept at 50c for 2 min after sample injection, increased to 150c at 50c / min, kept at 150c for 1 min, and then increased to 250c at 4c / min. maldi - tof - ms spectra were measured using a shimadzu - kratos mass spectrometer (kompact probe) in positive ion mode with 2.5%-dihydroxybenzoic acid as a matrix. ions were formed by a pulsed uv laser beam (nitrogen laser, 337 nm). 2 mg) was dissolved in 0.06 ml (saccharide 1) and 0.4 ml (saccharide 2 and 3) d2o. nmr spectra were recorded at 27c with a bruker amx 500 spectrometer (1 h 500 mhz, 13c 125 mhz) equipped with a 2.5 mm c / h dual probe (saccharide 1), a 5 mm diameter c / h dual probe (1d spectra of saccharide 2 and 3), and a 5 mm diameter txi probe (2d spectra of saccharide 2 and 3). chemical shifts of h (h) and c (c) in ppm were determined relative to an external standard of sodium [2, 2, 3, 3-h4]-3-(trimethylsilyl)-propanoate in d2o (h 0.00 ppm) and 1, 4-dioxane (c 67.40 ppm) in d2o, respectively. h - h cosy, hsqc, hsqc - tocsy ch2-selected e - hsqc - tocsy, hmbc and ct - hmbc spectra were obtained using gradient selected pulse sequences. for preparation of the initial juice, 50 kinds of fruits and vegetables were used to produce the final extract as shown in a previous paper. the 50 fruits and vegetables were cut, sliced or diced into small pieces, mixed and put in cedar barrels. afterwards, an equivalent weight of sucrose was added to the samples, mixed well to allow high contact between the samples and sucrose, and then the barrels were left for one week at room temperature. the fermented beverage was obtained by incubation of the juice at 37c in the dark by natural fermentation using yeast (zygosaccharomyces spp. and pichia spp.) and lactic acid bacteria (leuconostoc spp.). after 7 days, the fermented beverage was kept in a closed enameled tank at 37c for 180 days for additional maturation and ageing, finally obtaining a brown and slightly sticky liquid. the oligosaccharides were analyzed using a dionex bio lc series apparatus equipped with an hplc carbohydrate column (carbo pack pa1, inert styrene divinyl benzene polymer) and pulsed amperometric detection (pad). the mobile phase consisted of eluent a (150 mm naoh) and eluent b (500 mm sodium acetate in 150 mm naoh) with a sodium acetate gradient as follows : 01 min, 25 mm ; 12 min, 2550 mm ; 220 min, 50200 mm ; 2022 min, 500 mm ; 2230 min, 25 mm ; using a flow rate through the column of 1.0 ml / min. the applied pad potentials for e1 (500 ms), e2 (100 ms), and e3 (50 ms) were 0.1, 0.6, and -0.6 v respectively, and the output range was 1 c. the fermented beverage of plant extracts (1000 g) was loaded onto a carbon - celite [1:1 ; charcoal (wako pure chemical industries, ltd ; osaka, japan) and celite-535 (nacalai tesque inc, osaka, japan) ] column (4.5 35 cm), and was successively eluted with water (14 l), 5% ethanol (30 l) and 30% ethanol (10 l). almost all of the glucose and fructose were eluted with water (4 l), and then saccharides 1, 2 and 3 were eluted with 30% ethanol (12 l). the 30% ethanol fraction containing saccharides 1, 2 and 3 was concentrated in vacuo and freeze - dried to give 894 mg of sample. subsequently, the 30% ethanol fraction was successfully repeatedly purified using an hplc system (tosoh, tokyo, japan) equipped with an amide-80 column (7.8 mm 30 cm, tosoh, tokyo, japan) at 80c, and eluted with 80% acetonitrile at 2.0 ml / min, and using refractive index detection. furthermore, the saccharides were purified by hplc with the ods-100 v column (4.6 mm 25 cm, tosoh, tokyo, japan) at room temperature, and eluted with water at 0.5 ml / min. purified saccharides 1 (2.5 mg), 2 (2.2 mg) and 3 (2.0 mg) were obtained as white powders. the permethylated saccharides were methanolyzed by heating with 1.5% methanolic hydrochloric acid at 96c for 10 or 180 min. the reaction mixture was treated with amberlite ira-410 (oh) to remove hydrochloric acid, and evaporated in vacuo to dryness. the resulting methanolysate was dissolved in a small volume of methanol and analyzed using gas liquid chromatography. for the analysis of the methanolysate, glc was carried out using a shimadzu gc-8a gas chromatograph equipped with a glass column (2.6 mm 2 m) packed with 15% butane 1,4-diol succinate polyester on acid - washed celite at 175c. gc - ms analysis was performed using a jms - ax500 mass spectrometer (jeol, japan) using a db-17ht capillary column (30 m 0.25 mm i.d., j & w scientific, usa). injection temperature was 200c. the column temperature was kept at 50c for 2 min after sample injection, increased to 150c at 50c / min, kept at 150c for 1 min, and then increased to 250c at 4c / min. maldi - tof - ms spectra were measured using a shimadzu - kratos mass spectrometer (kompact probe) in positive ion mode with 2.5%-dihydroxybenzoic acid as a matrix. ions were formed by a pulsed uv laser beam (nitrogen laser, 337 nm). the saccharide (ca. 2 mg) was dissolved in 0.06 ml (saccharide 1) and 0.4 ml (saccharide 2 and 3) d2o. nmr spectra were recorded at 27c with a bruker amx 500 spectrometer (1 h 500 mhz, 13c 125 mhz) equipped with a 2.5 mm c / h dual probe (saccharide 1), a 5 mm diameter c / h dual probe (1d spectra of saccharide 2 and 3), and a 5 mm diameter txi probe (2d spectra of saccharide 2 and 3). chemical shifts of h (h) and c (c) in ppm were determined relative to an external standard of sodium [2, 2, 3, 3-h4]-3-(trimethylsilyl)-propanoate in d2o (h 0.00 ppm) and 1, 4-dioxane (c 67.40 ppm) in d2o, respectively. h - h cosy, hsqc, hsqc - tocsy ch2-selected e - hsqc - tocsy, hmbc and ct - hmbc spectra were obtained using gradient selected pulse sequences. ho, ay and nk performed data analysis, and contributed to drafting the manuscript. ns and so conceived of the study, participated in its design and contributed to drafting the manuscript. gas - liquid chromatographic analysis of methanolysates of permethylated saccharides 1, 2 and 3. | backgrounda fermented beverage of plant extracts was prepared from about fifty kinds of vegetables and fruits. natural fermentation was carried out mainly by lactic acid bacteria (leuconostoc spp.) and yeast (zygosaccharomyces spp. and pichia spp.). we have previously examined the preparation of novel four trisaccharides from the beverage : o--d - fructopyranosyl-(2->6)-o--d - glucopyranosyl-(1->3)-d - glucopyranose, o--d - fructopyranosyl-(2->6)-o-[-d - glucopyranosyl-(1->3)]-d - glucopyranose, o--d - glucopyranosyl-(1->1)-o--d - fructofuranosyl-(21)--d - glucopyranoside and o--d - galactopyranosyl-(1->1)-o--d - fructofuranosyl-(21)- -d-glucopyranoside.resultsthree further novel oligosaccharides have been found from this beverage and isolated from the beverage using carbon - celite column chromatography and preparative high performance liquid chromatography. structural confirmation of the saccharides was provided by methylation analysis, maldi - tof - ms and nmr measurements.conclusionthe following novel trisaccharides were identified : o--d - fructofuranosyl-(2->1)-o-[-d - glucopyranosyl-(1->3)]--d - glucopyranoside (named " 3g--d - glucopyranosyl, -isosucrose "), o--d - glucopyranosyl-(1->2)-o-[-d - glucopyranosyl-(1->4)]-d - glucopyranose (41--d - glucopyranosyl sophorose) and o--d - fructofuranosyl-(2->6)-o--d - glucopyranosyl-(1->3)-d - glucopyranose (62--d - fructofuranosyl laminaribiose). |
development of nonviral gene - delivery devices as alternatives to the viral vectors is essentially needed for the safe implementation of gene delivery concepts in clinical medicine (luo and saltzman 2000). despite existence of a wide variety of nonviral techniques, there has been limited focuses on synthesis of inorganic nano - size dna carriers which could have better performances for trans - gene delivery compared with the organic materials being extensively used so far (chowdhury and akaike 2005a). as inorganic materials, gold, silica, iron - oxide, carbon nanotube, clay minerals, and apatite have been used so far to carry genetic materials to mammalian cells (luo and saltzman 2000 ; thomas and klibanov 2003 ; chowdhury and akaike 2005). however, among the synthetic inorganic carriers, apatite particles have received considerable attention by virtue of their biodegradability and resemblance to body hard tissue components (chowdhury and akaike 2005b ; chowdhury 2006). in fact, calcium phosphate precipitation, one of the pioneering gene delivery methods, is based on generation of hydroxyapatite particles (chowdhury 2006). low - level of transgene expression due to the inefficiency in the cellular uptake and the endosomal escape of dna (loyter 1982 ; orrantia and chang 1990 ; jordan 1996 ; lee and welsh 1999) has limited the potential applications of this traditional technique. we have recently focused on the generation of carbonate apatite particles which, like hydroxyapatite particles, adsorbed dna, but unlike the latter, could enhance transgene expression to a significant extent in both cancer and primary cells (chowdhury and akaike 2005b ; chowdhury 2006). here we describe that a number of factors influence the generation of carbonate apatite particles by either inducing or inhibiting the development of supersaturation in the bicarbonate - buffered solution of calcium, phosphate, and dna. thus, a number of routes have been demonstrated for the synthesis of high transfec - tion - promoting nanoapatite particles for a particular cell culture environment. moreover, we proposed that efficient endocytosis of nano - size dna / apatite particles, followed by endosomal release of dna contributed to the remarkable transfection efficiency of carbonate apatite. plasmid pgl3 (promega, tokyo, japan) containing a luciferase gene under sv40 promoter was propagated in the bacterial strain xl-1 blue and purified by qiagen plasmid kits (qiagen, tokyo, japan) for being used in gene delivery and subsequently quantitative measurement of luciferase protein expression. propidium iodide (pi), a dna - labelling dye and lysosensorgreen dnd-189, an endosome - specific marker were purchased from sigma - aldrich (tokyo, japan) and molecular probes (invitrogen, tokyo, japan), respectively. lipofectamine 2000 and dmem were purchased from invitrogen and gibco brl (tokyo, japan), respectively. hela, a human cervical cancer cell widely used in gene delivery study, was cultured in 75-cm flasks in dulbecco s modified eagle s medium (dmem, gibco brl) supplemented with 10% fetal bovine serum (fbs), 50 g penicillin ml, 50 g streptomycin ml, and 100 g neomycin ml at 37 c in a humidified 5% co2-containing atmosphere. cells from the exponential growth phase were seeded at 50,000 cells per well into 24-well plates the day before transfection. three to 6 l of 1 m cacl2 was mixed with 2 g of plasmid dna in 1 ml of fresh serum - free hco3 buffered (ph 7.5) medium (dmem) followed by incubation for 30 min at 37 c for complete generation of dna / carbonate apatite particles. medium with generated dna - containing particles was added with 10% fbs to the rinsed cells. after 4-h incubation, the medium was replaced with serum supplemented medium and the cells were cultured for 1 day. luciferase gene expression was monitored by using a commercial kit (promega) and photon counting (td-20/20 luminometer, usa). each transfection experiment was done in triplicate and transfection efficiency was expressed as mean light units per mg of cell protein. transfection by calcium phosphate - dna co - precipitation was performed according to jordan and colleagues (1996). briefly, 12 g of plasmid dna was added to 300 l of a solution containing 250 mm cacl2. this solution was added to 300 l of a 2 hbs (50 mm hepes, 140 mm nacl, 1.5 mm na2hpo4.2h2o, ph 7.05) and mixed rapidly by gentle pipetting twice. the dna / capi mixture was incubated at room temperature for the period of time indicated. after addition of 100 l of the incubated mixture drop - wise to 1 ml serum supplemented media of each well, cells were grown for 4 h in the incubator. after removal of the particle - containing medium, fresh medium was added to the cells which were subsequently grown for 1 day. for lipofectamine - mediated transfection, protocol provided by invitrogen was followed. a 1:6 ratio of dna (2 g) to lipofectamine provided the best transfection efficiency in our study. cells were incubated with dna / lipofactamine complexes in serum media for 4 h and like above, grown for 1 day after replacement with fresh serum media. for visualization by a scanning electron microscope (sem), a drop of dna - carbonate apatite suspension prepared according to the instructions in transfection protocol, was added to a carbon - coated sem stage and dried by keeping the stage at 50c for 10 min, followed by observation by a high resolution sem (s-800, hitachi, japan). dynamic light scattering (dls) measurement for particle suspension was carried out with a super - dynamic light scattering spectrophotometer, pgl3 vector was labeled with pi at a pi / dna ratio of 1:1 and particles generated with this labeled plasmid were incubated with hela cells. membrane - bound precipitates were removed by 5 mm edta in pbs before observation by leica tcs - nt. plasmid pgl3 (promega, tokyo, japan) containing a luciferase gene under sv40 promoter was propagated in the bacterial strain xl-1 blue and purified by qiagen plasmid kits (qiagen, tokyo, japan) for being used in gene delivery and subsequently quantitative measurement of luciferase protein expression. propidium iodide (pi), a dna - labelling dye and lysosensorgreen dnd-189, an endosome - specific marker were purchased from sigma - aldrich (tokyo, japan) and molecular probes (invitrogen, tokyo, japan), respectively. lipofectamine 2000 and dmem were purchased from invitrogen and gibco brl (tokyo, japan), respectively. hela, a human cervical cancer cell widely used in gene delivery study, was cultured in 75-cm flasks in dulbecco s modified eagle s medium (dmem, gibco brl) supplemented with 10% fetal bovine serum (fbs), 50 g penicillin ml, 50 g streptomycin ml, and 100 g neomycin ml at 37 c in a humidified 5% co2-containing atmosphere. cells from the exponential growth phase were seeded at 50,000 cells per well into 24-well plates the day before transfection. three to 6 l of 1 m cacl2 was mixed with 2 g of plasmid dna in 1 ml of fresh serum - free hco3 buffered (ph 7.5) medium (dmem) followed by incubation for 30 min at 37 c for complete generation of dna / carbonate apatite particles. medium with generated dna - containing particles was added with 10% fbs to the rinsed cells. after 4-h incubation, the medium was replaced with serum supplemented medium and the cells were cultured for 1 day. luciferase gene expression was monitored by using a commercial kit (promega) and photon counting (td-20/20 luminometer, usa). each transfection experiment was done in triplicate and transfection efficiency was expressed as mean light units per mg of cell protein. transfection by calcium phosphate - dna co - precipitation was performed according to jordan and colleagues (1996). briefly, 12 g of plasmid dna was added to 300 l of a solution containing 250 mm cacl2. this solution was added to 300 l of a 2 hbs (50 mm hepes, 140 mm nacl, 1.5 mm na2hpo4.2h2o, ph 7.05) and mixed rapidly by gentle pipetting twice. the dna / capi mixture was incubated at room temperature for the period of time indicated. after addition of 100 l of the incubated mixture drop - wise to 1 ml serum supplemented media of each well, cells were grown for 4 h in the incubator. after removal of the particle - containing medium, fresh medium was added to the cells which were subsequently grown for 1 day. for lipofectamine - mediated transfection, protocol provided by invitrogen was followed. a 1:6 ratio of dna (2 g) to lipofectamine provided the best transfection efficiency in our study. cells were incubated with dna / lipofactamine complexes in serum media for 4 h and like above, grown for 1 day after replacement with fresh serum media. for visualization by a scanning electron microscope (sem), a drop of dna - carbonate apatite suspension prepared according to the instructions in transfection protocol, was added to a carbon - coated sem stage and dried by keeping the stage at 50c for 10 min, followed by observation by a high resolution sem (s-800, hitachi, japan). dynamic light scattering (dls) measurement for particle suspension was carried out with a super - dynamic light scattering spectrophotometer, pgl3 vector was labeled with pi at a pi / dna ratio of 1:1 and particles generated with this labeled plasmid were incubated with hela cells. membrane - bound precipitates were removed by 5 mm edta in pbs before observation by leica tcs - nt. generation of carbonate apatite particles was governed by exogenously added ca concentrations, ph of the hco3 buffered media and incubation temperatures. we have investigated a long range of ph (7.0 to 7.9) of the hco3 buffered medium as well as incubation temperatures (25 c to 65 c) in order to make particles by exogenously added ca and subsequently transfect hela cells using the generated particles. interestingly, the optimal ca concentrations required for generation of effective number of dna / carbonate apatite particles leading to the high transfection efficiency, were inversely related to the phs of the media (figure 1) and the incubation temperatures (figure 2). thus, while 4 mm ca was sufficient to induce particle formation at ph 7.4 by incubating the ca - supplemented buffered medium for 30 min at 37 c, only 1 mm was enough to stimulate particle generation to the similar level at ph 7.9. like ph, incubation temperatures have also profound and sensitive effects on particle formulation and subsequent trans - gene delivery. thus, at the incubation temperature of 37 c, 3 mm ca was able to induce the proper supersaturation whereas at 65 c, only 1 mm cacould stimulate supersaturation development to a similar extent a prior need for generation of the particles (jordan 1996 ; chowdhury, kunou, 2004). the decline below the high efficiency level of transfection (figures 1 and 2) was due to the formation of too few particles (microscopically observed) since increase in ph or temperature contributed to the development of supersaturation by increasing the ionization of phosphate and carbonate in the solution. the new system of particle synthesis is, therefore, very flexible since it allows us to make particles at a wide range of ph and temperatures. the analysis also indicates that induction of supersaturation as required for particle formation, can be delicately controlled by manipulating the parameters. thus, one of the big challenges of using inorganic particles to regulate supersaturation development for reproducing particle synthesis and trans - gene delivering efficacy, could be resolved. in addition to ph and temperature, concentrations of initially added ca, inorganic phosphate and hco3. should have profound influences on particle synthesis as the major constituents of carbonate apatite particles. generation of dna / apatite particles was usually performed in a ready - to - use cell culture medium (dmem) already containing sufficient amount of phosphate (0.9 mm) and insufficient amount of ca. we investigated the effects of externally added caas well as hco3. on the synthesis of particles as evaluated by the subsequent particle - mediated trans - gene expression efficiency. a significantly high number of particles resulted from the excessive induction of supersaturation by too high ph, temperature and ca concentrations, could adversely affect transfection of the cells being cultured in a dish of particular size (24-well plate) and so a 5x dilution of prepared particle suspension was used to transfect hela cells for interpreting the effects of ca and hcso3. as shown in figure 3, above 6 mm ca, a gradual decline in transgene expression was observed with a drastic reduction at 15 mm ca. this could be explained by the fact that higher concentrations of ca could accelerate development of supersaturation leading to formation of too many particles and growth of big size particles with the consequence of gradually reduced dna uptake by the cells. the relatively lower gene expression by the particles formed at 3 mm ca could simply be explained by the presence of very low number of the particles with insufficient amount of bound dna as expected for the dilution of particle suspension prior to transfection in a 24-well plate. thus, the transfection potential of carbonate apatite sustained even at higher doses of ca (up to 12 mm). on the other hand, keeping ca concentration constant (3 mm), increasing hco3 concentrations from 40 to 80 mm caused a shift to the synthesis of higher number of particles (microscopically observed) probably due to reaction accelerating effect of hco3 as one of the reactants and at a sufficiently higher dose (160 mm), fewer particles appeared due to the continuous inhibition of particle growth at higher hco3 concentrations (80 to 160 mm) as also evident from the particle size profiling (not shown). thus, higher gene expression at 80 mm hco3 was indicative of higher number of very small particles and reduced expression at 160 mm was reminiscent of very few small particles (figure 4). next, we explored the contribution of sizes of carbonate apatite particles generated under standard conditions, to the delivery of a trans - gene. carbonate when present in the apatite structure is known to limit the size of the growing apatite crystals (legeros and trautz 1967). we carried out scanning electron microscopy of generated carbonate apatite (figure 5a) which revealed reduced growth of the crystals, most of which had diameters of 50 to 300 nm. similar results were found by dynamic light scattering measurement (figure 5b). we verified this size limiting effect of carbonate by observing cellular uptake of the pi - labeled plasmid dna adsorbed to the apatites, since large particles are phagocytosed less efficiently than small ones (jordan 1996). dna was carried into the cells by carbonate apatite (figure 6c) at least 10 times more efficiently than hydroxyapatite, generated by 1 min incubation (jordan 1996) (figure 6d). longer period (30 min) incubation resulted in large hydroxyapatite particles (jordan 1996), showing extremely low cellular uptake of dna (not shown). our findings, therefore, clearly suggest that carbonate apatite is superior over hydroxyapatite for its intrinsic property of preventing crystal growth (legeros and trautz 1967), leading to high efficiency cellular uptake of dna. to elucidate the role of endosomal escape of dna in transgene expression, following endocytosis of pi - labeled plasmid dna for different period of time, we labelled endosomes with lysosensor (a fluorescence probe for endosomes). a significant portion of plasmid dna (red color) appeared to be released from the endosomes (green color) after 6 h of dna uptake by cells, as shown by colocalization of dna with endosomes (figure 7). the high dissolution rate of carbonate apatite (chowdhury 2006) might contribute to the destabilization of endosomes releasing dna to the cytoplasm, since v - atpase - driven massive proton accumulation for crystal dissolution could lead to passive chloride influx to endosomes and subsequent endosome swelling and rupture (boussif 1995). to evaluate the role of carbonate apatite as a powerful carrier of genetic material, we compared transfection efficiency of different techniques including two frequently used ones : cap co - precipitation method and lipofection. in hela cell, luciferase expression level for carbonate apatite - mediated transfection was over 25-fold higher than for lipofection and cap co - precipitation method (figure 8) in 10% serum - containing medium. nano gram level of dna was even sufficient for efficient transgene expression (figure 8). however, in the case of conventionally used cap precipitation technique, as mentioned before, with passage of time, particles continue to grow, creating large size particles which are extremely inefficient for cellular endocytosis (jordan 1996 ; chowdhury, zohra, 2004). thus, with increasing the incubation time from 1 min to 30 min, particles become larger resulting in lower cellular uptake of dna and gradually reduced transfection efficiency (figure 8). since endocytosed apatite particles are dissolved in endosomes finally releasing ca in cytoplasm, it is necessary to address the possible cellular mechanisms for processing these additional ca ions. indeed, cells possess machinery to precisely regulate cytoplasmic ca level (blaustein and lederer 1999). in order to remove excessive ca from the cytosol, there are atp - driven ca pumps in the endoplasmic reticulum for sequestering ca and in the plasma membrane for extruding ca. in addition, the plasma membrane of most animal cells also contain another ca transport system, the na / ca exchanger, that operates in parallel with the ca - selective channels and the atp - driven ca pump. thus both atp - driven ca pumps and na / ca exchanger might actively be engaged to maintain ca homeostasis and functionality of a cell after transfection with apatite nanocrystals. thus, we have unveiled the basic parameters which regulate delicately the generation of carbonate apatite nanoparticles in a wide range of experimental conditions, leading to the development of a highly simplified, flexible, and efficient gene delivery technology having wide applications from laboratories to clinical medicine (fasbender 1998 ; toyoda 2004). | increasing attention is being paid on synthetic dna delivery systems considering some potential life - threatening effects of viral particles, for development of gene - based nano - medicine in the 21st century. in the current nonviral approaches, most of the efforts have been engaged with organic macromolecules like lipids, polymers, and peptides, but comparatively fewer attempts were made to evaluate the potential of inorganic materials for gene delivery. we recently reported that biodegradable nanoparticles of carbonate apatite are highly efficient in transfecting a wide variety of mammalian cells. here we show that a number of parameters actively regulate synthesis of the nanoparticles and their subsequent transfection efficacy. development of supersaturation, which is the prerequisite for generation of such particles, could be easily modulated by reactant concentrations, ph of the buffered solution, and incubation temperatures, enabling us to establish a flexible particle generation process for highly productive trans - gene delivery. carbonate incorporation into the particles have been proposed for generating nano - size particles resulting in cellular uptake of huge amount of plasmid dna as well as endosome destabilization facilitating significant release of dna from the endosomes. |
amyloid- (a) peptides having a length of 4042 amino acids are naturally secreted as a cleavage product of the amyloid precursor protein. formation of a aggregates in patient s brain is a hallmark of alzheimer s disease. although the pathogenic identities and roles of a aggregates are still under debate, fibrillar aggregates formed by a likely play a critical role in a s cytotoxicity. kinetic experiments have established that formation of a fibrils include nucleation and elongation of fibrils. after nucleation, a monomers in solution are added to fibril tips to elongate fibrils. kinetics of a fibril elongation has been the subject of numerous experimental studies. on the basis of the interpretation of kinetic data, these studies proposed a two - step dock lock mechanism for fibril elongation : a monomers in solution first dock quickly to fibril tips ; then in a locking step, they undergo structural reorganization to assume fibril structures, probably with fibrils acting as templates. it has been suggested that the locking step involving structural transitions of a is likely the rate - limiting step. therefore, it is important to characterize, in molecular detail, not only various forms of a, both in solution and in fibrils, but also kinetics of the structural transitions during fibril elongation. significant progress has been achieved in the characterization of atomic structures of a fibrils through solid - state nmr experiments and modeling. it is now known that a fibrils mainly adopt cross- structures which are rich in parallel, in - register -sheets formed between peptides and aligned along the fibril axis. though different in detail, the fibril models determined by several laboratories share a similar feature : there is a bending region located within residues 2030 which brings into close contacts the two -sheets adjacent to the bending region, providing additional stabilization to fibrils (figure 1). furthermore, the models reveal an internal staggering between the two -sheets, leading to structural distinction between the two fibril tips (figure 1). similarly, structures of a peptides in solution were also intensively characterized through a combination of solution nmr experiments and computer simulations, exhibiting an ensemble of heterogeneous, compact structures. however, experimental information on structural transitions of a during fibril elongation is still scarce except for the case of local conformational change of a probed in a recent study combining two - dimensional ultraviolet spectroscopy and computer simulations. complementary to experiment, molecular dynamics (md) simulation has been a valuable tool to characterize the structural transitions involved in a fibril elongation at various levels of detail. a major challenge in simulating fibril elongation of a arises from the slow elongation kinetics that requires ms to s long simulations to be reproduced. to overcome this challenge, several coarse - grained (cg) models, which reduce the spatial resolution and thereby speed up simulations, have been employed to simulate a fibril elongation starting with dissociated monomers, shedding light on the dock lock mechanism of fibril elongation. despite some insight gained from the cg simulations, it is critical to model a fibril elongation in atomic detail. all - atom simulations of a fibril elongation are too daunting a task computationally. fortunately, several short segments of a, having length of 510 amino acids located within region k16-a42, are experimentally known to form fibril by themselves. for these segments, all - atom simulations of their fibril elongation become computationally practical, allowing observation in atomic detail of structural transitions arising in both the docking and locking steps. an attempt has also been made in simulating docking of a with fibril tips, revealing heterogeneous binding conformations. the observations of fibril elongation of short a segments are intriguing and perhaps relevant to a fibril elongation. however, considering that a includes several aggregation - competent short segments and exhibits a complex architecture, the following questions regarding structural transitions during a fibril elongation still need to be answered : (1) in which regions of a do initial fibril contacts form ? is there any region of a particularly favorable or unfavorable for initial fibril contacts ? (2) once initial fibril contacts form, how do fibril structures propagate through the remaining parts of a ? (3) a, compared to the short segments, is supposed to exhibit more complex monomeric structures. what is the impact of these a monomer structures on fibril elongation ? (4) the intersheet staggering, which is seen in fibrils formed by a, leads to the two tips of a fibril, the top and bottom tip, assuming actually different local structures. can the difference in the top and bottom tip lead to different fibril elongation kinetics and, thereby, account for unidirectional growth of a fibrils as seen in experiment ? addressing these questions requires simulations probing dynamics on a long time scale. to achieve such computation, we employ here a hybrid - resolution model, namely, pace, which combines models at two resolutions, with proteins represented in a united - atom model and with solvent described in a coarse - grained solvent model, the maritni solvent. pace has been shown to accelerate simulations significantly while folding proteins into their native structures. to enhance sampling, we also adopt an approach combining umbrella sampling and replica exchange molecular dynamics (remd) employed in previous studies of protein protein interactions and aggregation. biased simulations are further used to construct a kinetic network by following previous studies on folding and conformational transitions of proteins. the resulting kinetic network allows us to identify transition pathways using the recently developed transition path theory. by combining pace, enhanced sampling and transition path theory, we determine ensembles of pathways and, thus, establish the kinetics of structural transitions of a1742 during fibril elongation at both fibril tips. rate analysis on the pathways reveals that formation of fibril structures by a1742 indeed arises through a dock the pathways leading to formation of fibril structures reveal that hydrophobic regions l17-a21 and, to a lesser extent, g37-a42 exhibit a particular propensity for initial fibrillar contacts, consistent with previous all - atom simulation studies showing that region 1622 is key to fibril formation. we find that hairpin - like structures formed in monomers, similar to those observed in previous simulations, are dominant on - pathway intermediates arising upon the initial fibrillar contacts. we further find that due to the local u - shape structures of fibrils and the need to unfold the prior hairpin - like structures of monomers, fibril structures propagate to the remaining parts of a not in the expected zipper fashion, namely, do not propagate immediately to the parts adjacent in sequence to the initial contacts. finally, comparison of the results of fibril elongation at the two structurally different fibril tips suggests that the observed unidirectional growth of a fibrils is mainly due to distinct growth rates at the two tips. the above findings not only provide experimentally testable predictions regarding fibril growth, but also reveal important details needed for modeling fibril growth at the level of overall kinetics. the present study focuses on fibril elongation of a1742. to investigate the roles of different parts of a in fibril elongation, the peptide is divided into four regions (figure 1a), including the central hydrophobic region (chc, residues l17-a21), the c - terminal hydrophobic region (cthr, g37-a42) and the n- and c - terminal parts of the middle region (nmid, e22-a30 and cmid, i31v36). this model exhibits the u - shaped, cross- structures usually seen in a fibrils. two -sheets in the model, involving regions 1726 and 3142, form intersheet packing through side - chain contacts. interestingly, due to the internal staggering of the two -sheets, region 1726 of a monomer forms side - chain contacts mostly with region 3142 of the adjacent monomer, rather than with the region of the same monomer (figure 1b). as a result, at the fibril tips one of the two regions, namely, region 1726 or region 3142, is left unpaired, leading to two structurally different tips, namely, one (called even tip) with its chc region (part of 1726) exposed and the other (called odd tip) with its chc region buried (see figure 1c). we note that the geometry involved makes it impossible for an even tip to become an odd tip and vice versa ; one end of a fibril sports an even tip throughout elongation, the other always an odd tip. (a) amino acid sequence of a1742 and definition of four regions, namely, chc, nmid, cmid and cthr, investigated in the present study. (b) experimental fibril structures of a1742 (pdb i d : 2beg). shown in orange, green, purple and red are the chc, nmid, cmid and cthr regions, respectively. the side chains wrapped in the fibril are shown in stick representation. shown in gray and blue all the side chains are shown in ball representation. in (b) and (c), the fibril axis points from the odd tip to the even tip. (d) close - up view of fibril tip regions surrounding f19 as indicated by dashed boxes in (c). in the following sections, we present first simulation results of the binding of a monomers to both fibril tips. we compare then the kinetics, through kinetic network analysis, of the binding and actual formation of a fibril structures, the latter process leading to fibril elongation. we further present the identified pathways and associated kinetics of structural transitions during fibril elongation at both tips and compare then the elongation kinetics. finally, we discuss the possible factors that cause distinct kinetics at the two tips. to investigate the docking of a monomers to fibril tips during fibril elongation, we first characterize the thermodynamics of a docking. by definition, the docking step involves binding of a monomers to fibril tips irrespective of the detailed conformation that the a monomer assumes in the process. thus, the center - of - mass (com) distance, rcom, between a monomers and fibril tips was chosen as the reaction coordinate for docking. on the basis of biased simulations (see methods), we calculated the potential of mean force (pmf) profiles with respect to rcom for binding of a monomers to both fibril tips, namely, the even and odd tip, at 332 k by means of the temperature - weighted histogram analysis method (t - wham). the resulting pmfs, as shown in figure 2a, are flat at large com distances (rcom > 20), gradually decrease when the monomers are approaching the fibril tips and, eventually, exhibit a wide well at short distances (3 20) to fibrillar states, the latter involving at least 12 edge residues in fibrillar -sheets. the decomposition analysis identified a large number of pathways, each involving 2039 intermediate states. these pathways are heterogeneous, as revealed by their projection on the first two most significant principle components arising from the principal components analysis described in si (figure 3). to gain insight into the elongation mechanism represented by the pathways we simplified the pathways by grouping them according to the order of formation of -sheet structures in different regions of a (see si). the simplified pathways, as summarized in figure 4, reveal that in the majority (97%) of transition pathways fibril structures start to arise in the chc region (figures 1 and 3), while in some (38%) of these pathways formation of antiparallel -sheets in this region precedes formation of fibril structures (figure s1a (si)). the transitions from the antiparallel to fibrillar -sheets in the chc region appear to be similar to the antiparallel parallel transitions observed in previous md studies of a1622 dimers, arising through rotation of the chc region of a monomer about a hydrogen bond (hb) formed between the amide hydrogen of f19 in the monomer and the carbonyl group of v18 at the even tip (figure s1b (si)). in subsequent steps, the fibril structures extend to the cthr region, then to the cmid region, and finally to the nmid region. beside the major transition pathways, there is a small chance that extension of the fibril structures follows a slightly different pathway in which structures are formed initially in the cthr region and then propagate to the chc region (figure 4). the chance of these minor pathways is low (3%) at 332 k, but increases to 14% at high temperature (370 k) (figure 5a). the major pathways (white lines) accounting for 50% of transitions and their starting points (white dots) were projected onto the potential of mean force (pmf) profile (colored contour map) with respect to the first two principal components (pc1 and pc2), namely, those with the two largest eigenvalues, obtained through principal component analysis (pca) based on a covariance matrix of 625 cc distances between the incoming monomer and the fibril even tip and 231 cc distances within the monomer (see si). pc1 and pc2 account for 54% of total variance in the distances used in the pca. shown are also select intermediates of this pathway ; represented in orange, green, purple and red are the a chc, nmid, cmid and cthr regions, respectively. the network was generated (see si) on the basis of the order of -sheet formation in four regions, namely, chc, nmid, cmid and cthr. shown as text boxes are all the intermediates where either antiparallel (anti) or fibrillar (fib) -sheets form in one of the four regions. black arrows denote fluxes of reactive transitions with the arrow thickness proportional to the probabilities of the transitions. yields a possible sequence of -sheet formation observed in the present study. the five color maps shown nearby the network represent the probabilities (p) of inter - residual contact within the incoming monomer at different stages of fibril formation, including unbound states (a), initial contact states (b and d) and states where fibril structures form both in the chc and cthr regions (c and e). the probability of contact between residues i and j within the incoming monomer was calculated as the probability of c atoms of the two residues being within a cutoff of 6.5, averaged over all states that belong to the same stage of fibril formation. the axes of map a are shown as a chain of orange, green, purple and red arrows, which denote the positions of regions chc, nmid, cmid and cthr on the map, respectively. all residual contact probabilities (p) are scaled as ln p. the color bar on the top of map a denotes the ln p scale in units rt. the red dashed boxes in the maps indicate the contact patterns of the incoming monomer that exhibits the strand loop strand structures (sls). (a) probabilities of pathways initiated with formation of fibril structures in the cthr region during fibril elongation at the even and the odd tip. (b) temperature dependence of fibril formation at the even and the odd tip. rates were fitted to the arrhenius relationship (eq 1) with fitting quality r shown nearby. in all the pathways analyzed the fibril structures are initiated either in the chc region or, to a lesser extent, in the cthr region, but never in the middle regions (nmid and cmid) of a (figure 4). one wonders whether this result arises from a bias introduced by the enhanced simulations used here for the network analysis. to address this question, we performed, as described in methods, multiple 100 ns unbiased simulations of the association of a with the fibrils. although it is unlikely to observe the formation of fibril conformations on such a short time scale, the formation of initial hb contacts between monomers and the even fibril tip indeed occurred in a large number (164) of simulations. if initial hb contacts form randomly, the chance to observe the formation of hb between a specific pair of residues of monomers and fibril tips is roughly 1/25 = 0.16% for a1742. on the basis of this probability, we estimated for each region of a the expected numbers of trajectories in which at least one fibrillar hb could form in this region (figure 6a). our simulations show that the nmid and cmid regions are highly unfavorable for initial fibril contacts while the chc and cthr regions are involved in initial fibril contacts more frequently than expected on average. in particular, the trajectories leading to fibril formation in the chc region are six times more frequent than the mere average. altogether, our simulations suggest that formation of initial fibril contacts is sequence - specific and favorable in the hydrophobic regions. in the process of fibril elongation, the incoming monomers need to bind to the tip, but also need to undergo conformational transitions. to characterize the conformational transitions, we monitored structural features of a monomer at different stages of fibril formation, carrying out cluster and contact analysis of monomer trajectories. the cluster analysis of unbound monomers (rcom > 20) (figure s2 (si)) shows that these monomers adopt heterogeneous structures, the most populated five of which account for only 20% of total populations. the tertiary structures of the monomers were further examined by monitoring contact maps of backbone c atoms of a monomer, using a 6.5 distance cutoff for contacts. two major types of tertiary contacts emerge from the contact map (a in figure 4), one formed between the chc and cmid regions and the other formed between the cmid and cthr regions, both consistent with previous all - atom md studies. comparison between number of standard simulations in which fibril contacts arise (red bars) and expected numbers of simulations in which initial fibril contacts form randomly (black bars) at the even (a) and the odd tip (b). the expected numbers for any region were estimated as the number of simulations observed to form a contact the number of residues in this region 1/25. when initial fibrillar contacts form in the chc region, the monomer tends to adopt hairpin - like structures involving antiparallel contacts between the chc and cmid regions and a reversed loop spanning the nmid region, as revealed by the corresponding contact maps (b and d in figure 4) showing an off - diagonal band of high contact probabilities spanning residues 1736. these structures, known as strand loop strand (sls) structures (figure s3a (si)), have been suggested both in experimental and theoretical studies as important monomer intermediates for fibril elongation. to quantify the involvement of the strand loop strand (sls) structures in fibril elongation, we calculated, as described in si, the probability (ponpath) of the slss being present in the transition pathways. the on - pathway probability of slss turns out to be 53%, much higher than the probability (1013%) for either unbound or bound monomers to adopt the slss, suggesting, therefore, that the slss are indeed important intermediates for monomers during fibril elongation. eventually, the monomer loses most of its internal contacts involved in the slss when fibril structures have been achieved in the chc and cthr regions (b c and d e in figure 4). the simulations suggest that the slss need to be disrupted for fibril structures to extend, often prior to fibril contact extension to the middle regions (nmid and cmid) of a. of great practical concern regarding fibril formation are the rates of elongation kinetics. in order to determine the rates we performed the network analysis as described in methods on the constructed kinetic network to estimate the macroscopic rate of transitions from soluble a (rcom > 20) to either bound or fibrillar forms of a, the former representing the docking transitions and the latter representing the actual elongation of fibrils. the rates of the two types of transitions (table 1) were calculated to be 3 10k0 and 8 10k0, respectively, where k0, as described in si, is a base rate constant for the network. the time scales of the docking transitions and fibril elongation were thus estimated to be 0.5 s and 23 ms, respectively, revealing a large time scale gap (1010) between the two transitions. all rates were calculated at 332 k. the calculation procedure is described in si. the rate values reported in this table are given in terms of a reduced unit of k0, the base rate constant used to estimate transition rates in rate matrices. a rough estimate suggests k0 (20 ps) (see si). our kinetic network analysis revealed a complex energy landscape governing multitime scale processes involving fibril elongation. one may wonder whether fibril elongation corresponds to the slowest kinetic process of the fibril - monomer system. to address this question, the relaxation rates k of the slowest kinetic processes were estimated by calculating the smallest (in terms of magnitude) nonzero eigenvalues of the rate matrix associated with the kinetic network and employing then the relationship k =. the calculation revealed that the rates of the eight slowest relaxation modes range from 8 10k0 to 8 10k0, all smaller than kfibril (8 10k0). apparently, fibril elongation is not one of the slowest transition processes that the fibril - monomer system undergoes. a elongation kinetics is known to follow the arrhenius law1where a is a pre - exponential factor and h is the activation enthalpy. it has been shown that a elongation exhibits a large positive activation enthalpy (h), indicating a significant structural reorganization arising during fibril growth. therefore, it is essential to examine the temperature dependence of the kinetic model derived for formation of fibril structures. for this purpose, the network models were reconstructed at various temperatures based on the same biased simulations using t - wham. the rates of fibril formation (kfibril) at 332370 k obtained thus agree well with the arrhenius law, exhibiting a fitting correlation coefficient r = 0.98 (figure 5b). the activation enthalpy h extracted from the fitting is 22 kcal / mol, higher than, but still comparable to, the value (15.8 kcal / mol) reported for a142 obtained through quartz crystal microbalance measurements. the large activation enthalpy obtained is consistent with the large structural reorganization of monomers observed in our kinetic model (figure 4). to identify the difference in kinetics between fibril elongation at the even and the odd fibril tip, the network analysis applied for fibril elongation at the even tip the pathway analysis revealed that the elongation at the even and the odd tip proceed largely in a similar manner in regard to the order in which monomer regions attach to fibrils (figures s4 and s5 (si)). at 332 k, in most pathways (98%), the fibril structures are initiated in the chc region and then extend to the cthr and cmid regions (figure s5 (si)). in minor pathways (2%), the fibril structures start either in region cthr or cmid and extend then to the chc region (figure s5 (si)). the probability of the minor pathways increases (813%) at elevated temperature (345370 k) (figure 5a). strand structures for the incoming monomer appear to a major degree (89%) on - pathway (figure s3b (si)). however, despite the similarity observed, the formation of fibril structures was found to be about 40 times slower at the odd tip than at the even tip (table 1). moreover, the corresponding activation enthalpy h, estimated to be 34 kcal / mol (figure 5b), is much higher than that for fibril growth at the even tip (22 kcal / mol). taken together, our results suggest that fibril elongation at the odd tip is kinetically unfavorable. to find out what causes slower fibril elongation at the odd tip, kinetics of fibril formation in the chc region was first investigated as this region was found to be key for the initial fibril elongation step at both tips. since the chc region of the fibril edge is highly exposed at the even tip, but partially shielded at the odd tip (figure 1b), it is possible that initial fibril formation in the chc region is hindered at the odd tip. to examine this possibility, we compared the rates (kchc) of formation of fibril structures only in the chc region at both tips (table 1). the comparison showed that the fibril structures in the chc region arise about five times more slowly at the odd tip than they do at the even tip. moreover, out of 173 unbiased simulations in which hbs formed between monomers and the odd tip, only three showed the formation of fibrillar hbs in the chc region, which is less often than found (8 out of 164) in the simulations of a binding to the even tip (figure 6). taken together, the less accessible chc region at the odd tip does account, though only partially, for the slower formation of fibril structures at this tip. apart from initial fibril formation, the subsequent structural transitions of monomers were also investigated. the detail of the transitions of the monomer were examined by monitoring the monomer s internal contacts and its contacts with fibrils for all intermediates in the pathways to fibril formation, employing the number of hydrogen bonds formed between the monomer and the fibrils as the reaction progress variable. at early stages of the elongation at both tips (nhb = 5) the monomer loses most (70%) of its internal contacts (figure 7a), mainly due to the loss of contacts in region l17-v36 where the strand loop strand (sls) structures form (figure 7c). in the meanwhile, considerable side chain contacts between the monomer and the fibrils arise, presumably compensating the loss of contacts in the monomer (figure 7b). moreover, there are more contacts formed at these early stages (nhb = 5) during the transitions arising at the even tip than at the odd tip (figure 7b). to examine why more contacts are formed for the transitions at the even tip, we inspected the intermediates arising at this early stage highlighted by the shaded region in figure 7c. the structures of the intermediates reveal that the cmid region of the monomer forms contacts with side chains of f19 located at the even tip. the same contacts are absent in the intermediates arising at the odd tip (figure 7d). these contacts were further monitored during the entire course of fibril formation (figure 7e). the analysis shows that for fibril elongation at the even tip the contacts gradually increase at the early stage (nhb 10) between the monomer and the fibril arise. the same contacts, on the other hand, do not arise throughout fibril formation at the odd tip. therefore, our results suggest that the exposed side chains of f19 at the even tip may transiently stabilize the disrupted structures of the monomer and, thereby, facilitate formation of fibril structures. losses and gains of contacts for the incoming monomer during formation of fibril structures. (a) plot of the number of internal residual contacts of the monomers against the number of hydrogen bonds (nhb) formed between monomer and fibril tips for all on - pathway intermediates. (b) plot of the number of side chain contacts formed between monomer and fibril tips against nhb. (c) plot of the number of internal contacts of the monomer formed between l17-a21 and i31v36 against nhb. the shaded region highlights the states in which the monomer involves only about five hbs with the fibril tip but loses most of its internal contacts. (d) representative structures of major intermediates of fibril elongation at the even (top) and the odd (bottom) tip as highlighted in the shaded region in panel (c). shown in orange, green, purple and red are the chc, nmid, cmid and cthr regions, respectively. (e) plot of the number of side chain contacts formed between i31v36 of the monomer and f19 of the fibril against nhb. in panels (a c) and (e), the intermediates arising from fibril elongation at the even and the odd tip are plotted as blue and red circles, respectively, with the radii of circles proportional to ln ponpath. the shaded regions in (a), (b) and (e) denote the stages where drastic change of contacts arises. in the present study, we thought to describe a fibril elongation in atomic detail. to this end we performed molecular dynamics (md) simulations of fibril elongation by a1742. to overcome difficulties in simulating slow fibril elongation, both a hybrid - resolution model and an enhanced sampling method combining umbrella sampling and remd were employed. kinetic network analysis was then applied to furnish a kinetic model for fibril elongation, allowing us to identify structural transitions of a involved in fibril elongation. the dock lock mechanism for fibril elongation proposed earlier on the basis of experiments has received support from numerous md studies focusing on fibril elongation by short a segments, including a1622, a1528, a3540 and a3742. the kinetic model derived in the present study reveals that fibril elongation by a1742, which comprises basically all residues essential for fibril formation, also follows this mechanism. moreover, our rate calculation (table 1) reveals a large time scale gap (lock/dock = 10) between the dock (docking of monomer to fibril tip) and the lock (conformational transition of monomer to fibril structure) steps. our result agrees well with recent kinetic experiments by qiang., who showed that the measured rate of fibril elongation for full - length a is 10 times slower than expected for fibril growth by diffusion - limited attachment of monomers to fibril tips. notably, the time scale gap calculated for a1742 is much larger than that (lock/dock 10) for a short segment like a1622 as reported in previous simulations, suggesting that the length of a segments has a profound effect on separating the time scales of the docking and locking steps during fibril elongation. the fibril formation pathways identified in the present study reveal that different regions of a vary in their involvement in initial fibril formation. in particular, the nmid region (e22-a30) does not participate in initial fibrillar contacts (figures 4, 6 and s5 (si)). the following factors seem to contribute to the lack of involvement of the nmd region. one factor is that the nmid region includes largely polar and charged amino acids which may incur desolvation penalty against association ; in fact, previous all - atom simulations of a fibrils have shown that the same region of fibril edges gradually leaves fibrils driven by solvation. the second factor is that the nmid region adopts tight loop structures and appears to be the most structured part of the peptide according to both nmr experiments and simulations of a monomers by others as well as those in the present study. involvement of the nmid region in fibril formation would be highly unfavorable as it requires disruption of the loop structures formed in this region. in contrast, initial fibril formation in the chc region is more favorable compared to the other regions, highlighting the important role of the hydrophobicity of chc in driving initial fibril formation. the analysis based on energy - landscape theory by massi and straub proposed that certain conformations of monomers could undergo little structural reorganization upon binding to fibrils, thereby serving as important intermediates for fibril elongation. several experimental and computational studies, in seeking such intermediates, discovered monomeric structures in a hairpin - like motif of residues 1635, called strand loop strand (sls) motif. on the basis of their structural resemblance with the u - shape topology of a seen in fibrils, our simulations show that the sls structures arise in a majority (5090%) of the identified pathways when the initial fibrillar contacts are formed in the chc region. on the one hand, this finding provides direct support to the notion that the slss are on - pathways intermediates in fibril elongation as suspected previously ; on the other hand, the hydrogen bonds formed within the sls structures need to also be broken to allow other parts of peptides to participate further in hydrogen bonds with fibrils. the breaking of the sls structures could be energetically unfavorable, as indicated by a large positive activation enthalpy (22 kcal / mol) of fibril elongation estimated in the present study, comparable to that (15.8 kcal / mol) reported in kinetic experiments. interestingly, a similar conclusion regarding the role of hairpin - like monomeric intermediates in fibril growth has also been reached in a previous all - atom study for a shorter segment, namely, a2535. the key role of slss in fibril elongation raises the possibility that the sls structures can serve as a target for fibril inhibition. destabilizing sls formation in a or preventing a with the sls structures from binding to fibrils may slow down fibril formation. despite the significant involvement of the sls structures in fibril formation, our results do indicate a non - negligible chance of fibril formation which does not rely on slss and, thereby, may not be affected by inhibiting slss. therefore, it remains an open question how effective the sls structures are as an inhibition target. it has been assumed that after the initial contact, fibril structures propagate immediately to the regions adjacent in sequence to the initial contacts. indeed, this assumption has been supported by all - atom simulations of fibril elongation by short a segments like a1622, a3540 and a3742. if the same assumption is also true for a1742, one would expect that fibril structures extend to the middle regions (nmid and cmid) after the initial contact form in region chc or cthr. our simulations reveal instead that once fibril contacts are initiated in either region chc or region cthr, the structures continue to arise mainly in the other of the two regions (figures 3 and s5 (si)). regions chc and cthr, though distant in sequence, are spatially close to each other in the u - shaped fibril structures (figure 1). thus, fibril extension in the way stated above may allow the monomer to maintain at least partly its hairpin - like structures arising upon the initial contacts (figure 3). on the other hand, if fibril structures extend to the nmid region as expected, the loop of the hairpin - like structures would participate in fibril formation, leading immediately to deformation of the entire hairpin - like structures. our results disagree with the a locking mechanism suggested on the basis of a coarse - grained md study. according to this mechanism, when a monomer makes initial fibrillar contact with one of the two -sheets in fibrils, the remaining part of the monomer still moves freely and, thus, fibrillar contacts can propagate along the remaining part of the monomer. instead, the route of fibril extension observed in the present study agrees with a transition state ensemble model for a fibril elongation as reported recently on the basis of all - atom simulations. the reported model, derived indirectly through unfolding simulations of a fibril structures, develops intact fibril contacts in the hydrophobic regions of both -sheets of fibrils, but exhibits disordered loops in the nmid region. during fibril elongation, such transition states could be reached by unbound a monomers only if their conformational transitions to fibril structures follow a route similar to the one observed in the present study. structural characterization of a fibrils reveal distinct structures of two fibril tips exposing either their n - terminal or c - terminal edges (figure 1), indicating different binding interfaces for incoming monomers. previous all - atom simulations aiming to link this finding with unidirectional fibril growth suggested that a peptides bind to the two fibril tips with different affinities. in contrast, a computational study employing coarse - grained (cg) models showed that a monomers bind to either fibril tip without apparent thermodynamic preference. moreover, another cg simulation study proposed that the fibril tip with its chc region more accessible can allow faster formation of initial fibril structures and, thereby, facilitates fibril growth at this tip. in the present study, we found that a binds to the two fibril tips with similar affinities, but that fibril structures form much faster (about 40 times) at the tip exposing the chc region (even tip) than they do at the other tip (odd tip). our analysis revealed that the exposed chc region at the even tip indeed promotes initial fibril formation as suggested in previous studies. however, this promotion contributes only partly (25 times) to the faster fibril formation at this tip. an additional speed - up comes about since during subsequent structural reorganization of the monomer, the exposed f19 at the even tip can form transient interactions with parts of the monomer when its compact structures are disrupted., we suggest that the even tip with the exposed chc region, and thus exposed f19, serves as a better template to catalyze fast fibril formation than does the odd tip. in the present study, we employed a hybrid - resolution model, namely, pace (available at www.ks.uiuc.edu/whan/pace/pacevdw/), to simulate a fibril elongation. pace s parametrization and application to protein folding have been discussed in detail in previous studies. in the present study, we demonstrate, as shown in si and figure s6 (si), that simulations of a140/142 with pace reproduce key experimental observables of a structures, including secondary structure content and jhnh coupling constants measured in nmr experiments, with an accuracy rivaling that of all - atom simulations. thus, pace can also be extended to simulations of disordered peptides like a. beside a monomer conformational features, we examined also, as described in results, the ability of pace to reproduce important quantities relevant to fibril elongation such as a binding affinity and activation enthalpy of fibril formation. we notice that although the two quantities are qualitatively reproduced, they are both overestimated in the present study. as these quantities are determined mainly by hb interactions, we suspect that the pace force field applied here slightly overestimates individual hb interactions and that the overestimate accumulates for fibril formation in which multiple hb interactions are involved. we built an initial fibril model based on a part of the fibril structure reported by lhrs. the initial model had its fibril axis aligned to the z - direction, and then was solvated in a box of martini water and neutralized with 0.15 m nacl solution, leading to a system of 3000 particles. the resulting structure was used to prepare starting conformations of incoming monomers and fibrils for production runs in two approaches. in a first approach, the monomer on either accessible edge of fibrils was pulled away from the remaining three, which are positionally fixed, at a speed of 0.25 /ns by applying a force in the z - direction to the center - of - mass (com) ; the force was generated by pulling the end of an attached spring with spring constant k = 2.4 kcal / mol, a standard procedure in steered molecular dynamics. in a second approach, we replaced the edge monomer with one randomly selected from a remd simulation of a1742 (see si). runs, we performed umbrella sampling simulations with 17 windows whose z - com distances ranged from 4.8 to 20.8 at 1 intervals. in each window, a harmonic potential with a force constant of 2.4 kcal / mol was applied to maintain the respective z - com distance. also, positional restraints were applied to backbone atoms of the three peptides representing fibril tips and their side - chain atoms sandwiched by the two -sheets of fibrils. the force constant for the positional constraints was 2.4 kcal / mol. for each window a remd simulation was performed with 64 nvt replicas at temperatures chosen in the range of 320650 k. the starting structures for one - half of the replicas were selected from the first approach as discussed above ; those for the other half were selected from the second approach. the time step of simulation was chosen to be 4 fs, a value typical for pace simulations. exchanges between replicas were attempted every 8 ps and the acceptance ratio was 4050%. each replica ran for 0.6 s and only the last 0.4 s of simulation was used for analysis. the convergence of sampling is discussed in si (see figure s7 (si)). in addition to the biased simulations, we also performed 480 100 ns unbiased simulations of the association of a with fibrils. the starting structures of these simulations were generated in the second approach and placed halfway between the two fibril tips under periodic boundary conditions. previous remd simulations with a monomers moving freely have shown that a can not only bind to fibril tips, but can also bind, though with a much smaller affinity, to fibril sides. however, due to the restraints applied in our umbrella sampling simulations, we were unable to observe the weaker binding of a to fibril sides. consequently, the kinetic network model built upon the simulation results (see below) does not consider the binding to fibril sides as a part of fibril elongation. a class of methods have been developed recently for the study of long - time conformational transitions of proteins through md simulations. these methods are based on kinetic network models which assume that conformational space can be discretized into states and that conformational transitions correspond to hopping of systems between the states. to determine pathways of a structural transitions, we employed one variant of these methods which had been applied successfully to produce plausible pathways for both protein folding and large conformational change of proteins. in this method, kinetic network models are constructed on the basis of states sampled in biased simulations which allow a better sampling of conformations in transition regions that are usually high in energy and, thereby, rarely accessible in unbiased simulations. briefly, all sampled conformations are first clustered into microstates, employing principal components to measure structural similarity between conformations. the statistical probability of the microstates connectivity between these microstates is further established assuming that transitions arise between microstates with similar structures. the kinetic rates kij of transition from connected microstates j to i were assigned as2where peq(j) is the equilibrium probability of microstate j and k0 is a base rate constant assumed to be the same for transitions involving any pair of microstates. two important parameters are needed for construction of kinetic network models as described above, namely, a cutoff distance for clustering conformations and a cutoff distance for establishing connectivity between states ; in the present study, the two cutoff distances are 2.5 and 3.0, respectively. we also examined, as explained in si and figure s8 (si), other choices for these parameters and demonstrated that key observations of fibril elongation in the present study are not sensitive to the cutoff parameters selected. according to transition path theory (tpt), a kinetic network { kij } can be used to investigate transitions between two groups of microstates a and b by determining first the reactive flux jji from microstates i to j, defined as the net contribution to a b transitions via the transitions between the two microstates. jji is calculated according to the equation3where pfold(i) is the committor probability of state i, defined as the probability that the system, when being in state i, hits group b states before reaching group a states. the calculation of pfold(i) is described in si. using an iterative algorithm from tpt, the network { jij } of reactive fluxes can be decomposed into individual pathways ranked by their fluxes. many important kinetic properties can be derived according to the calculated network { jij } and the pathways identified, including the macroscopic rate of the a b transition, the probability of a particular pathway to be taken (ppath) and the probability of a microstate participating in any of the reactive pathways (ponpath). the tpt analysis on kinetic network models | a critical step of -amyloid fibril formation is fibril elongation in which amyloid- monomers undergo structural transitions to fibrillar structures upon their binding to fibril tips. the atomic detail of the structural transitions remains poorly understood. computational characterization of the structural transitions is limited so far to short a segments (510 aa) owing to the long time scale of a fibril elongation. to overcome the computational time scale limit, we combined a hybrid - resolution model with umbrella sampling and replica exchange molecular dynamics and performed altogether 1.3 ms of molecular dynamics simulations of fibril elongation for a1742. kinetic network analysis of biased simulations resulted in a kinetic model that encompasses all a segments essential for fibril formation. the model not only reproduces key properties of fibril elongation measured in experiments, including a binding affinity, activation enthalpy of a structural transitions and a large time scale gap (lock/dock = 103104) between a binding and its structural transitions, but also reveals detailed pathways involving structural transitions not seen before, namely, fibril formation both in hydrophobic regions l17-a21 and g37-a42 preceding fibril formation in hydrophilic region e22-a30. moreover, the model identifies as important kinetic intermediates strand loop strand (sls) structures of a monomers, long suspected to be related to fibril elongation. the kinetic model suggests further that fibril elongation arises faster at the fibril tip with exposed l17-a21, rather than at the other tip, explaining thereby unidirectional fibril growth observed previously in experiments. |
the world professional association for transgender health (wpath) is an international multidisciplinary professional association that publishes recognized standards for the care of transgender and gender variant persons. wpath recommends1in concert with policy statements from the american medical association,2 the american psychiatric association,3 the american psychological association,4 the american college of obstetricians and gynecologists,5 and the center of excellence for transgender health at the university of california, san francisco6that the healthcare needs of transgender people should be openly and properly addressed, at the same level of quality and thoroughness as is afforded to any other person. failure to collect data on and provide systematic inclusion in health delivery systems of transgender persons has negative impacts on health ; in order to receive appropriate and meaningful care, it is essential that individual populations be recognized and counted.7 a 2010 institute of medicine (iom) report on the health of lesbian, gay, bisexual and transgender (lgbt) persons recommends that data on gender identity be collected in electronic health records (ehrs) and that this goal be incorporated into meaningful - use objectives.8 data are severely lacking with respect to the use of ehr systems in transgender care. a pubmed search conducted on january 7, 2013 using a variety of combinations of the terms transgender, electronic, and record as well as (electronic health records[mesh]) and (transgendered persons[mesh]) yielded no relevant results. therefore, in september 2011, the wpath executive committee convened an electronic medical records working group, comprised of both expert clinicians and medical information technology specialists, to make recommendations for developers, vendors, and users of ehr systems with respect to transgender patients. transgender people experience their gender identity as different from the sex which was assigned to them at birth.1 transgender people may seek medical care such as hormone therapy or surgery to effect changes in their secondary sex characteristics toward those of the gender with which they identify, as part of a process referred to as gender transition.9 it should be noted that the terms sex and gender, while often used interchangeably, have specific medical and psychological meanings that may differ from general social or even legal usage. sex commonly refers to one 's physical sex characteristics (eg, facial hair, body fat distribution, breasts), whereas gender transition may be thought of as the process through which one aligns one 's physical sex (through hormones, surgery, etc) with one 's gender identity, keeping in mind that not all transgender people will seek a medical transition but may simply focus on a social one ; for any given individual, transition may or may not have a specific end point and may represent a continued state of flux and exploration that varies by the individual.10 transgender people may have preferred names and/or pronouns that differ from those listed on government - issued documents or health insurance policies. furthermore, transgender people may obtain name and/or sex / gender designation changes on identity or other documents (eg, passport, birth certificate, driver 's license, financial accounts, etc) at various stages of their transition. in the usa, specific guidelines exist for identity document changes both at the federal level as well as state by state. failing to be identified by the preferred name and pronoun in a medical setting has been shown to impact patient satisfaction and quality of care for transgender people.11 for example, a patient with an outwardly female appearance may be called by a male name in a crowded medical office reception area, or a patient listed as female who has a male appearance and identity may be referred to with a feminine pronoun by a healthcare provider unfamiliar with the patient 's history and preferences. this may negatively impact the patient provider relationship, as well as put patients at risk for verbal or even physical abuse from other patients in the waiting area. a recent non - peer reviewed report on transgender discrimination showed some 28% of respondents had experienced harassment in a clinic setting and that 2% had been subject to physical abuse.12 it may also lead to the avoidance of care or loss of retention of the patient if they feel that their gender identity is not being acknowledged or respected. a qualitative study of hiv+ low income transgender women of color found that culturally sensitive practices influenced linkage to a variety of healthcare services.13 in addition to concerns about demographic information such as listed versus preferred name, gender, and pronouns, providers require a means to maintain an accurate record of what organs a patient may or may not have ; this record can not be limited or defined by the patient 's assigned or apparent sex / gender as entered into the ehr. for example, a patient may have been assigned female at birth, and have transitioned to male through the use of testosterone and surgical removal of the breasts ; they may also have obtained a court ordered name and sex or gender change and are registered in the ehr system under a male name and gender. however, since this patient still has a cervix, ovaries, and uterus, health care providers will require the ability to enter pelvic exam findings and gynecologic review of systems, and to order a cervical pap smear within the ehr system. ehr products that restrict or pre - populate an individual encounter with sex - specific history, exam, or ordering templates will prevent this patient 's provider from accurately and efficiently documenting their care. as another example, a patient may have been assigned male at birth and have transitioned to female through the use of estrogen ; however, this patient has not yet changed her government - issued identity documents and is currently listed with a male name and sex or gender. the patient has an outwardly female appearance and wishes to be referred to using a feminine name and pronouns. the patient also has breasts as a result of their hormone treatment and will require a breast examination and ordering of a mammogram. an ehr system should guide the administrative and clinical staff to use the patient 's chosen name and pronoun, which should serve to improve patient engagement and comfort while improving retention in care. an ehr system should also allow the provider to document a breast examination and order a mammogram even though the patient remains registered as male. given these concerns, the working group recommends an individualized approach in the care of each transgender patient, based on a current inventory of organs as well as the patient 's own transgender status and sex- or gender - specific history. drawing on methods first developed by the center of excellence for transgender health at the university of california, san francisco, the working group adopted a two - step question technique (table 1) to allow the collection and documentation of gender identity information.14 this technique involves first querying the patient 's gender identity, followed by a query of the sex assigned at birth ; by querying gender identity first, the importance of this parameter from the perspective of a transgender person is emphasized over that of the assigned birth sex. furthermore, this technique provides more detailed and accurate demographic and historical information, and also increases overall rates of identification of transgender patients as compared to older one - step methods (ie, choices of male, female, or this information could be collected via a face - to - face interaction with clinic staff or a provider ; some evidence suggests such information would be more accurately collected via self - registration kiosks or patient portals.15 two - step method for the collection of sex and gender identity information ftm, female to male (ie, female assigned at birth, male - spectrum identity) ; mtf, male to female (ie, male assigned at birth, female - spectrum identity). using a two - step method has been shown to increase the identification of transgender patients in demographics studies as compared to a one - step method. the two - step method was adopted in 2011 by the us centers for disease control and prevention for use in their adult case report form as well as their electronic surveillance system, the enhanced hiv / aids reporting system (ehars).16 this method has been found to be superior at identifying transgender individuals in comparison to other methods, according to a recently published study.17 the working group was composed of wpath members from a range of disciplines with a variety of backgrounds, including medicine, nursing, pharmacy, social work, policy, law, and public health ; all members had experience in ehr use. members were selected for participation via a written application process overseen by the wpath board of directors. initial draft recommendations were developed via an informal consensus process among several clinician working group members ; this consensus process was directed by a physician experienced in ehr use in transgender care as well as in directing informal consensus guideline development, who also holds a degree in computer information systems. all clinicians who contributed to the initial draft guidelines were experienced in both transgender clinical care and ehr use and implementation. these initial draft recommendations were then circulated among the entire working group membership for further comment ; an updated draft was then sent to the wpath board of directors for final approval. it is recognized that the overwhelming majority of patients are not transgender, which has led to implementation of a binary male / female oriented system across multiple platforms such as ehr systems, billing and coding systems, and laboratory systems ; however, this structure inhibits the collection of accurate medical information, and therefore such systems should be modified.preferred name, gender identity, and pronoun preference, as identified by patients, should be included as demographic variables (such as with ethnicity). these would be captured in readily amendable, optional fields that are separate from the patient 's state - listed name and sex or gender designation, which may continue to be used for billing purposes in circumstances when the patient has not yet obtained legal change of name and/or sex or gender designation. note that some patients may identify as genderqueer and prefer the use of neither pronoun. while lists of current common gender identities, sex options (table 2), and pronoun options (box 1) are provided, ideally field parameters would be easily amended to reflect changing paradigms and social trends within transgender communities.provide a means to maintain an inventory of a patient 's medical transition history and current anatomy. an anatomical inventory would allow providers to record into the chart (and/or update as needed) the organs each individual patient has at any given point in time ; this inventory would then drive any individualized auto - population of history and physical exam templates. this inventory should be uncoupled from the patient 's recorded gender identity, assigned sex, or preferred pronouns. a list of recommended organs for inventory in transgender patients appears in box 2, and commonly sought treatments and procedures which may not be listed in current systems but should be included as selectable items in the medical or surgical history, appear in box 3. the following non - exhaustive list of procedures are not transgender - specific procedures and are omitted from box 3 as they are already listed in existing systems : hysterectomy, oophorectomy, vaginectomy, orchiectomy, breast augmentation. these procedures, however, also should also be un - coupled from any gender - coded template so that an individual coded as male who has had a hysterectomy, for example, could have that history documented. in addition, sex - specific organ procedures and diagnoses relating to these organs should be un - coupled, so that (as an example) a prostatic ultrasound may be ordered on a patient registered as female, or a cervical pap smear ordered on a patient registered as male. such practices would allow enhanced decision support for transgender - specific care, such as medication interactions, organ- and sex - specific preventive health alerts, or accommodations for sex - specific laboratory normal value ranges. for example, a patient with a female birth sex and male gender identity, currently registered as a female, who is taking testosterone, may have a hemoglobin of 17 g / dl flagged as a local flag driven by the patient 's birth sex, gender identity, and current testosterone prescription could alert clinicians to reconsider this high flag and review laboratory male reference ranges.the system should allow a smooth transition from one listed name, anatomical inventory, and/or sex to another, without affecting the integrity of the remainder of the patient 's record. it should be noted that, in some cases, changes in name and sex or gender designation of record will come at different times, and that in some jurisdictions official recognition of a change of sex or gender designation is not possible.a system should exist to notify providers and clinic staff of a patient 's preferred name and/or pronoun (if either or both of these differ from the current legal documented name / sex). systems should include an easily recognized notification or alert flag which appears at a time most consistent with the end - user 's workflow (figure 1). box 1suggested optional pronoun data collection fields masculine pronouns feminine pronouns neutral pronouns no pronouns something else (please specify) : box 2organs for inventory penis testes prostate breasts vagina cervix uterus ovaries box 3common treatments and procedures cross - sex hormone therapy, current user cross - sex hormone therapy, past user vaginoplasty, penile inversion vaginoplasty, colon graft phalloplasty, abdominal flap phalloplasty, free flap metoidioplasty scrotoplasty urethroplasty scalp advancement forehead reconstruction reduction thyrochondroplasty laryngeal feminization surgery soft tissue filler injections bilateral total reduction mammoplasty voice surgery other unlisted surgical procedure it is recognized that the overwhelming majority of patients are not transgender, which has led to implementation of a binary male / female oriented system across multiple platforms such as ehr systems, billing and coding systems, and laboratory systems ; however, this structure inhibits the collection of accurate medical information, and therefore such systems should be modified. preferred name, gender identity, and pronoun preference, as identified by patients, should be included as demographic variables (such as with ethnicity). these would be captured in readily amendable, optional fields that are separate from the patient 's state - listed name and sex or gender designation, which may continue to be used for billing purposes in circumstances when the patient has not yet obtained legal change of name and/or sex or gender designation. note that some patients may identify as genderqueer and prefer the use of neither pronoun. while lists of current common gender identities, sex options (table 2), and pronoun options (box 1) are provided, ideally field parameters would be easily amended to reflect changing paradigms and social trends within transgender communities. provide a means to maintain an inventory of a patient 's medical transition history and current anatomy. an anatomical inventory would allow providers to record into the chart (and/or update as needed) the organs each individual patient has at any given point in time ; this inventory would then drive any individualized auto - population of history and physical exam templates. this inventory should be uncoupled from the patient 's recorded gender identity, assigned sex, or preferred pronouns. a list of recommended organs for inventory in transgender patients appears in box 2, and commonly sought treatments and procedures which may not be listed in current systems but should be included as selectable items in the medical or surgical history, appear in box 3. the following non - exhaustive list of procedures are not transgender - specific procedures and are omitted from box 3 as they are already listed in existing systems : hysterectomy, oophorectomy, vaginectomy, orchiectomy, breast augmentation. these procedures, however, also should also be un - coupled from any gender - coded template so that an individual coded as male who has had a hysterectomy, for example, could have that history documented. in addition, sex - specific organ procedures and diagnoses relating to these organs should be un - coupled, so that (as an example) a prostatic ultrasound may be ordered on a patient registered as female, or a cervical pap smear ordered on a patient registered as male. such practices would allow enhanced decision support for transgender - specific care, such as medication interactions, organ- and sex - specific preventive health alerts, or accommodations for sex - specific laboratory normal value ranges. for example, a patient with a female birth sex and male gender identity, currently registered as a female, who is taking testosterone, may have a hemoglobin of 17 g / dl flagged as high by the interfacing laboratory system. a local flag driven by the patient 's birth sex, gender identity, and current testosterone prescription could alert clinicians to reconsider this the system should allow a smooth transition from one listed name, anatomical inventory, and/or sex to another, without affecting the integrity of the remainder of the patient 's record. it should be noted that, in some cases, changes in name and sex or gender designation of record will come at different times, and that in some jurisdictions official recognition of a change of sex or gender designation is not possible. a system should exist to notify providers and clinic staff of a patient 's preferred name and/or pronoun (if either or both of these differ from the current legal documented name / sex). systems should include an easily recognized notification or alert flag which appears at a time most consistent with the end - user 's workflow (figure 1). something else (please specify) : cross - sex hormone therapy, current user cross - sex hormone therapy, past user vaginoplasty, penile inversion vaginoplasty, colon graft phalloplasty, abdominal flap phalloplasty, free flap forehead reconstruction reduction thyrochondroplasty laryngeal feminization surgery soft tissue filler injections bilateral total reduction mammoplasty other unlisted surgical procedure suggested optional gender identity and sex data collection fields data should be able to be collected for any patient (ie, transgender / gender - non - conforming persons) but would not be required fields for the majority of (non - transgender) patients whose birth and current listed sex as well as gender identity are identical. these data could be housed in a special supplemental demographics section or in the medical or social history sections of the chart. additionally, the patient 's current listed sex (ie, the sex / gender as they are listed on their insurance documents, current state - issued identity documentation, etc) would be collected and stored in the primary sex / gender demographics field for billing purposes. ftm, female to male (ie, female assigned at birth, male - spectrum identity) ; mtf, male to female (ie, male assigned at birth, female - spectrum identity). implementation in allscripts professional ehr using the nickname field and a coding system. the preferred name (michael) is seen in parentheses after the patient 's legal name (mary) in the heading of every screen by every viewer. the preferred pronoun (m for male) is indicated by a second - order parenthesized letter after the preferred name. as the care of transgender patients moves into the mainstream, the medical informatics field will be asked to respond to the unique needs of this demographic through the implementation of more accurate and appropriate data collection methods in a range of products and systems. it is hoped that these user - driven recommendations will better inform health information technology research and ehr vendors on the specific needs of transgender patients in this context. future research should aim to explore current practices among both clinicians and vendors ; ultimately this information would be used to drive developer implementation of feasible models which satisfy the recommendations presented here. these recommendations of the working group have been reviewed and endorsed by the wpath board of directors. | transgender patients have particular needs with respect to demographic information and health records ; specifically, transgender patients may have a chosen name and gender identity that differs from their current legally designated name and sex. additionally, sex - specific health information, for example, a man with a cervix or a woman with a prostate, requires special attention in electronic health record (ehr) systems. the world professional association for transgender health (wpath) is an international multidisciplinary professional association that publishes recognized standards for the care of transgender and gender variant persons. in september 2011, the wpath executive committee convened an electronic medical records working group comprised of both expert clinicians and medical information technology specialists, to make recommendations for developers, vendors, and users of ehr systems with respect to transgender patients. these recommendations and supporting rationale are presented here. |
the reaction of 1,1-bis(pinacolboronate) esters with alkyl halides can be effected by metal alkoxides and provides a strategy for the construction of organoboronate compounds. the reaction is found to occur by alkoxide - induced deborylation and generation of a boron - stabilized carbanion. |
|
human metapneumovirus (hmpv) is a negative single - stranded rna virus of the family paramyxoviridae and closely related to the avian metapneumovirus (ampv) subgroup c [1, 2 ]. hmpv is an important aetiological agent of respiratory tract infection (rti) in infants, or senior and immunocompromised individuals. this infection caused different symptoms ranging from influenza like syndromes (i.e., fever, cough, and rhinorrhea) to severe lower respiratory tract infection. previous studies have shown that many children exposed to this virus and also easily to be reinfected as common [35 ]. therefore, hmpv is becoming as a major concern in child respiratory tract viral infection. genome sequencing and comparative analysis provides us a useful approach to analyze the pathogenicity of organisms. moreover, this analysis might also provide us an approach to understand its evolution history and cell - host interaction. as previously reported hmpv genome is approximately 13 kb in length, and the gene composition from 3 terminal to 5 terminal is n - p - m - f - m2 - 1/m2 - 2-sh - g - l [6, 7 ]. comparative analysis suggests that its genomic organization is similar to human respiratory syncytial virus (hrsv), which just lacks 2 nonstructural genes, ns1 and ns2. moreover, hmpv has been demonstrated the existence with two main genetic lineages termed as subtype a and b, which also containing within them the subgroups a1/a2 and b1/b2, respectively. the genetic diversity analysis shows the a2 sublineage exhibits the greatest diversity among all the sublineages of hmpv. as we all know, there are differences in the frequency of occurrence on synonymous codons in coding dna, which termed as synonymous codon usage bias. briefly, there are 64 different codons (61 codons encoding for amino acids plus 3 stop codons) in each organism, but only 20 different translated amino acids. these alternative codons for the same amino acids are termed as synonymous codons. in general, codon usage variation may be the product of natural selection and/or mutation pressure for accurate and efficient translation in various organisms [810 ]. synonymous codon usage bias on virus can provide us with a better understanding on the evolution profile, gene expression, and virus - host interaction [1114 ]. however, there is still lacking about codon usage pattern of hmpv genome and its major influence factors. herein, we firstly performed the comparative analysis of synonymous codon usage in hmpv genomes and analyzed their influencing factors. this study will provide a new insight to understand the pathogenicity and its evolution history of hmpv. in this study, a total 17 complete hmpv genomes which representing two genotypes were retrieved from ncbi (http://www.ncbi.nlm.nih.gov/) until december, 2011. the serial number (sn), genbank number, genotype, and other information moreover, 10 ampv genomes sequences were retrieved from ncbi database as reference frame (in supplemental table 1 supplementary material available online at doi:10.1155/2012/460837). to investigate the characteristics of synonymous codon usage, relative synonymous codon usage (rscu) values of each complete coding region in 17 hmpv genomes and the rscu value of each codon for their amino acid was calculated as previously described. a codon with an rscu value of more than 1.0 has a positive codon usage bias, while a value of less than 1.0 has a negative codon usage bias. when the codon with rscu values close to 1.0, it means that this codon is chosen equally and randomly. the codon usage data of human cell and bird cell were obtained from the codon usage database online (http://www.kazusa.or.jp/codon/). the effective number of codons (enc) is used to measure deviation from expected random codon usage of hmpv and is independent of hypotheses involving natural selection. if only one codon is used for each amino acid, this value would be 20, while all of codons are used equally, it will be 61. moreover, the index of gc3s was used to calculate the fraction of the nucleotides g + c content at the synonymous third codon position (excluding aug [met ], ugg [trp ], and the termination codons). multivariate statistical analysis can be used to explore the relationships between variables and samples. in this study, correspondence analysis was used to investigate the major trend in codon usage variation among genomes. in this study, the complete coding region of all 17 hmpv genomes was represented as a 59 dimensional vector, and each dimension corresponds to the rscu value of one sense codon (excluding met, trp, and the termination codons). correlation analysis was used to identify the relationship between nucleotide composition and synonymous codon usage pattern. in order to investigate the synonymous codon usage of hmpv, we calculated various rscu values of various codons from 17 different strains, including different genotypes. as shown in table 3, the preferred codons in hmpv are gca, aga, aau, gau, ugu, caa, gaa, gga, cau, cau, auu, uua, aaa, uuu, cca, uca, agu, aca, uau, guu. interestingly, all preferred codons in hmpv genomes are ended with a / u, while none of them is ended with g / c. this result suggests that hmpv genome has a great synonymous codon usage bias, and this phenomenon might highly associate with the nucleotide composition in its genomes. as shown in table 2, the g + c content of hmpv genome is 36.91%, which shares similar extent with another rna virus. there are over 68.57% codons are ended with a / u among hmpv genomes, and 40.87% codons are a3 end, and 27.7% codons are u3 end. this high abundance of a / u nucleotides is consistent with all preferred codons, which are ended with a / u. this phenomenon reflects that nucleotide composition is the main force to affect the codon usage bias in hmpv genome. moreover, as shown in table 2, the enc values among hmpv genomes show a range from 45.127 to 48.28, and its average value of 45.785 and sd value of 0.8458. the stable enc values suggest that their genomic compositions are much conserved among hmpv genomes. natural selection and mutation pressure have been considered to be two key factors which have effect on codon usage patterns of organisms. in order to investigate whether mutation pressure or natural selection as a determinative factor for codon usage mutation in hmpv, we calculated correlation relation between a%, u%, g%, c%, gc% and a3%, u3%, g3%, c3%, gc3%. as shown in table 4, there exhibits a very complex correlation map observed in nucleotide compositions. in detail, u3% has a significant positive correlation with u% (r = 0.7821, p < 0.01), while shared negative correlation with c% (r = 0.7153, p < 0.01) and gc% (r = 0.7474, p < 0.01). c3% has positive correlation with c% (r = 0.8331, p < 0.01) and gc% (r = 0.7880, p < 0.01), and negative correlation with a% (r = 0.6199, p < 0.01) and g% (r = 0.5846, p < 0.05). gc3% has significant positive correlation with c% (r = 0.7178, p < 0.01) and gc% (r = 0.8052, p < 0.01), but has negative correlation with u% (r = 0.7342, p < 0.01). interestingly, the gc3% shows positive correlation with c%, while shows negative correlation with g%. we calculated the gc3 skew by using formula as cg3 skew = (c3 g3)/(c3 + g3). the gc3 skew of hmpv range s from 0.371 to 0.592, which reveals that gc composition involved in the codon usage bias. these data suggest that the nucleotide constraint might play an important role in influencing synonymous codon usage bias. furthermore, the correlation analysis of first two principle axes (f1 and f2) in hmpv and its nucleotide contents were performed (table 5). apparently, the first principle axis (f1) has a significantly negative correlation with u3%, and negative correlation with gc3%. this result suggests that nucleotide u3% and gc3% are the major factor influencing the synonymous codon usage pattern in hmpv genome. moreover, we observed the second principle axis (f2) shared a significant positive correlation with g3%, and negative correlation with c3% and gc3%. therefore, compositional constraint is a major factor which is involved in shaping the pattern of synonymous codon usage bias in hmpv genome. to investigate the effect of different hpmv genotypes on synonymous codon usage, we analyzed the codon usage bias of different genotypes with correspondence analysis. from the correspondence analysis, the first dimension variable f1 and the second dimension variable f2 can reflect 43.27% and 33.38% of total mutation, respectively. as the plot of correspondence analysis shown (figure 1), each genotype is mainly separated and clustered into two clades. however, the sublineage of each genotype did not exhibit any significant difference among them. but this might be due to the limited number of hpmv genomes available in current study. therefore, the phylogenetic distant might effect on the variation of synonymous codon usage in hmpv, and this difference might reflect on their biological effect, such as viral replication, virulence, and so forth. from the enc - gc3% plot analysis (figure 2), the plots of each hmpv genomes are all under the expected curve, none of them shows above the curve. moreover, there are still some other factors that can effect on the codon usage bias of hmpv. in the current study as shown in table 3, the pattern of synonymous codon usage in hmpv shows a complementary profile, which shows in human cell. in detail, hmpv and human host cell shared only 1 preferred codon (aga), which encoded for arginine, while there are 17 different preferred codons between them. as a reference frame, ampvs were enrolled in this study, and it also shows a complementary pattern with its host, bird cell. the comparative analysis among hmpv and human host cell, hmpv and ampv host bird cell, and hmpv and ampv were analyzed. to compare the complementary ability of hmpv with bird cell, there are more overlays (6 preferred codons overlay) than hmpv with its human host cell (only 1 preferred codons overlay). this result shows human cell has much more complementary pattern with hmpv than bird cells. this might be more suitable for hmpv survive and persist infect in human host environment. this result also suggests that host factor plays an important role in codon usage bias in hmpv. this complementary trend will benefit for virus replication instead of competitive with its host and it might help us to understand the mechanism of hmpv persistent infection. interestingly, hmpvs are shares with more than 15 preferred codons to ampv, which might be due to a close phylogenetic distance. synonymous codon usage analysis can reveal much about virus genome. to understand the extent and causes of codon usage bias is essential for studying the viral evolution, particularly the interaction between viruses and host immune response. in this study as we know, the variation and evolution of virus generally happened in the changes of nucleotide composition. therefore, the nucleotide composition bias is the main force to influence the synonymous codon usage patterns. in this study first of all, in hmpv, all preferred codons are ended in a / u nucleotide, which occupied the majority of nucleotide composition in hmpv genome. this phenomenon confirmed that nucleotide composition was the main force in shaping the pattern of codon usage. secondly, enc was used to quantify the codon usage bias, which is one of the best overall estimators of absolute synonymous codon usage bias. in this study, we observed enc of these genomes fluctuated from 45.13 to 48.28 with a mean 45.78 0.85. this enc value of hmpv is consistent with other previously reported rna virus in the same family paramyxoviridae, ranging from 43.8 to 55.1, that is, measles virus 55.1, mumps virus 54.3, parainfluenza-3 virus 43.8, and respiratory syncytial virus 44.3 [8, 24, 25 ]. moreover, the enc of hmpv is more close to respiratory infection agents, rsv and parainfluenza-3, which reveals that the similar extent of codon usage bias among viruses might have similar infection syndrome. this observation helps us to address an interesting assumption that synonymous codon usage bias of virus might associate with its pathogenicity. mutation pressure and natural selection are generally treated as the main factors that account for codon usage bias in different organisms. enc - gc plot was considered as a part of the general strategy to investigate patterns of synonymous codon usage [9, 18, 26 ]. herein, all the plots are laid below the expected curve, suggesting that codon usage bias in all these 17 hmpv genomes was principally influenced by mutation bias, which consistent with that mutation pressure rather than natural selection is the most important determinant of the codon usage in human rna virus [8, 2731 ]. this observation can be explained as the mutation rates in rna viruses much higher than those in dna viruses. mutation pressure is the main force in shaping synonymous codon usage bias of rna virus. however, based on correspondence analysis, we observed an interesting phenomenon that codon usage bias in hmpv showed distinct differences among different phylogenetic types. it might suggest that codon usage bias plays an important role in hmpv evolution history. this similar phenomenon also observed in several other viruses, this might reflect that phylogenetic difference is a common influencing factor in shaping codon usage bias [25, 27, 3034 ]. therefore, we hypothesize that the difference of codon usage bias might influence its virulence in different genotypes. in this study, we also observed that hmpv showed a complementary trend with human cells by comparing the codon usage. this complementary will be benefit for the survive of virus, which can keep replication by using the nonpreferred codons in the host cell without competition, and this could be one of the mechanisms of virus persistent infection in the human environment. moreover, this pattern might also be caused by the longitude selection and evolution between human hosts with virus. therefore, this characteristic is important for hmpv keeping balanced with their host on the codon usage side, and also for understanding the cell - host interaction and viral evolution. in summary, we firstly reported the synonymous codon usage pattern in hmpv genomes and revealed that mutation pressure is the main force in shaping its codon usage bias. phylogenetic difference and host factors are also discussed, and this information can provide better understanding on the molecular evolution and its pathogenicity of hmpv. | human metapneumovirus (hmpv) is an important agent of acute respiratory tract infection in children, while its pathogenicity and molecular evolution are lacking. herein, we firstly report the synonymous codon usage patterns of hmpv genome. the relative synonymous codon usage (rscu) values, effective number of codon (enc) values, nucleotide contents, and correlation analysis were performed among 17 available whole genome of hmpv, including different genotypes. all preferred codons in hmpv are ended with a / u nucleotide and exhibited a great association with its high proportion of these two nucleotides in their genomes. mutation pressure rather than natural selection is the main influence factor that determines the bias of synonymous codon usage in hmpv. the complementary pattern of codon usage bias between hmpv and human cell was observed, and this phenomenon suggests that host cells might be also act as an important factor to affect the codon usage bias. moreover, the codon usage biases in each hmpv genotypes are separated into different clades, which suggest that phylogenetic distance might involve in codon usage bias formation as well. these analyses of synonymous codon usage bias in hmpv provide more information for better understanding its evolution and pathogenicity. |
the knhanes is a nationwide, population - based, cross - sectional health examination and survey conducted regularly by the division of chronic disease surveillance of the korea centers for disease control and prevention, under the ministry of health and welfare. the survey has three parts : health interview, health examination, and nutrition survey. the household component is based on information provided by an adult respondent aged 19 years from a sampled household ; it surveys the demographic characteristics of all members of the sampled household, as well as their income. the individual component questionnaire collects information on medical conditions, education and occupation, use of healthcare services, activity limitation, and quality of life and injury using a face - to - face interview conducted by trained interviewers. the content of the individual component questionnaire differs among age groups, because the risk and prevalence of many diseases vary with age. for participants aged 5 to 11 years, the health interview questionnaire was completed by a legal guardian. to select which households are to participate in the survey, a stratified, multistage, probability - sampling design is used ; this ensures that each year 's survey result represents the entire general population of south korea. in the 2010 to 2012 survey, all members of each selected household were asked to participate in the survey, and the participation rate was 75.9% to 77.5%. our cross - sectional study included data from a representative sample of south koreans aged 5 years ; the data were collected during the fifth knhanes (knhanes v 1, 2, and 3, conducted in years 2010, 2011, and 2012, respectively). a total of 23,376 subjects had completed the health examination survey and undergone ophthalmologic examinations ; after those aged < 5 years (n = 601) and subjects who had no visual acuity data (n = 679) were excluded, the remaining 22,096 were included in this study (fig. the survey adhered to the tenets of the declaration of helsinki for biomedical research and was approved by the institutional review board of the korea centers for disease control and prevention. the design of this specific study was approved by the institutional review board of kim 's eye hospital, seoul, south korea. participants aged 5 to 11 years were asked, have you visited a healthcare provider for an eye examination within the past year ? participants who had undergone eye examination specified whether it had taken place in a pediatric clinic affiliated with the national health insurance (nhi) corporation, an ophthalmology clinic affiliated with the nhi corporation, an opticians ' shop, or a non - nhi corporation ophthalmologist, during the course of vision screening in school, or other. participants aged 12 years or older were asked, when was the last time you had your eyes examined by an ophthalmologist, pediatrician, or other eye care provider (e.g., an optician or a provider of vision screening in school) ? they responded with one of the following options : within the past month, within the past year, within the past 3 years, 3 or more years ago, or never. we then categorized all respondents in this older age group into two groups : those who had visited an eye care provider in their lifetime and those who had not. participants were categorized into two main age groups : 5 to 11 years (young children) and 12 years (older population). the young children were then further categorized into two subgroups : 5 to 6 and 7 to 11 years ; the older population was subcategorized into seven age groups (1219, 2029, 3039, 4049, 5059, 6069, and 70 years). the sociodemographic covariates were sex (men / women), monthly house income (lowest, medium highest, highest quartiles), residential area (urban / rural), nhi status (nhi holder who was not a recipient of national basic livelihood security [nbls ], or recipient of nbls), private health insurance (phi) status (phi holder or not), and best - corrected visual acuity (bcva, 20 / 40 and < 20 / 40) in the better - seeing eye and also in the worse - seeing eye were included as covariates that might affect eye care service utilization. residential area was classified as urban in the cities of seoul, busan, daegu, incheon, daejeon, gwangju, and ulsan and all other provinces were classified as rural (gyeonggi, gangwon, chungnam, chungbuk, jeonnam, jeonbuk, gyeongnam, gyeongbuk, and jeju). education was categorized into four levels of elementary school graduate or lower, middle school graduate, high school graduate, and university graduate or higher. the overall weighted prevalence rates for eye care services utilization were expressed as percentages of the study population, with 95% confidence intervals (cis). because the results of the knhanes v had been weighted to compensate for the complex sampling design and allow for approximations of the korean population, weighted analyses were performed using spss ver. 20.0 software (ibm co., armonk, ny, usa). the associations between the use of eye care services (whether or not the participant had visited an eye care provider) and age, sex, education level, monthly household income, residential area, and the bcva values of the better - seeing eye and worse - seeing eye were analyzed using logistic regression. odds ratios (ors) and 95% cis were calculated : the or was used to assess the mutually confounding effect of these variables on the likelihood of eye care utilization. the knhanes is a nationwide, population - based, cross - sectional health examination and survey conducted regularly by the division of chronic disease surveillance of the korea centers for disease control and prevention, under the ministry of health and welfare. the survey has three parts : health interview, health examination, and nutrition survey. the household component is based on information provided by an adult respondent aged 19 years from a sampled household ; it surveys the demographic characteristics of all members of the sampled household, as well as their income. the individual component questionnaire collects information on medical conditions, education and occupation, use of healthcare services, activity limitation, and quality of life and injury using a face - to - face interview conducted by trained interviewers. the content of the individual component questionnaire differs among age groups, because the risk and prevalence of many diseases vary with age. for participants aged 5 to 11 years, the health interview questionnaire was completed by a legal guardian. to select which households are to participate in the survey, a stratified, multistage, probability - sampling design is used ; this ensures that each year 's survey result represents the entire general population of south korea. in the 2010 to 2012 survey, all members of each selected household were asked to participate in the survey, and the participation rate was 75.9% to 77.5%. our cross - sectional study included data from a representative sample of south koreans aged 5 years ; the data were collected during the fifth knhanes (knhanes v 1, 2, and 3, conducted in years 2010, 2011, and 2012, respectively). a total of 23,376 subjects had completed the health examination survey and undergone ophthalmologic examinations ; after those aged < 5 years (n = 601) and subjects who had no visual acuity data (n = 679) were excluded, the remaining 22,096 were included in this study (fig. the survey adhered to the tenets of the declaration of helsinki for biomedical research and was approved by the institutional review board of the korea centers for disease control and prevention. the design of this specific study was approved by the institutional review board of kim 's eye hospital, seoul, south korea. participants aged 5 to 11 years were asked, have you visited a healthcare provider for an eye examination within the past year ? participants who had undergone eye examination specified whether it had taken place in a pediatric clinic affiliated with the national health insurance (nhi) corporation, an ophthalmology clinic affiliated with the nhi corporation, an opticians ' shop, or a non - nhi corporation ophthalmologist, during the course of vision screening in school, or other. participants aged 12 years or older were asked, when was the last time you had your eyes examined by an ophthalmologist, pediatrician, or other eye care provider (e.g., an optician or a provider of vision screening in school) ? they responded with one of the following options : within the past month, within the past year, within the past 3 years, 3 or more years ago, or never. we then categorized all respondents in this older age group into two groups : those who had visited an eye care provider in their lifetime and those who had not. participants were categorized into two main age groups : 5 to 11 years (young children) and 12 years (older population). the young children were then further categorized into two subgroups : 5 to 6 and 7 to 11 years ; the older population was subcategorized into seven age groups (1219, 2029, 3039, 4049, 5059, 6069, and 70 years). the sociodemographic covariates were sex (men / women), monthly house income (lowest, medium highest, highest quartiles), residential area (urban / rural), nhi status (nhi holder who was not a recipient of national basic livelihood security [nbls ], or recipient of nbls), private health insurance (phi) status (phi holder or not), and best - corrected visual acuity (bcva, 20 / 40 and < 20 / 40) in the better - seeing eye and also in the worse - seeing eye were included as covariates that might affect eye care service utilization. residential area was classified as urban in the cities of seoul, busan, daegu, incheon, daejeon, gwangju, and ulsan and all other provinces were classified as rural (gyeonggi, gangwon, chungnam, chungbuk, jeonnam, jeonbuk, gyeongnam, gyeongbuk, and jeju). education was categorized into four levels of elementary school graduate or lower, middle school graduate, high school graduate, and university graduate or higher. participants aged 5 to 11 years were asked, have you visited a healthcare provider for an eye examination within the past year ? participants who had undergone eye examination specified whether it had taken place in a pediatric clinic affiliated with the national health insurance (nhi) corporation, an ophthalmology clinic affiliated with the nhi corporation, an opticians ' shop, or a non - nhi corporation ophthalmologist, during the course of vision screening in school, or other. participants aged 12 years or older were asked, when was the last time you had your eyes examined by an ophthalmologist, pediatrician, or other eye care provider (e.g., an optician or a provider of vision screening in school) ? they responded with one of the following options : within the past month, within the past year, within the past 3 years, 3 or more years ago, or never. we then categorized all respondents in this older age group into two groups : those who had visited an eye care provider in their lifetime and those who had not. participants were categorized into two main age groups : 5 to 11 years (young children) and 12 years (older population). the young children were then further categorized into two subgroups : 5 to 6 and 7 to 11 years ; the older population was subcategorized into seven age groups (1219, 2029, 3039, 4049, 5059, 6069, and 70 years). the sociodemographic covariates were sex (men / women), monthly house income (lowest, medium highest, highest quartiles), residential area (urban / rural), nhi status (nhi holder who was not a recipient of national basic livelihood security [nbls ], or recipient of nbls), private health insurance (phi) status (phi holder or not), and best - corrected visual acuity (bcva, 20 / 40 and < 20 / 40) in the better - seeing eye and also in the worse - seeing eye were included as covariates that might affect eye care service utilization. residential area was classified as urban in the cities of seoul, busan, daegu, incheon, daejeon, gwangju, and ulsan and all other provinces were classified as rural (gyeonggi, gangwon, chungnam, chungbuk, jeonnam, jeonbuk, gyeongnam, gyeongbuk, and jeju). education was categorized into four levels of elementary school graduate or lower, middle school graduate, high school graduate, and university graduate or higher. the overall weighted prevalence rates for eye care services utilization were expressed as percentages of the study population, with 95% confidence intervals (cis). because the results of the knhanes v had been weighted to compensate for the complex sampling design and allow for approximations of the korean population, weighted analyses were performed using spss ver. 20.0 software (ibm co., armonk, ny, usa). the associations between the use of eye care services (whether or not the participant had visited an eye care provider) and age, sex, education level, monthly household income, residential area, and the bcva values of the better - seeing eye and worse - seeing eye were analyzed using logistic regression. odds ratios (ors) and 95% cis were calculated : the or was used to assess the mutually confounding effect of these variables on the likelihood of eye care utilization. in total, 22,096 koreans (2,232 young children and 19,864 older people), representative of 47,990,761 koreans (based on the 2010 census of korea), were included in this study. the mean age of the study participants was 39.4 years (95% ci, 39.039.9 years), and 797 subjects who had not undergone ophthalmologic examination, 601 subjects under 5 years of age, and 679 subjects who had no visual acuity data were excluded. the nonparticipants were more likely to be in the older age groups, less educated, and in the lower household income group than the participants. however, the distributions of sex, residential area, nhi status, and possession of phi to supplement nhi coverage were not significantly different between participants and nonparticipants. in young children, the weighted prevalence of the eye care service utilization within the past year was 61.1% (95% ci, 58.1%64.1%). more than half of the population (56.3% ; 95% ci, 52.6%59.9%) had received eye care services by visiting an ophthalmology clinic (including nhi corporation clinics) ; the next largest proportion had undergone school vision screening (19.8% ; 95% ci, 17.0%23.0%). a smaller portion of subjects visited an optician 's shop (7.4% ; 95% ci, 5.7%9.5%), and the smallest portion of subjects attended a pediatric clinic (4.1% ; 95% ci, 2.8%6.0%). fig. 2 shows the types of eye care services that were used by young children within the past year. the associations between various factors and the use of eye care services within the past year among young children are described in table 2. the use of eye care services was significantly associated with age, sex, and monthly household income, but not with residential area, nhi status, phi status, or bcva of either the better - seeing eye or the worse - seeing eye in univariate analysis. in multivariate analysis, no association was found between the use of eye care services and sex, but there was a significant association with age, monthly household income, nhi status, and phi status. notably, the odds of eye care service utilization increased as monthly household income increased and were 14.6 times higher in the highest monthly household income group than in the lowest one (95% ci, 2.4587.03). a strong association between eye care services utilization was also found with nhi status (or, 10.18 ; 95% ci, 1.01102.32) and phi status (or, 2.37 ; 95% ci, 1.065.39) : nhi holders who were not non - nbls recipients were 10.18 times more likely to have used an eye care service in the past year than recipients of nbls, and phi holders were 2.4 times more likely to have used one in the past year than non - phi holders. table 3 shows the associations between sociodemographic factors and eye care in an ophthalmology clinic during the previous year for young children. age was the only significant factor in univariate analysis, whereas age and monthly household income were both significant factors associated with ophthalmology clinic visits in the multivariate analysis. young children in the older age group (711 years) were significantly more likely to have visited an ophthalmology clinic than children in the younger age group (56 years), even when controlling for other factors ; conversely, children in the highest monthly household income group were more likely to have received eye care in an ophthalmology clinic than those in the lowest group, but only when age and other variables had been controlled (or, 8.42 ; 95% ci, 1.0169.93). the weighted prevalence of lifetime use of eye care services in the older population was 73.5% (95% ci, 72.4%74.6%). the age group - specific weighted prevalence of lifetime use of eye care services is shown in fig. lifetime use of eye care services was highest in those aged 12 to 19 years (82.1% ; 95% ci, 79.8%84.2%) and lowest in those aged 30 to 39 years (69.0% ; 95% ci, 66.8%71.1%). the prevalence of eye care service utilization during the past year demonstrated similar patterns throughout all age groups : the highest prevalence was in those aged 12 to 19 years (47.1% ; 95% ci, 44.4%49.9%) and the lowest was found in those aged 30 to 39 years (23.7% ; 95% ci, 22.0%25.5%). the age group - specific weighted prevalence of eye care service utilization during the past year is shown in fig. the likelihood that a given participant had used eye care services gradually decreased through the ages of 12 to 19 years and 30 to 39 years and then gradually increased with advancing age. 4 shows the elapsed time from the last use of eye care services in the older population. the weighted prevalence in those who had never used eye care was 26.5% (95% ci, 25.4%27.6%). table 4 shows the results of the univariate and multivariate analyses for the associations between various sociodemographic factors and the likelihood of lifetime use of eye care services in the older population. univariate analysis indicated significant association between the use of eye care services and age, sex, residential area, bcva of the better - seeing and the worse - seeing eye, and education level. unlike young children, no association was found between lifetime use of eye care services and monthly household income, nhi status, or phi status in the older population. in multivariate analysis, there was a significant relationship between the use of eye care services and age, sex, residential area, bcva of the worse - seeing eye, and education level. women were more likely to have received eye care services than men (or, 1.78 ; 95% ci, 1.472.16), and urban residents were more likely to have received eye care than rural residents (or, 1.35 ; 95% ci, 1.041.73). the odds of elementary school graduates or lower having received eye care were 0.56 times lower than that of those with university or higher level education (95% ci, 0.370.85). in total, 22,096 koreans (2,232 young children and 19,864 older people), representative of 47,990,761 koreans (based on the 2010 census of korea), were included in this study. the mean age of the study participants was 39.4 years (95% ci, 39.039.9 years), and 797 subjects who had not undergone ophthalmologic examination, 601 subjects under 5 years of age, and 679 subjects who had no visual acuity data were excluded. the nonparticipants were more likely to be in the older age groups, less educated, and in the lower household income group than the participants. however, the distributions of sex, residential area, nhi status, and possession of phi to supplement nhi coverage were not significantly different between participants and nonparticipants. in young children, the weighted prevalence of the eye care service utilization within the past year was 61.1% (95% ci, 58.1%64.1%). more than half of the population (56.3% ; 95% ci, 52.6%59.9%) had received eye care services by visiting an ophthalmology clinic (including nhi corporation clinics) ; the next largest proportion had undergone school vision screening (19.8% ; 95% ci, 17.0%23.0%). a smaller portion of subjects visited an optician 's shop (7.4% ; 95% ci, 5.7%9.5%), and the smallest portion of subjects attended a pediatric clinic (4.1% ; 95% ci, 2.8%6.0%). 2 shows the types of eye care services that were used by young children within the past year. the associations between various factors and the use of eye care services within the past year among young children are described in table 2. the use of eye care services was significantly associated with age, sex, and monthly household income, but not with residential area, nhi status, phi status, or bcva of either the better - seeing eye or the worse - seeing eye in univariate analysis. in multivariate analysis, no association was found between the use of eye care services and sex, but there was a significant association with age, monthly household income, nhi status, and phi status. notably, the odds of eye care service utilization increased as monthly household income increased and were 14.6 times higher in the highest monthly household income group than in the lowest one (95% ci, 2.4587.03). a strong association between eye care services utilization was also found with nhi status (or, 10.18 ; 95% ci, 1.01102.32) and phi status (or, 2.37 ; 95% ci, 1.065.39) : nhi holders who were not non - nbls recipients were 10.18 times more likely to have used an eye care service in the past year than recipients of nbls, and phi holders were 2.4 times more likely to have used one in the past year than non - phi holders. table 3 shows the associations between sociodemographic factors and eye care in an ophthalmology clinic during the previous year for young children. age was the only significant factor in univariate analysis, whereas age and monthly household income were both significant factors associated with ophthalmology clinic visits in the multivariate analysis. young children in the older age group (711 years) were significantly more likely to have visited an ophthalmology clinic than children in the younger age group (56 years), even when controlling for other factors ; conversely, children in the highest monthly household income group were more likely to have received eye care in an ophthalmology clinic than those in the lowest group, but only when age and other variables had been controlled (or, 8.42 ; 95% ci, 1.0169.93). the weighted prevalence of lifetime use of eye care services in the older population was 73.5% (95% ci, 72.4%74.6%). the age group - specific weighted prevalence of lifetime use of eye care services lifetime use of eye care services was highest in those aged 12 to 19 years (82.1% ; 95% ci, 79.8%84.2%) and lowest in those aged 30 to 39 years (69.0% ; 95% ci, 66.8%71.1%). the prevalence of eye care service utilization during the past year demonstrated similar patterns throughout all age groups : the highest prevalence was in those aged 12 to 19 years (47.1% ; 95% ci, 44.4%49.9%) and the lowest was found in those aged 30 to 39 years (23.7% ; 95% ci, 22.0%25.5%). the age group - specific weighted prevalence of eye care service utilization during the past year is shown in fig. the likelihood that a given participant had used eye care services gradually decreased through the ages of 12 to 19 years and 30 to 39 years and then gradually increased with advancing age. 4 shows the elapsed time from the last use of eye care services in the older population. the weighted prevalence in those who had never used eye care was 26.5% (95% ci, 25.4%27.6%). table 4 shows the results of the univariate and multivariate analyses for the associations between various sociodemographic factors and the likelihood of lifetime use of eye care services in the older population. univariate analysis indicated significant association between the use of eye care services and age, sex, residential area, bcva of the better - seeing and the worse - seeing eye, and education level. unlike young children, no association was found between lifetime use of eye care services and monthly household income, nhi status, or phi status in the older population. in multivariate analysis, there was a significant relationship between the use of eye care services and age, sex, residential area, bcva of the worse - seeing eye, and education level. women were more likely to have received eye care services than men (or, 1.78 ; 95% ci, 1.472.16), and urban residents were more likely to have received eye care than rural residents (or, 1.35 ; 95% ci, 1.041.73). the odds of elementary school graduates or lower having received eye care were 0.56 times lower than that of those with university or higher level education (95% ci, 0.370.85). we classified our participants into young children (aged 511 years) and an older population (12 years) based on vision development and frequency of vision - screening examinations at specific ages. children tended to regularly use eye care services as part of health - screening programs for infants and children that are funded under the nhi act, as well as a vision - screening program that was introduced by the school health act in south korea. specifically, infants and children undergo health screenings that include examinations for normal healthy growth ; these include growth and development assessments and infant care consultations that reinforce health education. before the age of 6 years, children may undergo up to seven screening examinations, i.e., at 4, 9, 18, 30, 42, 54, and 66 months. under the school health act, first and fourth graders in elementary school, first year middle schoolers, and high school students are eligible for school - based vision - screening examinations at designated clinics ; the examinations include tests for visual acuity, color vision, and common ocular diseases such as conjunctivitis, epiblepharon, and strabismus. in addition, school health staff check children 's visual acuity in the second, third, fifth, and sixth grades of elementary school, so that all grades in elementary school, the first year in middle school, and high school students are assessed regularly. moreover, school health staff recommend that students undergo a more thorough examination if visual acuity in either eye is less than 20 / 30. eye care service at ophthalmology clinics can be distinguished from pediatric clinics and optician shops or any other screening eye care services. uncorrected visual acuity within the normal range does not guarantee that the eye is free of ophthalmologic disorders, and visual acuity can not be fully corrected in those with amblyopia or other eye disorders. the korean ophthalmological society suggests that everyone between 3 to 4 years of age should go through ophthalmologic examinations even if they do not have symptomatic ophthalmologic problems. the estimated prevalence of eye care service utilization in young children during the previous year was 61.1% (95% ci, 58.1%64.1%), which is higher than in the united states and taiwan, where the annual rate of eye care service use was 57.4% and 43.5%, respectively, in children with ocular disease and 49.3% and 22.5% in children without ocular disease. south korea has a nationwide universal health system, the nhi corporation, which is a single insurer that provides health insurance for most citizens living in the country. the nhi covers the majority of citizens, while the nbls covers the poor and other specified groups. people who receive coverage pay a certain portion of their healthcare costs as a co - payment ; this portion is determined differently depending on whether the individual is using inpatient or outpatient services, as well as the type and level of the healthcare institution. people who want coverage of services that the nhi system does not cover can get additional phi. recent research has indicated that screening leads to improved visual outcomes due to consequent early treatment in children aged 18 months to 5 years ; however, in this study, the prevalence of eye care service utilization in children aged 5 to 6 years (39.4% ; 95% ci, 34.0%45.1%) was significantly lower than in those aged 7 to 11 years (68.0% ; 95% ci, 64.7%71.2%). this discrepancy by age group may have occurred because children aged 7 to 11 years can complain about the loss of vision or visual symptom compared with younger children (aged 56 years). however, our data indicate no association between visual acuity and the use of eye care services in young children ; this discrepancy may have occurred because the school vision - screening program is compulsory, whereas the health - screening programs for children aged 5 to 6 years are not mandatory. our results suggest that many south koreans do not follow the recommendations to visit an eye care professional every 1 to 2 years, especially for younger children. su. reported that lack of parental awareness is an important barrier to children receiving vision screening, and williams. reported that family unawareness of the need for care is a major obstacle to ophthalmologic care in at - risk pediatric populations. a nationwide intervention program aimed at increasing awareness of the importance of eye care in children aged 5 to 6 years coupled with the rapid increase in the number of health facilities in recent decades, the public health financing policy has improved access to healthcare facilities in south korea ; however, there are significant economic and insurance factors associated with eye care utilization by young children. an individual nhi holder is about 10 times more likely to have had eye care in the previous year, and those having phi are about twice as likely to have sought eye care for young children in the past year. however, no difference in receiving eye care at ophthalmology clinic was revealed by nhi status or phi status. moreover, the younger children in the highest monthly household income group were more likely to have had eye care at an ophthalmology clinic in the previous year. this indicates that having national and/or phi could lead to increased screening, but not to increased ophthalmology clinic visits. a patient might visit an ophthalmology clinic only if they were recommended for further evaluation for abnormal results in screening ; additionally economic barriers might prevent care at an ophthalmology clinic, regardless of health insurance status. since eye care for children is initiated by their caregivers, an intervention program and insurance policy should be put in place to ensure that children receive the eye care they need at the appropriate times, regardless of family income status. in this study, women in the older population were more likely to have attended eye care services than men ; this corroborates with reports from other countries. for example, lee. reported that women in the united states use eye care services more often than men, and this is consistent with the beaver dam eye study.. reported that men were more likely to have received eye care services in hospitals in rural india and northern iran. furthermore, a sex difference in the prevalence of visual impairment has been noted in both high - income and low - income countries. the main cause of this disparity is that, in low - income countries, women use eye care services much less frequently. park. reported in another study that used the knhanes phase v survey that the prevalence of low vision and blindness was generally higher among women than men in south korea. in the present study, the odds of eye care service utilization was higher in women than man even after adjusting the vision by bcva 20 / 40 or < 20 / 40 in the better - seeing eye and also in the worse - seeing eye. additional research is required to determine whether the more frequent use of eye care services among women can be explained by their higher prevalence of low vision and blindness. this study found that south korea individuals in the older population with higher education levels tended to use eye care services more often than those with lower education ; this is consistent with other reports. parental educational level is also important, because it affects not only personal use of eye care services, but also children 's access to care. several studies have shown that children with eye diseases whose parents have a lower level of education receive a lower level of eye care. furthermore, numerous studies have shown that the quality of physician - patient communication is lower among the less educated than among more educated patients. in our study, the aforementioned economic and insurance factors were not related to the use of eye care services in the older age population. this suggests that, in contrast with some other reports, financial constraints were not significant barriers to eye care in older individuals in our population because most are covered by the nhi system. in contrast with our study, several studies have shown that eye care is less common among uninsured adults. specifically, lee. reported that adults who had experienced an interruption in their health insurance coverage in the previous 12 months were less likely to have reported visiting an ocular healthcare provider than those who were consistently insured. they estimated that 3.8% (7.8 million) of community - dwelling adults in the united states reported an insurance - coverage gap in the past 12 months. they also found that adults with concurrent private and public healthcare coverage were generally more likely to have reported an ocular healthcare visit in the preceding 12 months than were adults who had private insurance only, even after adjusting for sex, level of visual impairment, and education. together, these results indicate that, while economic and insurance factors were not related to the use of eye care services in the older population of south korea, men, rural residents, and those with better visual acuity in the worse - seeing eye were less likely to have had an eye examination during their lifetime. one possible explanation for this is that the older population may encourage their children to receive eye care, because of their economic status, but they may not have time or accessibility for their own eye care ; in addition, accessing facilities may be difficult in terms of time and geographic distance, and these individuals may not prioritize eye care in the absence of serious vision impairment. therefore, different intervention programs or campaigns are needed and should be customized for specific age groups. education or campaigns to emphasize the importance of regular eye check - up are needed for the older south korean population rather than encouraging them to buy phi. first, the non - response rate in the knhanes ranged from 24.1% to 22.5% between 2010 and 2012, and non - response to the survey introduced a bias. researchers conducting the knhanes must devise and implement methods for reducing this bias during the knhanes data analysis. similarly, our data may have some degree of recall bias, and the correlations identified in this cross - sectional study do not necessarily ensure causal relationships. second, comparison of our study participants and nonparticipants revealed significant differences : nonparticipants were more likely to be older and less educated than participants. these differences in age and education between participants and non - participants in our study may have led to an underestimation of eye care services utilization. our results show that the elderly population (aged 70 years) tended to use eye care services more than younger age groups (or, 0.46 ; 95% ci, 0.280.74 for age 3039 years ; or, 0.62 ; 95% ci, 0.390.99 for age 4049 years), whereas the least educated group (elementary school or lower) tended to use eye care services less than the more highly educated population (or, 0.56). third, we did not compare individuals diagnosed with a specific eye condition, e.g., diabetic retinopathy or glaucoma, to those without eye disease. individuals with diabetic retinopathy or glaucoma may be more likely to have used eye care services, because these conditions necessitate regular check - ups. to assess eye health, we analyzed the bcva values in the better - seeing and the worse - seeing eye ; however, glaucoma patients with peripheral visual field defects and central vision sparring may have good bcva. additional research is needed to assess whether specific eye conditions are related to the use of eye care services. despite the above limitations, one notable strength of this study was the large sample size (n = 22,096), which is representative of the general south korean population in all age groups older than 5 years. furthermore, the validity of our results was controlled ; we used a standardized protocol, and our examiners were periodically trained by acting staff members of the epidemiologic survey committee of the korean ophthalmological society. our findings could be used to promote the use of eye care in south korea and to identify specific sub - groups that should receive additional education about care resources. in conclusion, in south korea, there are sociodemographic disparities that correlate with the use of eye care services. our current study has demonstrated that there are significant differences in socioeconomic factors of household income, nhi status, and phi status in children aged 5 to 11 years. females, who are generally considered to have a more flexible work schedule, may have more time to use eye care services, as well as people who living in urban areas, where they can easily access eye care service. people that have difficulty with daily activity with a bcva less than 20 / 40 in the worse - seeing eye are more likely to use eye care services during their lifetime ; however, people with low education levels and those 12 years of age are not likely to use eye care service during their lifetime. awareness must be increased to address these disparities, and targeted intervention programs must be established to increase access to eye care services. the findings of this population - based study will provide useful information for policy makers and program planners to promote the use of eye care services in south korea. | purposeto estimate the factors and prevalence of eye care service utilization in the south korean population.methodsthis cross - sectional, population - based study included data from 22,550 koreans aged 5 years who participated in the korea national health and nutrition examination survey from 2010 to 2012. for people aged 5 to 11 years (young children), information was based on self - reports of contact with eye care service in the past year ; for people aged 12 years (older population), the information was based on the self - reported lifetime contact with eye care service. univariate and multivariate logistic regression analyses of the complex sample survey data were performed.resultsthe prevalence of eye care service use in young children during the past year was 61.1% (95% confidence interval, 58.1%64.1%), while that in the older population during their lifetime was 73.5%. subjects aged 7 to 11 years were more likely to have had an eye examination in the past year than subjects aged 5 to 6 years (odds ratio, 3.83 ; 95% confidence interval, 2.376.19). multivariate logistic regression analysis indicated that higher monthly household income, being a national health insurance holder, and having private health insurance were related to more frequent use of eye care services in young children. for the older population and women, those living in an urban area and those with a best - corrected visual acuity less than 20 / 40 in the worse - seeing eye were more likely to have had an eye examination during their lifetime. low education level was associated with low lifetime use of eye care services in the older population.conclusionsthere are sociodemographic disparities with use of eye care services in south korea. this population - based study provides information that is useful for determining different intervention programs based on sociodemographic disparities to promote eye care service utilization in south korea. |
we report a case of residual neuromuscular blockade lasting more than 5 h following an intravenous intubating dose of 50 mg of rocuronium, followed by 10 mg of the same neuromuscular blocking agent given an hour later. we have also discussed possible causes of prolonged neuromuscular antagonism in our patient, the importance of neuromuscular assessment, and the use of post - tetanic count when receiving a surgeon s request for additional relaxation. an 82-year - old 74 kg woman presented for completion of a right extended hemicolectomy for colonic dysplasia. her past medical history included long - standing hypertension, obesity, dyslipidemia, type 2 diabetes mellitus, and glaucoma. her surgical history included bilateral eye procedures for glaucoma, left achilles tendon repair, and a colonic resection for dysplasia 4 years ago. she denied any anesthesia - related complications with her previous procedures, family history of anesthetic complications, or family history of neuromuscular disorders. her medications at the time of surgery included aspirin, bimatoprost - timolol ophthalmic solution, calcium with vitamin d, dorzolamide ophthalmic solution, ezetimibe, glyburide, hydrochlorothiazide, insulin glargine, lisinopril, pravastatin, and prednisolone ophthalmic solution. the patient received 2 mg of intravenous midazolam pre - operatively, and induction was accomplished with 100 mcg of fentanyl, followed by 150 mg of propofol. the patient received 0.4 mg of hydromorphone shortly after induction as well as 3 g of ampicillin - sulbactam intravenously. anesthesia was maintained throughout the case at 1mac of end - tidal desflurane with 50/50 air and oxygen. esophageal temperature was kept above 35c throughout the case using active warming with a forced air warming device. our involvement in the case begins at 1.5 h after induction, at which point an anesthesia personnel change occurred. it was noted that 10 mg of rocuronium was given 15 min prior to hand - off communication due to the surgeon s monitoring of neuromuscular function was not performed at that time and the surgeon s assessment was made by tactile feel in the surgical field. shortly after hand - off, a nerve stimulator (tof - watch, bluestar enterprises, omaha ne) was attached over the facial nerve using electrocardiogram pads and it was set to 60 ma, revealing a train - of - four (tof) count of 0 at the corrugator supercilii. at 1.75 h after induction, tof count was still 0 and the post - tetanic count (ptc) was 0. the post - tetanic count was determined by 5 sec of 50 hz tetanic stimulation followed 3 sec later by 1 hz single stimulation. an additional nerve stimulator was attached over the ulnar nerve giving the same results. surgery was completed at approximately 2 h post - induction, and tof count remained 0 with a ptc of 0. desflurane was discontinued and an infusion of propofol 25 - 50 mcg / kg / min was started. a mapleson f circuit was constructed and the patient was transferred to the post - anesthesia care unit (pacu) while delivering positive pressure ventilation using 100% oxygen. consideration was made for a selective relaxant binding agent (srba) such as sugammadex, however, it is not currently available in the united states. on arrival to the pacu the laboratory results were similar to pre - operative values except for serum potassium, which was 3.6 the patient received 40 meq of intravenous potassium given at 10 meq / h to correct the hypokalemia. hepatic enzymes had been assessed within recent months and were found to be within normal limits. tof count was 0, ptc was 1, and no spontaneous respiratory effort was noted. at 4.5 h, tof count was 1. at 5.5 h, tof ratio was 90%. since the tof - watch was not placed or calibrated at the beginning of surgery, and due to prolonged neuromuscular blockade, it was decided to give an acetylcholinesterase inhibitor prior to extubation. the patient was given 5 mg of neostigmine with 0.6 mg of glycopyrrolate and extubated without difficulty 10 min later. evaluation of the degree of neuromuscular blockade by clinical criteria alone does not exclude clinically significant curarization. anesthesia personnel should take into consideration the assessment of neuromuscular blockade by the surgical team, but should not administer additional neuromuscular blocking agents based on this assessment alone. similar cases[24 ] have demonstrated that even a single intubating dose of an intermediate - acting neuromuscular blocking agent can cause prolonged neuromuscular blockade, which should mandate the need for neuromuscular assessment every time additional neuromuscular blocking agent is considered. failure to objectively assess neuromuscular function may result in prolongation of a profound neuromuscular blockade, as was seen in our case. liver disease could decrease drug metabolism and increase the volume of distribution, which could prolong the duration of action. renal failure prolongs the duration of action and the effect could range from insignificant to an 84% increase in mean residence time with a 0.6 mg / kg bolus dose. volatile anesthetics may slightly reduce neuromuscular transmission, even in the absence of neuromuscular blockade. volatile anesthetics prolong the duration of action of neuromuscular blockers and decrease the dose required for blockade.[1012 ] potentiation is greatest with desflurane, followed in order by sevoflurane, isoflurane, halothane, and propofol. antibiotics that are known to potentiate neuromuscular blockade include aminoglycosides, polymyxins, lincomycin, clindamycin, and tetracyclines. there is a long list of drugs that can augment blockade including lithium, local anesthetics, cardiac antidysrhythmics, diuretics, antiestrogens, and magnesium sulfate. when assessing a profound neuromuscular blockade, the choice of muscle to monitor as well as the type of stimulation to deliver should be taken into account. the corrugator supercilii is useful as a guide to assess deep blockade when compared to the orbicularis oculi and adductor pollicis muscles.[1618 ] assessment of tof at the corrugator supercilii better reflects abdominal muscle relaxation. post - tetanic count can provide useful information about deep neuromuscular blockade.[2022 ] a post - tetanic count of <3 demonstrates over 5 - 10 min of additional deep blockade with rocuronium, vecuronium, atracurium, or cisatracurium and over 30 min of additional deep blockade with pancuronium. it is important to note that tetanic stimulation may produce lasting antagonism of neuromuscular blockade at the site of testing, and tetanic stimulation should not be performed more often than every 6 min at a given site. it is probable that a single factor can not explain the prolonged neuromuscular blockade in our patient. these factors include female gender, increased age, mildly reduced renal function, hypokalemia, and the use of desflurane. however, there are many cases of patients with similar compromises who emerge from neuromuscular block without delay. the exact cause of our patient s prolonged blockade remains unknown and helps to emphasize that neuromuscular assessment should be used for all patients despite the presence or absence of factors that prolong neuromuscular blockade. figure 1 represents our algorithm to aid in clinical decision making when presented with a surgeon s perception of inadequate neuromuscular antagonism, leading to a request for additional neuromuscular blocking agent. given our patient s presentation, it is very likely that she had profound neuromuscular blockade at the time when additional relaxation was requested. qualitative confirmation of an appropriate surgical level of relaxation could have been achieved by following our proposed algorithm. this case illustrates the need to assess neuromuscular function prior to the use of additional neuromuscular blocking agent and not to rely on a surgeon s perception of inadequate blockade. numerous factors can lead to prolonged effect of neuromuscular blocking agents including increased age, female gender, renal failure, electrolyte disturbances, and concomitant use of various medications. train - of - four assessment combined with post - tetanic count can be used to provide objective evidence of profound blockade when the anesthesia provider is presented with perceived inadequate neuromuscular antagonism in the surgical field. | evaluation of the degree of neuromuscular blockade by the surgeon using clinical criteria alone is unreliable. we report a case of prolonged neuromuscular blockade lasting 5.5 h, where an additional intra - operative dose of neuromuscular relaxant was given at the request of the surgical team. possible causes of prolonged neuromuscular antagonism are discussed, as is the importance of neuromuscular assessment prior to the administration of additional neuromuscular blocking agents when receiving a surgeon request for additional neuromuscularblockade. |
a single egg contains all the information required for its proliferation and differentiation into a complete organism and accurate spatial distribution of maternal factors is a critical issue for early development, cell determination, differentiation and germ layers formation (1). all mrnas translated during the initial stages of development originate from the mother as transcription of new zygotic mrna is initiated only after 12 cell divisions during what is called the midblastula transition (mbt). the cellular distribution of maternal factors and their functions are usually studied in model organisms such as drosophila melanogaster, caenorhabditis elegans and mus musculus. these studies are hampered by the very small amounts of rna (pg of total rna) in invertebrate and mammalian cells. in contrast, the egg from the african clawed frog xenopus laevis contains a microgram of total rna. the dark pigmented animal hemisphere derives its colour from the pigmented melanosomes and contains the egg nucleus (2), whereas the opposite light vegetal hemisphere contains yolk platelets. during early development, the vegetal hemisphere follows endodermal fate (gut) and the marginal zone forms mesoderm layer with blood, bone and muscle cell types. some are transcription factors, while others are signalling factors or regulators of activity of signalling molecules (3). two groups of mrna molecules have been reported to localize in the vegetal hemisphere during oogenesis. germ cell determinants such as xcad2 (nanos related, zn finger protein), xpat (unknown function), deadsouth (rna helicase) and mrnas for the wnt11 (wnt family member) gene are vegetally localized in early stages 1 and 2 by the metro (messenger transport organizer) pathway (28). a second group of vegetal genes includes vegt (t - box transcription factor), otx1 (a homeobox gene) and vg1 (tgf - beta family member). these localize vegetally by cytoskeletal - based transportation during later stages of oogenesis (8,9). other genes, such as oct60 (transcription factor, pou family), an1 (ubiquitin like fusion protein), an2 (mitochondrial atpase subunit), ets1 and ets2 (transcription factors, ets family members) and xpar-1 (serine / threonine kinase) have been found localized to the animal pole (3,911). xenopus sperm enters the egg through the animal hemisphere and the point of entry can be distinguished by a change in cortex cytoskeleton structure that leads to a local change in pigmentation. the cortex rotates by about 30 and alters a v organization through cytoskeletal and cytoplasmic rearrangements (12,13). the cortex movement induces local redistribution of -catenin protein and the -catenin stabilizing agent to a site opposite to the sperm 's entrance (14,15). accumulated -catenin proteins then induce local gene expression of some zygotic factors including siamois and xnr3. these are important for the formation of the organizer, which defines the future dorsal site of the embryo. we have previously shown that there is substantial variation in gene expression among seemingly homogeneous cells (16). in this work we describe a novel approach to study intracellular expression profiles by real - time pcr tomography (17,18). xenopus laevis females were stimulated by hcg (human chorionic gonadotrophin) injection and in vitro fertilized (ivf) eggs were incubated at 22c. the eggs were not treated with cystein, which is common procedure, because the treatment compromises manipulation of the eggs and rna stability after defrosting of the material. only eggs that turned round with the animal pole on the top were harvested for sectioning. more than 90% of the turned eggs divided within 90 min following ivf. four types of eggs were collected : unfertilized eggs and eggs collected at 25, 50 and 85 min post- ivf. the eggs were embedded in optimum cutting temperature (oct) compound and dissected into 35 slices (30 m) across the a v axis (figure 1). consecutive slices were pooled into five groups with seven slices in each. from each group, rna concentrations were determined with the nanodrop nd1000 quantification system (nanodrop inc.) and rna quality was assessed with the 2100 bioanalyzer using the rna pico chip (agilent). in general rna quality was very high. total rna was reverse transcribed (high capacity cdna archive kit- applied biosystems) using 100 ng total rna with 2.5 l of random primers in water in a total volume of 16.5 l. the mixture was incubated for 10 min at 72c. after cooling to room temperature, 1 l of dntps (25), 2.5 l of 10 reverse transcription buffer and 2.5 l of multiscribereverse transriptase (50 u/l) were added. real - time pcr assays had a final volume of 25 l and contained 3 l of cdna, 1u surestart taq dna polymerase (stratagene - europe), 2.5 l of reaction buffer (10), 3 mm mgcl2, 0.4 mm dntp mix, 50 000-fold diluted sybrgreen i (molecular probes), 25 000-fold diluted rox reference dye and 0.3 mm primers. pcr was run in a mx3005p (stratagene) with cycling conditions : 95c for 12 min, 45 cycles at 95c for 20 s, 60c for 25 s, 72c for 30 s. after cycling the samples were heated to 95c for 1 min, and melting curve was recorded between 65 and 95c. (a) the xenopus laevis oocyte imbedded in oct is mounted in a cryostat. (b) the material is sliced for subsequent analysis by real - time rt - pcr. (a) the xenopus laevis oocyte imbedded in oct is mounted in a cryostat. (b) the material is sliced for subsequent analysis by real - time rt - pcr. gene - expression data were analyzed using genex software from multid analysis (www.multid.se) and prism4 from graphpad (www.graphpad.com). it was not possible to use any internal reference genes for normalization, since this is the first time intracellular mrna levels are being quantitated and there is no information on what mrnas might be homogeneously distributed within the cell. consequently, we normalized individual mrnas against the total amount of rna used for reverse transcription, essentially measuring gene - expression levels relative to the total amount of rna in each section. since rna yield is rather uniform, we assume that total rna is distributed homogenously in the cell. consequently, normalization is against the volume of the segments, thus accounting for the differences in segment sizes due to the spherical shape of the cell. although the data are perfectly comparable within each section, there may be bias across sections due to variations in the density of total rna and in reverse transcription yields. these are caused by the sample matrix, which is quite different in the animal and the vegetal poles of the oocyte. the real - time pcr ct values were converted to relative quantities assuming 100% pcr efficiency, and the amounts of transcripts in the five egg sections were expressed as the fractions of the mrna molecules found in each of five segments along the a v axis in the xenopus oocyte : ctj is the ct determined for section j of the oocyte and xj is the fraction of the mrna found in this section. since the amounts of mrnas in the five sections are of the same order of magnitude, the assumption of 100% pcr efficiency will have little effect on the calculated intracellular mrna profiles. the initial normalization against total rna ensures that the profiles reflect true variations in the levels of the mrnas along the a v axis of the cell. the conventional real - time pcr results were confirmed for selected genes with digital pcr using the biomark digital array from fluidigm (www.fluidigm.com). the array is designed to accept 12 sample mixtures, which each is partitioned into a different 765-chamber grid. ten - microliter reaction mix was loaded onto the chip, containing 3.4 l of total rna, 0.5 l superscript rt / taq (cellsdirect qpcr - rt kit, invitrogen), 1 l buffer containing rox, 1 l of primers (9 m) and fam - labelled taqman probe (2 m) and 0.1 l of tween (10%). the input amount of total rna was tuned to produce less cdna molecules than the number of chambers. the mixture was distributed into the 765 chambers, incubated for 15 min at 50c for reverse transcription and then analyzed by pcr, starting with hotstart activation at 95c for 2 min followed by 45 pcr cycles at 95c for 15 s and 60c for 30 s. fam / rox fluorescence signal was collected at the end of each cycle, and the number of chambers that gave positive fluorescence signal after 40 cycles was registered. assuming poisson distribution of the cdna molecules in the chambers, the average number of cdna molecules per chambers is given by in{[1p(x1) ] }, where p(x1) is the fraction of positive pcr reactions. a sample distributed into 765 chambers thus contained a total of 765 in{[1p(x1) ] } cdna molecules. the number of mrna molecules in the sample can then be grossly estimated assuming 80% cdna synthesis yield in the reverse transcription reaction (19). expression levels of mrnas specified by the wnt11, foxh1, vegt, vg1, oct60, gsk-3, dishevelled, elongation factor-1 (ef-1), xdazl, xmam, tcf-3, gapdh, -catenin, xcad2, otx1, xpar-1, deadsouth and stat3 genes were all characterized by distinct and reproducible intracellular gradients. as an example, figure 2a and b shows vg1 and oct60 intracellular mrna gradients measured on eggs from four different females. oct60 is predominantly found at the animal pool, while vg1 is preferably found at the vegetal pool. although there is variation among individual cells, the intracellular gradients are clearly observed against the biological variation of the females, as reflected by the standard errors of the means. figure 2c and d also shows mrna intracellular distributions for vg1 and oct60 prior to ivf, and at 20, 55 and 85 min after ivf. statistical analysis using two - way anova with bonferroni post - test on a pairwise comparison of the profile of the unfertilized oocyte with mrna profiles collected at different time points after fertilization revealed that the correlation between segment and mrna level is extremely significant (p < 0.0001), but that there is no effect of fertilization and time following fertilization (p 1). distribution of (a) vg1 and (b) oct60 expression density along the oocyte animal vegetal axis. the rna was prepared from two to three individual eggs (standard error of the means indicated by error bars) from four different females (indicated by regular bars). distribution of (c) vg1 and (d) oct60 along the animal vegetal axis. rna was prepared from at least six eggs before ivf and at 20, 50 and 85 min after fertilization. distribution of (a) vg1 and (b) oct60 expression density along the oocyte animal vegetal axis. the rna was prepared from two to three individual eggs (standard error of the means indicated by error bars) from four different females (indicated by regular bars). distribution of (c) vg1 and (d) oct60 along the animal vegetal axis. rna was prepared from at least six eggs before ivf and at 20, 50 and 85 min after fertilization. the mrna profiles of 15 genes characterized in at least six eggs are shown in figure 3a. the profiles fall into two distinct classes, and are characteristic of animal and vegetal locations, respectively. the mrnas located preferentially at the animal pole are foxh1, oct60, gsk-3, dishevelled, ef-1alpha, xmam, tcf-3, gapdh, -catenin and xpar-1. those located at the vegetal pole are vegt, vg1, xdazl, wnt11 and otx1. in addition, stat3 was measured in four cells and found to be located in the animal hemisphere, while xcad2 (measured in four cells) and deadsouth (measured in three cells) were vegetally located (data not shown). for oct60 (animal) and wnt11 (vegetal), the intracellular expression profiles measured by qpcr tomography were confirmed with digital pcr (figure 4). oct60 shows highest expression in sections 2 and 3 from the animal pole, while wnt11 expression is largest in section 5, which is closest to the vegetal pole. qualitatively, this is in agreement with the real - time pcr results in figure 3. assuming there are no important differences, we calculated the average vegetal and animal mrna profiles also shown in figure 3b. the error bars represent 1 sd, within which 68% of the measured values should be found. the standard errors of the means were insignificant, and the average values shown by the symbols have negligible errors. figure 3.(a) averaged intracellular mrna concentration profiles (av) for genes studied in at least six eggs. animal genes (foxh1, oct60, gsk-3, dishevelled, ef-1alpha, xmam, tcf-3, gapdh, -catenin and xpar-1) are shown in red and vegetal genes (vegt, vg1, xdazl, wnt11 and otx1) are shown in blue. (b) average expression profiles of all vegetal (red) and all animal (blue) genes. the error bars indicate 1 sd. figure 4.digital pcr of wnt11 and oct60 showing abundance of transcripts in the five oocyte segments (av). mrna from each segment was distributed into 765 chambers that were analyzed by rt - pcr. (a) averaged intracellular mrna concentration profiles (av) for genes studied in at least six eggs. animal genes (foxh1, oct60, gsk-3, dishevelled, ef-1alpha, xmam, tcf-3, gapdh, -catenin and xpar-1) are shown in red and vegetal genes (vegt, vg1, xdazl, wnt11 and otx1) are shown in blue. (b) average expression profiles of all vegetal (red) and all animal (blue) genes. digital pcr of wnt11 and oct60 showing abundance of transcripts in the five oocyte segments (av). mrna from each segment was distributed into 765 chambers that were analyzed by rt - pcr. this is the first report of sub - cellular expression profiling and quantification of mrna within a single cell. using real - time pcr, which is currently the most sensitive and reliable technique for quantitative mrna analysis, we measured the intracellular profiles of selected developmental mrnas within the x. laevis oocyte. our results reveal the existence of characteristic expression gradients, and demonstrate that real - time pcr tomography is highly suitable for measuring them quantitatively. out of the 18 genes studied, 11 were found preferentially located at the animal pole (animal genes), while seven were preferentially located at the vegetal pole (vegetal genes). animal genes were foxh1, oct60, gsk-3, dishevelled, ef-1, xmam, tcf-3, gapdh, -catenin, xpar-1 and stat3. oct60 has previously been found located at the animal pole by in situ hybridization (10). ef-1 and gapdh have been ascribed housekeeping functions and used as reference genes (20). however, they show clear animal location. interestingly, apc, -catenin, fz7, gsk-3, dishevelled and tcf-3, which specify components of the wnt pathway are animal genes, whereas wnt11 itself shows vegetal location. the genes found to have vegetal location were vegt, vg1, xdazl, wnt11, otx1, deadsouth and xcad2. within the resolution of our technique the animal genes were preferentially found in the second and third (central) sections of the oocytes, while the vegetal genes were found preferentially in the fourth and fifth sections. however, although the polarization of the vegetal genes is much stronger than the (opposite) polarization of the animal genes, that polarization is not total. about 5% of mrna molecules of the vegetal genes were found in the first section taken from the opposite pole and another 10% in the second section (figure 3b). hence, the extreme polarization of both animal and vegetal genes seen by in situ hybridization techniques, where virtually all genes are located at either pole (9), is not supported by our observations. instead our data suggest that although there is a distinct bias to the location of the mrna, it is distributed more evenly. the reason for this discrepancy is unclear ; however, we note that the cell nucleus is expected to be located in sections two and three, which is where we find the animal genes to be most abundant. perhaps most of the animal mrnas are still located within the nucleus and are held there until their translation is required. interestingly, fertilization of the oocytes and the cortical rotation that follows has no detectable effect on the intracellular mrna gradients. in summary, real - time pcr tomography can measure intracellular mrna gradients more sensitively and with greater resolution than traditional in situ hybridization. in the present work, each cell was cut into 35 30 m slices, yielding up to 75 ng of rna per slice. this is not close to any limit, since a regular cryostat can easily cut slices of 10 m, yielding some 100 slices from a single x. laevis oocyte. this would allow the generation of mrna profiles with much higher resolution, the only potential constraint being the amount of rna extracted from each slice. however, the use of appropriate multiplexing and/or pre - amplification techniques should help overcome this limitation. other applications of real - time pcr tomography are readily envisaged : the localization of nuclei through genomic dna, of mitochondria through mitochondrial dna and of translationally active sites through ribosomal rna. | real - time pcr tomography is a novel, quantitative method for measuring localized rna expression profiles within single cells. we demonstrate its usefulness by dissecting an oocyte from xenopus laevis into slices along its animal vegetal axis, extracting its rna and measuring the levels of 18 selected mrnas by real - time rt - pcr. this identified two classes of mrna, one preferentially located towards the animal, the other towards the vegetal pole. mrnas within each group show comparable intracellular gradients, suggesting they are produced by similar mechanisms. the polarization is substantial, though not extreme, with around 5% of vegetal gene mrna molecules detected at the animal pole, and around 50% of the molecules in the far most vegetal section. most animal pole mrnas were found in the second section from the animal pole and in the central section, which is where the nucleus is located. mrna expression profiles did not change following in vitro fertilization and we conclude that the cortical rotation that follows fertilization has no detectable effect on intracellular mrna gradients. |
ultrasound elastography (eus) is an ultrasoundbased method for the assessment of the mechanical properties of tissue. the method was initially introduced in vitro in the 90s and later evolved into an imaging tool for in vivo applications. eus is based upon the principle that stress applied to tissue causes changes within it, which depend on the elastic properties of tissue. free hand strain (compression) eus is the commonest technique that has been employed mainly in the field of oncology. degenerative and traumatic muscle and tendon disease results in biomechanical changes. since the commercial availability of eus, there has been a lot of interest for potential clinical applications of the method, such as for early diagnosis of muscle and tendon disease, for guiding and monitoring therapy, for predicting the risk of injury in athletes and for assessing the effect of physiotherapy or training in healthy and diseased musculotendinous tissue. this review aims at presenting the main indications of eus for muscle and tendon disease based on the published evidence, at presenting the technical difficulties and limitations of the method and finally at discussing the future perspectives of eus for assessing the musculoskeletal system. depending on the way of stress application and displacement detection, there are several eus techniques, including strain eus, shear wave eus, transient eus and acoustic radiation force eus. the most commonly used method is free - hand strain eus, also found in the literature as compression elastography, sonoelastography, and real - time elastography. this is performed by manually compressing the tissue using the hand - held us transducer. strain eus is based upon hook 's law for the calculation of young 's elastic modulus (e), which is a physical quantity measuring elasticity. by assuming that the applied stress is uniform, the elastic moduli are inversely proportional to the measured strain (e = stress / strain). strain represents the amount of deformation and can be calculated is as the change in distance between two points (displacement) divided by the initial distance. so the main principle of strain (compression) eus is that a compressive force (stress) is applied to tissue causing axial tissue displacement, which is lower in hard tissue and higher in softer tissue. the displacement data are used to construct strain distribution maps (elastograms) (fig. the elastogram is superimposed on the conventional b - mode image as a gray scale or color - coded image, displayed next to the conventional b - mode image on the screen in real time. although the gray / color scale encoding is elective by the user, usually red is used for encoding soft tissue, blue for hard tissue and yellow / green for tissue of intermediate stiffness (fig. 1). the elastogram is a relative image, where the strain of each area is displayed relative to the strain of other tissues within the region of interest. there is also a semiquantitative measurement method (the strain ratio), which represents the ratio of the strain of the area of interest (roi) to an equally measuring area in the reference tissue (usually fat). longitudinal (a, c) and transverse (b, d) strain elastograms of the middle third of asymptomatic achilles tendons (t) showing two distinct eus patterns : type 1 tendons (c, d) appear homogenously stiff (green / blue) ; type 2 tendons (a, b) appear considerably inhomogenous with soft (red) areas, which do not correspond to any changes in b - mode us. the retroachilles fat (f) appears as a mosaic of green, red and blue. note the red areas at the lateral and medial sides of the tendon in the transverse plane (b, d) which correspond to artifacts, secondary to difficulty in stabilizing the transducer most of the clinical research on musculoskeletal applications of strain eus focuses on the achilles tendon. it has been found that the normal achilles tendons in healthy volunteers may present with two distinct eus appearances : they may be either homogenously hard structures or, in the majority of cases (62%), they may be considerably inhomogenous with soft areas parallel to the long axis of the tendon (longitudinal bands or spots) (fig. 1). the areas of distinct softening may not correspond to any changes in b - mode or doppler us. the above findings are confirmed by two studies comparing normal (asymptomatic) and abnormal (symptomatic) tendons. the majority of asymptomatic tendons were hard (8693%), however, in 1.312% cases there was mild or discrete softening (orange or red areas). on the contrary, symptomatic tendons were characterised by discrete softening in 57% and mild softening in 11%. mild softening (yellow) did not correlate to b - mode us abnormalities, whereas discrete softening (red) was found mainly in cases with us pathology. therefore, it has been suggested that only discrete soft areas (red) in achilles tendon should be considered as abnormal (fig. 2). it is not yet clear what the alterations in asymptomatic and sonographically normal tendons represent ; it is suggested that they may either correspond preclinical changes or to false positive findings, possibly secondary to tissue shifting at interfaces between collagen fibers. however, a study of 12 athletes with achilles tendinopathy evaluated using us, eus and mri reported completely different findings. it seems that there is some discrepancy on the appearance of normal achilles tendons between the studies available so far, which points out the need for further research on the field. longitudinal free hand strain elastogram of a 34-year - old recreational runner with healed achilles tendon injury. there is hypoechogenicity at the superficial half of the achilles tendon at the area of the healed partial injury. the abnormal area appears softer (yellow with red areas) compared to the stiffer (blue / green) normal - appearing remaining tendon. the kager 's fat appears as a mosaic of various levels of stiffness free - hand strain eus for the achilles tendon has been found to have high accuracy and good reproducibility. the overall correlation between us and eus findings was found to be excellent (accuracy 97%), if mild softening (yellow) is considered physiological and only distinct red areas are considered abnormal. compared to clinical findings, eus has a mean sensitivity of 93.7% and a specificity of 99.2%. the inter- and intra - observer reproducibility of the eus was found to be good to excellent, if the evaluation of the achilles tendon elastogram was performed qualitatively and poor (variation 2937%), if semiquantitative software measurements (strain ratio) were employed. besides achilles tendon disease, lateral epicondylitis has also been evaluated in a single study, which showed significant softening in the abnormal extensor tendons with strong correlation with us and clinical findings. further applications of eus for tendon disease based on preliminary data and the authors personal experience include patella tendinopathy, regenerated tendons after harvesting and rotator cuff tendinopathy and tears ; however, there are no casecontrolled studies available in the published literature on these applications yet (figs. 3, 4). longitudinal free - hand gray - scale (b) and color - coded (c) strain elastogram and corresponding b - mode image (a) of a 22-year - old football player with proximal patella tendinopathy. the tendinopathic area appears hypoechoic (a) and softer than the remaining tendon (b, d). in this case soft is encoded as white (gray - scale) or blue (color scale) b - mode and free - hand strain elastograms of a normal supraspinatus tendon footplate (a) and a case with partial articular surface tear of the supraspinatus tendon (b). the normal tendon appears hard (blue) and can be easily differentiated from the normal bursa which appears softer (red). the tear () appears as a softer (yellow / red) area within the harder (blue) remaining tendon (b). note the presence of soft (red) lines beneath the greater tuberosity, corresponding to artifacts there are limited data available on the use of strain sel for normal and diseased skeletal muscle. eus is a feasible and accurate means to produce muscle elasticity maps and evaluate normal skeletal muscle. normal relaxed muscle is found to be an inhomogeneous mosaic of intermediate stiffness (green and yellow colors) with scattered softer and harder areas especially at the periphery near boundaries (fig. a few studies have showed differences in the elasticity (strain index) of the masseter muscles between sexes as well as differences in elasticity of periocular rectus medialis and lateralis muscles in various gaze positions. transverse free hand strain elastograms of normal relaxed vastus lateralis muscle, which appears as a mosaic of intermediate or increased stiffness (green or blue color respectively) with scattered softer (red) areas near muscle boundaries. the subcutaneous fat appears soft (red / yellow) degenerative and neuromuscular disease has been studied using strain eus. a study on inflammatory myositis showed changes in elasticity of the affected muscles (increased or reduced stiffness, due to fibrosis or fatty infiltration respectively). a correlation between quantitative strain eus parameters and elevated serum markers was also found and the study concluded that eus may be helpful in staging and monitoring inflammatory myopathies. similar findings are reported in a case study of a patient with congenital bethlem myopathy, where exact correlation between eus and us / mr imaging findings was found. eus also detected changes which were not evident on us and mr, possibly indicating increased sensitivity for myopathic changes. a study using vibration and doppler signal alterations reported that eus could be used for depicting myofascial trigger points. in cases of cerebral palsy spasticity, eus has been found useful to detect changes in contracted muscle and to establish the site of botolinum toxin injection. based on the above data, it seems that eus has a role in the early diagnosis and staging of dystrophic, myopathic and spastic muscle and in guiding intervention in muscle disease. although there are preliminary reports on the use of eus for diagnosis and staging of rotator cuff muscle atrophy and for depicting and staging muscle injury, there is no published evidence yet. one of the major issues lies in the fact that there are various eus techniques and processing algorithms available, therefore, the findings as well as the artifacts or limitations may be highly dependent on the technique used and may be specific to a certain system. most of the experience on technical problems and ways to overcome them has resulted from the use of free - hand compression eus. compression eus is technically very challenging in terms of producing artifact - free cineloops of decompression - compression cycles. a major issue is the correct amount of pressure to be applied, which ideally should be moderate, as in high or low levels of pressure the elastic properties of tissue become non - linear, and thus the calculation of strain is not correct. most eus systems now provide an on - screen indicator providing real - time feed back on the appropriate amount of pressure. to minimize intra - observer variation, the scoring or measurements should be based on evaluation of images derived after reviewing entire cineloops. another major problem in strain eus is the lack of quantitative measurements, leading to the need of alternative methods for the evaluation of the elastograms, including semiquantitative measurements either using the built - in software (strain ratio) or using external computer software or qualitative visual evaluation of elastograms. this has led to lack of reproducibility and difficulty in comparing the results among different studies. when using eus for examining musculoskeletal tissue, special issues should be taken into consideration. anisotropy should be avoided, as the b - mode appearance influences the acquisition of eus data. when examining the achilles tendon, longitudinal images are of better quality than transverse images, because of artifacts at the medial and lateral sides of the image. when examining a long structure (e.g. the achilles tendon), overlapping images should be acquired, to overcome the problem of artifacts at the borders of the elastogram caused by inhomogenous pressure. for the evaluation of soft tissue masses, eus may be challenging in cases of superficial protuberant masses, where the application of uniform compression may be difficult. the elastogram is a relative image where the elasticity of each tissue is displayed compared to the mean elasticity of all tissues. this fact is not a major issue in breast, as the surrounding tissue is fairly homogenous (fat and glandular tissue). in musculoskeletal eus though, the elastogram may include tissues with wider elasticity differences (fat, tendon, bone, muscle), and thus wider scatter in acquired elasticity data. for the achilles tendon, the suggested, but not universally applied, standard size is a depth of 3 times the tendon and about 3/4 of the screen in longitudinal scans. in transverse scans the paratenon should be included in the elastogram. in many musculoskeletal applications (e.g. achilles tendon), a minimum distance from surface (usually about 1 mm) is needed to place the elastogram, so the use of gel pads or probe adaptors is necessary in such cases to increase the distance between the skin and probe. in conventional musculoskeletal imaging, the use of large amounts of gel is common practice in order to create an even surface and to reduce the amount of pressure on the tissue. however, when performing eus for musculoskeletal applications, care should be taken not to include the gel in the elastogram, as it results in dramatic changes in the elastogram, making the tendons appear considerably stiffer compared to the gel (fig. 6). the impact of including gel in the strain elastograms. axial strain elastograms of the same asymptomatic achilles tendon including (a) and excluding (b) small amount of gel. the inclusion of some gel in the elastogram results in a homogenously stiffer tendon without areas of distinct softening (red), which are evident there is no gel included (b). the eus settings and the level of pressure were kept stable, as indicated on the screen several artifacts may lead to misinterpretation of the images. these include fluctuant changes at the edges of the elastogram, red (soft) lines around calcifications, behind dense bone, at the superficial margin of homogenous lesions and at the interfaces between tissues (e.g. between adjacent muscles). characteristic artifacts are also associated with cystic masses, which appear as a mosaic of all levels of stiffness and with lesions adjacent to major vessels present, due to pulsations. the above artifacts should be excluded from the qualitative or quantitative scoring of the elastograms. eus probably represents the most important technical development in the field of ultrasonography since doppler imaging. the advantages of eus include the low cost, and non - invasiveness and the potential of wider clinical availability than other methods of elasticity such as mr elastography. so far there is promising evidence that eus can be used to assess the mechanical properties of musculoskeletal tissues in the clinical setting and that eus may even be more sensitive than mr or us in detecting subclinical changes of muscle and tendon. therefore eus could potentially be valuable for early diagnosis, for monitoring during rehabilitation medicine and as a research tool for biomechanics and pathophysiology of musculotendinous disease. limitations include the limited amount of published evidence available and several technical issues limiting the reproducibility of the method, including lack of quantification, artifacts and variation in the application of the technique between users. moreover, considerable criticism focuses on its potential clinical utility, as in most cases eus showed changes already evident on conventional us or doppler imaging, whereas eus changes not evident on conventional imaging were clinically occult. for all the above reasons, we think that a more systematic approach for the investigation of this new diagnostic tool should be undertaken, including standardization of eus for soft tissue applications, selecting the proper indications for research and exploring newer software algorithms. standardization is of paramount importance in order to achieve consistency in the application of the technique, which would allow for comparisons between findings. the indications for eus should be established and would ideally focus on symptomatic but non - us evident disease or very early stages of disease, in order to investigate whether eus is more sensitive than conventional imaging. newer algorithms such as shear wave eus or arfi and quantitative eus should be evaluated in comparison to strain eus. eus in its current form remains a highly subjective technique with debatable clinical value, due to lack of standardization and limited evidence. further multicenter controlled studies are needed including large populations and long term follow up together with correlation with histology, conventional imaging, biomechanical and clinical data, in order to describe the pattern and clinical significance of eus findings. with proper standardization and further structured research | ultrasound elastography is a recently developed ultrasound - based method which allows the qualitative or quantitative evaluation of the mechanical properties of tissue. strain (compression) ultrasound elastography is the commonest technique performed by applying mild compression with the hand - held transducer to create real - time strain distribution maps, which are color - coded and superimposed on the b - mode images. there is increasing evidence that ultrasound elastography can be used in the investigation of muscle, tendon and soft tissue disease in the clinical practice, as a supplementary tool to conventional ultrasound examination. based on preliminary data, potential clinical applications include early diagnosis, staging, and guiding interventions musculotendinous and neuromuscular disease as well as monitoring disease during rehabilitation. ultrasound elastography could also be used for research into the biomechanics and pathophysiology of musculotendinous disease. despite the great interest in the technique, there is still limited evidence in the literature and there are several technical issues which limit the reproducibility of the method, including differences in quantification methods, artefacts, limitations and variation in the application of the technique by different users. this review presents the published evidence on musculoskeletal applications of strain elastography, discusses the technical issues and future perspectives of this method and emphasizes the need for standardization and further research. |
it is caused mainly by three species wuchereria bancrofti, brugia malayi and brugia timori. in india, filariasis has been reported in cytologic smears from various organs and sites like male genital organs, thyroid, breast, lymph node, liver, soft tissue swellings, bone marrow, cervical smears, body fluids etc.[17 ] detection of microfilaria in voided urine sediment, especially in achylous hematuria specimen, is extremely rare. we report a case with microfilariae of w. bancrofti in a 25-year - old patient who presented with achylous hematuria. a 25-year - old male patient presented in urology out - patient department (opd) with history of intermittent painless hematuria for three weeks. there was no history of fever, trauma, instrumentation, flank pain, ureteric colic or passage of milky white urine. a voided urine sample was collected and sent to the cytopathology laboratory for routine cytological examination. smears revealed urothelial cells along with neutrophils, lymphocytes, red blood cells and few microfilariae [figure 1 ]. high power examination of the microfilaria showed a sheathed parasite with central axis of nuclei which ended abruptly before the tip of tail [figure 1 ]. with this, characteristic morphology the microfilariae were identified as w. bancrofti. photomicrograph of urine sediment showing a sheathed microfilaria with central axis of nuclei ending abruptly before the tip of tail along with few urothelial cells, inflammatory cells and red blood cells (mgg, 400) subsequent to the cytological diagnosis patient was treated with 21 days course of diethylcarbazine (dec) after which he became asymptomatic. w. bancrofti accounts for majority of the filarial infection in india ; accounting for approximately 95% of cases. infection by this sheathed species is commonly seen in india, china, indonesia and eastern pacific. the common presentations include microfilaremia, lymphedema, hydrocele, acute adenolymphangitis (adl), chronic lymphatic disease and less common presentations are like chyluria and tropical eosinophilia. shedding of microfilaria in urine is possibly determined by local factors like inflammation, trauma or stasis, which mainly affect the lymphatics and small vessels causing either lymphatic blockage or damage to the vessel wall. to the best of our knowledge, only few cases of w. bancrofti have been reported in voided urine sample in literature. in most of these reported cases, there was a history of chyluria. webber. first reported microfilaria in a 23-year - old male patient during a routine workup for intermittent painless hematuria. reported w. bancrofti in a 45-year - old man who presented with painless hematuria and clinical suspicion of malignancy. in the present case, microfilaria was incidentally detected in a voided centrifuged urine sample of a young male patient who presented with complaints of intermittent painless hematuria. the explanation of non - chyluric hematuria may be that significant lymphatic obstruction may not have taken place ; therefore, patient did not present with chyluria. the microfilaria can be detected in blood, various body fluids, fine needle aspiration smears and histological examination as well as by serological and immunological tests. among these, elisa and rapid format immunochromatographic card test have a very high sensitivity and specificity. detection of parasite dna by polymerase chain reaction (pcr) is now considered as the most sensitive technique for definite diagnosis of this infection. diethylcarbamazine (dec, 6 mg/ kg daily for 12 days) remains the treatment of choice for the individual with active infection even after many decades since it was first used in w. bancrofti infection. most of the infected individuals do not have microfilaremia at the time of clinical manifestation, and definitive diagnosis in such cases can be difficult. it saved the patient from undergoing the trauma of invasive investigations and prevented further complications. | filariasis is a widespread public health problem seen commonly in tropical countries. microfilariae have been reported in aspiration smears from various sites. however, it is very rare to detect these organisms in voided centrifuged urine cytology. we, report this rare finding in a 25-year - old patient who presented with achylous hematuria. |
open fracture of distal femur with popliteal artery occlusion could be a challenge to many orthopedic surgeons (1, 2). moreover treatment of severe bone loss of distal femur would also be considerably more difficult because of complete loss of ligamentous stability of knee joint, and involvement of disruption to the knee extensor mechanism (3, 4). in this article, we report a case of severe bone loss of distal femur with popliteal artery occlusion. after revascularization of the limb, we successfully performed a reconstructive total knee arthroplasty with modular segmental endoprosthesis, and augmentation of patellar tendon using a semitendinosus allograft. paramedics brought a 45-yr - old man into our medical center immediately after a high - velocity crush injury. on admission to the emergency room, the patient was noted to have a large open wound in anterior aspect of left knee. distal pulses of dorsalis pedis artery and posterior tibial artery were absent, and only femoral pulse was palpable on the injured left lower extremity. he was alert with respiratory rate of 22, pulse rate of 98, and blood pressure of 100/80. the knee joint was open, and a portion of the patellar tendon was lost. about 50% of the patellar tendon was remained, but it was detached from the inferior pole of the fractured patella. all of the collateral ligaments and the cruciate ligaments of the knee were lost together. only the biceps tendon and a portion of the semimembranosus tendon were attached to the proximal tibia and fibula around the knee. plain radiography showed severe bone loss of distal femur, fractures of patella and proximal tibia (fig. then we repaired the ruptured patellar tendon with pull - out sutures through the patella. primary fracture fixation of the distal femur was impossible because expelled fragments of the distal femur were lost. prior to repair of the popliteal artery, we applied an external fixator across the knee joint. we made a small longitudinal incision to a suspected portion of injury of the popliteal artery. the intima of the popliteal artery was torn and dissected, and a large thrombus occluded the popliteal artery completely (5). we excised a segment of the contused and thrombosed popliteal artery in 1.5 cm length. we were able to easily perform an end - to - end repair of the artery only with mild mobilization of the proximal part of the popliteal artery because severe bone loss of the distal femur made approximation of ends of the popliteal artery to be ease. after the direct repair of the popliteal artery, distal pulse was palpable and limb circulation was recovered. then we waited for 30 min watching for circulation, swelling of the leg and compartment syndrome. we decided not to perform a fasciotomy of the low leg to prevent compartment syndrome because we could not find cyanosis, swelling or hardness of muscle compartments of the low leg, such as associated signs of compartment syndrome. at 4 days after the emergency operation, we performed debridement again and inserted antibiotics (vancomycin)-mixed cement beads into the defect of the distal femur to prevent infection. the patient received 2nd - generation cephalosporin and aminoglycoside antibiotics during the initial 2 weeks and he received more 2nd - generation cephalosporin antibiotics during the following 2 weeks (fig., we performed a reconstructive total knee arthroplasty with modular segmental endoprosthesis, mutars (implantcast, buxtehude, germany) to treat the large bone loss of the distal femur (fig. simultaneously, we carried out patellar tendon augmentation using a semitendinosus allograft because gracilis and semitendinosus were lost at the initial trauma (fig. furthermore, a medial gastrocnemius rotational flap with meshed skin graft was followed because conditions of the repaired patellar tendon and anterior skin of the knee were not healthy for rehabilitation of the knee joint (fig. one week after the reconstruction, physiotherapy team commenced continuous passive motion of the knee and crutch ambulation began 2 weeks after reconstruction. at his most recent follow - up visit, 36 months postoperatively, power of quadriceps muscle is 4/5, and the knee society knee score in pain is 79 and functional score is 50. the patient shows a mild limping gait because of 1.5 cm shortening of the left leg after the reconstruction arthroplasty. however, the patient can ambulate independently and is satisfied with the results (fig. popliteal artery is fixed to femur proximally by tendinous hiatus of adductor magnus and distally by a tendinous portion of soleus muscle. therefore, it is susceptible to shearing or stretching by fractures of the lower femur and dislocations of the knee (1, 5, 6). prompt recognition of associated popliteal artery disruption in damaged extremities and early revascularization are essential for good results as major factor favoring major limb salvage is a short preoperative ischemia time (2, 5, 6). massive trauma of lower extremity is particularly associated with vascular injuries and presents an immediate and complex decision - making challenge between limb salvage attempt and primary amputation. lange (2) suggests two absolute indications that would warrant primary amputation in massive lower extremity trauma. the first is complete posterior tibial nerve disruption in adults ; the second is crush injury with more than six hours of warm ischemic time. unfortunately, the literature to date is unable to provide sound guidelines for treatment of such injuries. individual patient variables, specific extremity injury characteristics, and associated injuries must all be weighed up before a treatment decision can be reached (2)., this normal nerve condition would contribute to good long - term results. in this case, multiple surgical options are available after revascularization, including arthrodesis, delayed amputation of a useless limb, allograft - prosthetic composite, and segmental prosthetic arthroplasty (2, 7, 8). reconstruction methods to maintain a mobile knee joint are allograft - prosthetic composite, and segmental prosthetic arthroplasty (7, 8). modular segmental endoprosthesis with rotating - hinged knee kinematics was designed to allow reconstruction of large femoral deficits after tumor surgery (7). a modular segmental endoprosthesis is readily available and can easily be modified to fit the skeletal defect. this prosthetic arthroplasty also involves a shorter operation time, immediate weight - bearing with early stability, and early return to activities of daily living (4, 7). there has been general reluctance to perform a total knee arthroplasty using constrained modular segmental endoprosthesis in young patients because of the variable results of a rotating - hinge knee design and potential complications, including aseptic loosening, infection, and component breakage (3, 4). otherwise, petrou. (9) reported good results in a primary total knee arthroplasty using endo - model rotating - hinge prosthesis with a 96.1% survival rate at 15 yr postoperatively. also, excellent long - term prosthetic survival for aseptic loosening was reported for 83 patients with a segmental rotating - hinge total knee arthroplasty of distal femur with 15-yr survival from the time zero point of 5 yr after the first operation noted to be 86% (7). another option, allograft prosthetic composite has many advantages, including biocompatibility, bone stock restoration, and potential for ligamentous reattachment. but disadvantages also exist ; for example, late resorption of allograft possibly secondary to immune reaction, allograft fracture, nonunion, malunion, collapse, and the risk of disease transmission (7, 8). this procedure is also technically more difficult and would require a longer recovery and healing period for host - graft junction (4, 8). we have described the case of severe knee joint injury asscociated with severe loss of the distal femoral bone and popliteal artery occlusion. prompt recognition of artery disruption, early revascularization with intact function of posterior tibial nerve, and consecutive reconstruction surgery are important for successful and functional results (2, 7). although we have used the modular segmental endoprosthesis, we would not advocate it as a routine procedure in cases when both allograft - prosthetic composite and modular segmental endoprosthesis were available. in our opinion, reconstructive surgery with modular segmental endoprosthesis was a suitable treatment option in terms of the short - term outcome in this case, where it was impossible to perform an internal fixation of fracture around the knee joint. however, we need to know and consider the long - term results of this case. | severe injury to the knee and the surrounding area is frequently associated with injury to ligaments of the knee joint and structures in the popliteal fossa. this case involved a popliteal artery occlusion, severe bone loss of distal femur, loss of collateral ligaments, and extensor mechanism destruction of the knee. initially, prompt recognition and correction of associated popliteal artery injury are important for good results after treatment. after successful revascularization, treatment for severe bone loss of distal femur and injury of the knee joint must be followed. we treated this case by delayed reconstruction using modular segmental endoprosthesis after revascularization of the popliteal artery. this allowed early ambulation. at 36 months after surgery, the patient had good circulation of the lower limb and was ambulating independently. |
according to the kidney disease quality outcome initiative (k / doqi), chronic kidney disease (ckd) is defined as the atrophy of the kidney or failure of renal function (1). the progression of renal disease can be separated into five stages, the criterion of which is glomerular filtration rate (gfr). patients in the first stage share similar gfr (90) to healthy counterparts (kidney damage with normal or increased gfr), making it difficult to identify the disease according to gfr alone. stage 4, or pre - end - stage renal disease (esrd), has a gfr level of 1529 ml / min/1.73 m2 (severely decreased gfr). finally, esrd is declared upon the fifth stage when the kidney is completely deprived of its function. if transplantation is not available, it is crucial to undergo dialysis at this point (2). hemodialysis (hd) process requires a vascular access (va) which can be perform by three common surgeries : arteriovenous fistula (avf), arteriovenous graft (avg) or central venous catheter (cvc) (3). according to kdoqi hd adequacy guidelines, the preference of avf over other forms of access arises from their functional advantages because of a lower rate of complications. fistulae have the lowest rate of thrombosis, and require the fewest interventions, providing longer survival of the access (4). catheters may cause central vein stenosis, which can adversely affect va outcomes (5). our criteria for avf maturation was : (i) easily palpable superficial vein, (ii) vein relatively straight, (iii) adequate diameter for easy cannulating needles (3 - 4 mm), (iv) adequate length (10 cm, for adequate distance between the cannulating needles), and (v) uniform thrill to palpation and auscultation. avgs require far more interventions than do avfs to maintain long - term patency for dialysis. on the average, the annual frequency of intervention (elective angioplasty, thrombectomy, or surgical revision) in mature accesses is approximately four - fold higher for avgs than for avfs (7). also the durability of avf is about 90% for a year after maturation for the reason of dialysis, while this rate is only 60% for avg (8). clinical guidelines established by the national kidney foundation cite these reasons in advocating use of avfs whenever possible (9). placement rates for each type of access have changed dramatically over the last 10 years (10), with a decrease in catheter placement rates and an increase in avf placement rates (11). in 2003, the using rates of avf has been reported in about 91% in japan, and between 70% and 90% in most european countries, compared with a dismal 30% in the united states (fig. 1) (12). data from the dopps. note the dismal prevalence rate for native avf and the high prevalence rate for ptfe grafts in the united states as compared with other industrialized countries. anz, australia and new zealand ; be, belgium ; ca, canada ; fr, france ; ge, germany ; it, italy ; jpn, japan ; sp, spain ; sw, sweden ; us, united states (data as of september, 2003 ; courtesy dr. rajiv saran, university of michigan, ann arbor, mi.) (12). note the dismal prevalence rate for native avf and the high prevalence rate for ptfe grafts in the united states as compared with other industrialized countries. anz, australia and new zealand ; be, belgium ; ca, canada ; fr, france ; ge, germany ; it, italy ; jpn, japan ; sp, spain ; sw, sweden ; us, united states (data as of september, 2003 ; courtesy dr. rajiv saran, university of michigan, ann arbor, mi.) (12). the avf using rate has reported to be 93.4% in iran (13). in the usa, avf use increased from 27.9% in 1998 to 55.0% in 2007 (14) and shows an increase from 32.4% in 2003 to 57.9% in 2011 (15) and has grown up into 61% in 2013 (16). finally, according the report fiscal year (fy) 2014 (16), increasing the rate of avf access for esrd patients improves the quality of hd treatment, while decreasing unnecessary complications and hospitalizations. since fy 2009, cms has increased avf access by over 6 percent, exceeding recent targets (table 1). the avf is the preferred type of vascular access because thrombosis rates, infection rates, access - related expenditures, and total health care expenditures all are lower for patients with fistulas than for those with either synthetic avg or cvc ; however, the advantages of fistulas are counterbalanced by the substantially higher proportion of fistulas than grafts that are never able to be used for dialysis because of failure to mature adequately to support effective hd (17). a much higher likelihood of fistula non - maturation, ranging between 20% and 60% (18) and recent studies have reported 20 - 50% of the fistulas failed to maturation (19). there are several variables interfering with the time takes for an avf to develop to become accessible for hd, which all together can be divided into four groups (20) : first, demographic factors, such as ; age, race, economic condition and geographic region (21), second, diagnostic exams and evaluations before surgery, such as, ultrasound results to examine the arteriovenus system and regional vascular anatomy (22), third, measuring the physiological variables during surgery, such as fistula blood flow and dimensions of arteries and veins (23), and the last group is rehabilitative exercises and post operation cares (24). the nkf - kdoqi guidelines provides an evidence based on clinical measurements that a reciprocal relationship exists between the blood pressure (bp) and ckd, in which, bp is a cause and a consequence for ckd. likewise, it has suggested the determination of the relation between the levels of bp against time in esrd patients (25). therefore, having information about the factors which have impact on the maturation time (mt) after avf creation, can help in screening and choosing the right patients for the procedure and thus offering better diagnostic services and treatments and finally expediting the healing and maturation period after avf creation as well as minimizing the side effects and eventually preparing the patient for hd (26, 27). for this reason, this study is prepared to investigate the impact of bp conditions on the period in which takes the avf to develop for hd in esrd patients. also, studies the interference of other variables such as age, diabetes and cardiac failures with the process. data mining is a process in extraction of interesting (non - trivial, implicit, previously unknown and potentially useful) patterns or knowledge from huge amount of data. (28) the outcome of data mining technologies are to provide benefits to healthcare organization for grouping the patients having similar type of diseases or health issues so that healthcare organization provides them effective treatments ; this analysis of health data improves the healthcare by enhancing the performance of patient management tasks. (29) these approaches have developing implication in the health area and have aided the clinical data description processing via transaction of health systems (30). in this cross - sectional and prospective study, the medical history of 87 patients who referred to the hasheminejad kidney center for avf creation in 2010 were analyzed. for all of the patients, the surgery was performed within the radio - cephalic area by one surgeon. the patient s information were recorded including personal descriptions such as age and gender, medical history before avf creation, monitoring during operation (the operation region) and potential risk factors such as bp, diabetes and cardiac failures. the avf accessibility was indicated by the length of the straight superficial vein (should be about 10 cm), palpable with proper diameter (about 4 mm) and having continues and uniform thrill and continues bruit in auscultation (20). in this way, the fistulas maturation times were recorded for a successful hd and then the data were processed with the data mining method. the maturation of a fistula (becoming accessible for dialysis) is described as a fistula condition which can be cannulated with two needle probes permanently or by routine and supply the adequate blood flow (min 350 - 450 ml / min) for a successful hd session (3 - 4 hours) each time (31). one of the data mining approaches is descriptive method, which a subdivision of that called clustering, was used in our previous study to explore the early failure of the inserted avf in 99 patients (32). another approach is predictive method which we have implemented in our recent researches on 193 hd patients (33, 34). in this study the data mining was performed by rapid miner, before starting the data processing. for this reason, the data were randomly sampled by bootstrapping algorism (70 samples). later, the decision trees (dt) were extracted, representing the impacts of different variables on avf maturation. there were quite a few trees which were extracted by this method, however, only those were chosen for our two groups of data, which according to the compatibility rules of the trees had better consistency with the medical instruction criteria (8, 22). to improve processing, instead of entering all the data into the software this definition is regarding the average age of the patients and their mt range. before the processing, we had data preparation and categorize the data into two frames : considering the average age of the patients which is 57 years, we had two subgroups : below-57 and above 57. another group - related subgroups are : 19 - 49, 50 - 64 and 65 - 83 years, labeled accordingly. the mt has also been classified as follow : class a : 3 - 4 weeks, class b : 5 - 7 weeks and class c : 8 - 10 weeks. there was only one case of 16 weeks mt, which we considered it as class c (fig. the medical characteristics of 87 patients (including 61 (70%) male and 26 (30%) female) diagnosed with ckd, was studied. the mean (sd) age of patients in this study was 57.0 (16.36) years. this tree is a continuous type of dt known as classification and regression trees (cart). (35) for clarificationof findings (a, b and c) in figures 3 and 4, see previous paragraph ; the words systolic and diastolic are literally the systolic blood pressure (sbp) and diastolic blood pressure (dbp), accordingly. also the labels a, b and c are shown in blue, red and green colors. fig. 3 the decision tree diagram displaying the data mining for 70 patients using the first method fig. 4 the decision tree on 70 patients classified on the basis of age into three subset of 19 to 49 years (with labels [19 - 49 ]), 50 to 64 years (with labels [50 - 64 ]) and 65 to 83 years (with labels [65 - 83 ]) table 2 displays the compatible laws with this tree, which indicates that the patient s gender does not have any impact on the mt of avfs on the other side, the systolic blood pressure (sbp) is differentially an important factor (table 2). the decision tree diagram displaying the data mining for 70 patients using the first method the decision tree on 70 patients classified on the basis of age into three subset of 19 to 49 years (with labels [19 - 49 ]), 50 to 64 years (with labels [50 - 64 ]) and 65 to 83 years (with labels [65 - 83 ]) while, the extracted knowledge from the second group tree indicates that the sbp and dbp have significant impact on the mt. for the systolic bps above 105 - 122.5mmhg, the maturation accomplished better in groups one and three than patients with ages 50 - 64 years. so far, few patients were randomly selected and the system trained the rules extracted from their attributes. at the next step of the data mining process, the quality of the mt was predicted on the remaining patients who were not selected in sampling stage. one study have evaluated potentially modifiable factors associated with dialysis access patency using statistical methods (36) and they have indicated the role of maturation period blood pressure as a risk factor. in this study we evaluated medical characteristics of the patients diagnosed with ckd. we divided patients information into two main groups based on their ages and the data mining was performed on both groups, followed by checking the compatibility of the study with the standard medical guidelines. decision trees. this method of study indicated that the gender has not any specific impact on the time needed for the fistula to mature and on the other hand, clearly displayed the significant impact of the patient s systolic (sbp) and diastolic (dbp) blood pressure on the avf mt. moreover, the effects of other factors such as ischemic heart disease (ihd) and diabetes mellitus (dm) has been analyzed in formulas (1) : sbp > ihd > dbp > dm > age (1) in addition to the descriptive results, the avf mt was evaluated with the 70.59% of precision in 17 patients whose data were not sampled in this investigation. regarding the high impact of the sbp and dbp in the avf mt, it is very important to constantly monitor this variable in the ckd patients before and during the avf creation procedure and likewise, considering this variable in the patient s medical plans for a successful hd. association of the blood pressure with the fistula maturation time, may play an important role in vascular access patency. the results of this study are not only important in improving the physicians and surgeon s knowledge of the time of the accessibility of the inserted fistulas, also, improves the medical care provided to the patients. this study also demonstrated that with the use of the data mining it is not only possible to uncover the unknown relations between the medical characteristics related to the disease with the patient s general condition, but also to predict the medical conditions related to the disease in future patients. thus, the knowledge extracted from the data mining method is considerably helpful in making medical decisions and predicting the patient s condition related to his treatment in similar situations. limitations of this study were limited number of patients and lack of evaluation of other factors which may have effects on maturation process. | background : chronic kidney disease (ckd) is a complicated kidney problem causing permanent renal failure in progressive stages. the final stage of ckd is called esrd in which most accepted management is hemodialysis (hd). arterio - venus fistula (avf) is the most practical way of making proper access to the blood circulatory system ; however, maturation of the avf is a challenge, since there are number of variables interfering with the whole process. the purpose of this study was to evaluate potentially modifiable factors associated with maturation time (mt) after creation of a vascular access (va). methods : in this cross - sectional study, a total of 87 patients referred to the hasheminejad kidney center for avf creation in 2010 were evaluated. patients were evaluated before and after the avf creation and risk factors such as history of blood pressure abnormalities, diabetes and congestive heart failure, as well as the successive development of avf was studied and finally processed using data mining technology. results : the " decision trees " indicated the significant impact of the systolic blood pressure (sbp) in the delay of the patient s avf maturation. also, prediction of avf maturation was made with 70.59% of precision in regard to their bp condition. conclusion : this study demonstrated that monitoring the sbp is one of the important steps in management of the cardiovascular variables producing any delay in the process of the patient s hd. also the data mining method can discover the hidden relationship between the patient s medical conditions in order to predict the potential disorders. |
to describe the case of a 74-year - old man who developed cytomegalovirus (cmv) retinitis after multiple ocular surgeries. a 74-year - old man who had a history of multiple ocular surgeries developed unilateral retinitis with whitening of the entire peripheral retina. a presumptive diagnosis of viral retinitis was considered, and polymerase chain reaction of the aqueous fluid was positive for cmv dna. moreover, the patient did not have any subtenon or intravitreal injection of triamcinolone acetonide (ta). cytomegalovirus (cmv) retinitis is a viral inflammation of the retina in immunosuppressed patients, which sometimes results in severe visual loss. a 74-year - old man who had a cataract surgery, 8 glaucoma surgeries for secondary glaucoma, a vitrectomy for retinal detachment, and a deep lamellar endothelial keratoplasty and 2 penetrating keratoplasties for bullous keratopathy for his right eye visited our clinic for a routine examination. he had used 0.05% cyclosporine eye drops once a day for over ten years after the keratoplasties. funduscopic examination revealed areas of confluent exudate in the entire peripheral retina in his right eye (fig. 1). however, slit - lamp examination revealed no inflammatory cells in the anterior chamber or the anterior vitreous. although the retinitis did not reach the macular area, the patient 's best corrected visual acuity (od) was hand motion because of optic nerve atrophy due to glaucoma. fluorescein angiography demonstrated dye leakage in the corresponding peripheral retina, and severely reduced retinal blood flow due to retinal atrophy caused by multiple ocular surgeries (fig. 2). a presumptive diagnosis of viral retinitis was considered, with possible causative organisms including herpes simplex virus (hsv), varicella - zoster virus (vzv), or cytomegalovirus (cmv). aqueous fluid was aspirated for use in real - time polymerase chain reaction (pcr) analysis, and prior to getting results, 1,500 mg acyclovir was administered intravenously. real - time pcr of the aqueous fluid was positive for cmv dna (3.89 10 copies / ml) and negative for hsv dna or vzv dna. laboratory examination revealed that the patient was negative for human immunodeficiency virus and had a normal cd4 cell count (611/l). there were no signs of systemic cmv infection or systemic disease such as diabetes mellitus except for a history of acute myocardial infarction. since the laboratory analyses indicated cmv retinitis, the cyclosporine eye drops were stopped and the patient was treated with 300 mg intravenous ganciclovir twice daily for 14 days, and two intravitreal ganciclovir injections (0.4 mg). the retinitis lesion slowly decreased in size, but real - time pcr of the aqueous fluid after intravenous ganciclovir administration revealed 9.37 10 copies / ml of cmv dna. thus, the patient was treated with oral valganciclovir (900 mg / day) for 8 weeks. after disappearance of the retinitis, real - time pcr of aqueous specimens did not detect cmv dna. however, a recurrence of cmv retinitis was observed two months after finishing oral ganciclovir. the left eye showed no cmv retinitis at any time during the follow - up period. cmv retinitis usually affects severely immunosuppressed individuals. however, previous studies have also reported cmv retinitis in immunocompetent patients after intravitreal or subtenon injection of triamcinolone acetonide (ta) [1, 2, 3 ]. interestingly, most patients who have developed cmv retinitis after using steroids had diabetes mellitus [1, 2, 3 ] and it has been suggested that cmv retinitis and diabetes mellitus may be related [3, 4 ]. our patient was immunocompetent based on the results of laboratory examinations and did not have systemic disorders such as diabetes mellitus, nor any history of ta injection. cmv retinitis in immunocompetent patients typically manifests with anterior and vitreous inflammation [1, 2, 3 ], but no inflammatory reaction was observed in our case, similar to the typical manifestation in an immunocompromised patient. thus, it seems likely that the immune reaction in our patient 's right eye was severely suppressed. although topical betamethasone was administered after the second keratoplasty, it was stopped about six months before the onset of the cmv retinitis. since the patient had received cyclosporine eye drops for over ten years after the first keratoplasty, one possibility is that long - term use of cyclosporine eye drops induced local immunosuppression, leading to cmv retinitis. however, previous work shows that penetration of cyclosporine eye drops into intraocular tissues is poor. although our patient had penetrating keratoplasty for the right eye, there is few epithelial damage of the cornea in his right eye, thus it is supposed that his right eye had almost normal corneal barrier function. moreover, since this patient exhibited recurrences of cmv retinitis even after discontinuing cyclosporine eye drops, other possibilities should be considered. retinal vasculopathy, such as damaged retinal vascular endothelium and reduced blood flow, is thought to promote leukocyte entrapment in the retina and reactivation of latently infecting cmv [5, 6 ]. in our patient, fluorescein angiography demonstrated that retinal blood flow in his right eye was extremely decreased due to multiple ocular surgeries, and this may have some relevance to local immunosuppression. | purposeto describe the case of a 74-year - old man who developed cytomegalovirus (cmv) retinitis after multiple ocular surgeries.methodsobservational case report.resultsa 74-year - old man who had a history of multiple ocular surgeries developed unilateral retinitis with whitening of the entire peripheral retina. a presumptive diagnosis of viral retinitis was considered, and polymerase chain reaction of the aqueous fluid was positive for cmv dna. laboratory examination revealed that the patient was completely immunocompetent. moreover, the patient did not have any subtenon or intravitreal injection of triamcinolone acetonide (ta). the patient responded well to intravenous ganciclovir and oral valganciclovir.conclusioncmv retinitis can occur to immunocompetent patients without local immunosuppression with ta injection. |
biological complexity is not only the consequence of the number of genes expressed in an organism but it is also the consequence of further modifications to the gene products. once a gene is transcribed into nuclear rna, this rna can be modified by alternative splicing to yield distinct mrnas, which may subsequently be translated into different protein isoforms. post - translational modification of each of these isoforms by phosphorylation (or other processes) can affect the behaviour of each isoform, producing a wide variation in the activity of the different isoforms generated from an unique gene such as tau. it has been suggested that the post - translational modifications that direct the diverse protein functions of a protein may reflect a code and for example, a histone code exists that directs the different functions of chromatin (jenuwein and allis, 2001 ; strahl and allis, 2000). in this review, i shall look at how a single tau gene can generate many tau isoforms, first through alternative rna splicing and afterwards, by different post - translational modifications, phosphorylation being the most important. finally, i will discuss whether these modifications could define a tau code that might explain the role of different tau isoforms in distinct cellular functions, or in the different neurodegenerative disorders known as tauopathies. tau plays a role in different cellular events and as a microtubule associated protein, tau is principally involved in microtubule dynamics, stabilizing the microtubule polymers (drubin and kirschner, 1986 ; feinstein and wilson, 2005 ; goode., 1997). however, it has also been proposed that the tau protein is a key component of axonal transport (dixit., 2008 ; ebneth., 1998 ; yuan., 2008) and more recently, tau was found to bind to and inhibit histone deacetylase hdac6 (ding and johnson, 2008 ; perez., 2009), impairing its activity and facilitating tubulin acetylation. in addition, tau can also bind to other proteins apart from tubulin, perhaps influencing their activity (avila., in neurodegenerative diseases, modifications to tau are important for the development of tauopathies (lee., 2001) and the presence of extracellular tau, as a consequence of neuron death, could result from its interaction with muscarinic receptors on surrounding neurons, provoking toxicity in those neurons (gomez - ramos. different functional tau isoforms arise through alternative splicing and their behaviour can be modified by different phosphorylation events tau is also subject to ubiquitination, glycation, nitration, truncation and other posttranslational modifications that could result in the appearance of additional tau isoforms (avila., 2004). thus, these different isoforms could fulfil a different role in all of the processes indicated above. although in this review i will focus on those tau isoforms arising from alternative splicing or phosphorylation. tau is a microtubule associated protein found in the brain that promotes microtubule polymerisation in vitro (weingarten., 1975). tau binds to the carboxy terminal region of tubulin (serrano., 1984), the main component of microtubules, through a region known as the microtubule - binding domain (goedert., 1989 ; lee., this region of tau contains three or four (depending on the specific tau isoform) similar but not identical motifs of 31 or 32 residues, known as the microtubule binding repeats. those tau isoforms with three repeats are described as 3rtau, whereas those with four repeats are defined as 4rtau. the unique human tau gene is located on chromosome 17q21 (andreadis., 1992 ; neve., 1986) and it can be transcribed into a nuclear rna with 16 exons that can yield different tau isoforms through alternative splicing (goedert., 1989). six different protein isoforms of tau have been described in the central nervous system (cns), three 3rtau and three 4rtau isoforms, the fourth microtubule binding repeat reflecting the alternative splicing of exon 10. alternative splicing of exon 10 in tau, results in isoforms that contain either three (3r tau) or four (4r tau) microtubule binding repeats. the ratio of tau 4r to tau 3r approximates to one in the normal brain, both in terms of mrna and protein, although in some tauopathies this ratio is affected and this might be sufficient to cause neurodegeneration. in frontotemporal dementia with parkinson 's linked to chromosome 17 (ftdp-17) and corticobasal degeneration (cbd), there is an increase in the 4r tau/3r tau ratio, while in pick 's disease there are no phosphorylated tau isoforms containing exon 10 (buee., 2000). furthermore, 3rtau is preferentially expressed in myotonic dystrophy type 1 (dm1), the main isoforms that are expressed in foetal neurons (ghanem., 2009). by contrast, exonic and intronic mutations in tau associated with ftdp-17 may result in the forced expression of exon 10 (grover., 1999 ; hutton., 1998 ; spillantini., 1998 ; varani., 1999). curiously, some tau mutations present in ftdp-17 patients could produce a change in tau phosphorylation. the k257 t mutation generates a new threonine residue although it remains unclear whether or not it may be modified. alternatively, the p301s mutation results in the appearance of a serine (underlined) in the sequence vsgggs, generating a sxxxs motif needed for gsk3 phosphorylation. finally, the s320y mutation prevents the phosphorylation of a serine that can be phosphorylated by pka or by mar kinase in vitro (schneider., 1999). the putative enzymes that could participate in establishing a tau code are the kinases and phosphatases that act on tau. there are 45 serine, 35 threonine and 5 tyrosine residues that can be phosphorylated in the longest cns tau isoform of 441 residues (goedert., 1989). in the most prevalent tauopathy, alzheimer 's disease (ad), 40 phosphorylation sites have been reported (hanger., 2009 ; wang and liu, 2008), two of which are tyrosine residues (lee., 2004 ; noble, 2004) and the rest are either serine or threonine residues. these serine and threonine residues can be classified into two groups : those that can be modified by proline directed kinases like gsk3, cdk5, p38map kinase or jnk ; and those that can be phosphorylated by non - proline directed kinases like pka, mar(par-1) kinase, ckii, pkc or calcium - calmodulin kinase (avila., 2004). among all of these kinases, gsk3 is that which can modify most sites in the tau molecule, although in some cases gsk3 modification requires prior phosphorylation of tau by another kinase (cho and johnson, 2003). on the other hand, several tau phosphatases have been described such as pp1, pp2a and pp2b (gong., 1994a, b), of which pp2a is capable of dephosphorylating most phosphorylated sites in the tau molecule (goedert., 1995 ; gong., 2000). tau phosphorylation is developmentally regulated, and it is more prevalent in foetal neurons and less intense in adult neurons (morishima - kawashima., 1995). moreover, different tau isoforms may be phosphorylated at different sites (hernandez., 2003), which could be the origin of the tau code. in addition, tau phosphorylation may facilitate the binding of the protein to other proteins that might regulate its activity, for example tau binding to pin-1, a chaperone of phosphorylated proteins, (lu. the activity of kinases on tau may also be modulated by different mechanisms, for example beta amyloid peptide can regulate gsk3 activity through the insulin and wnt signalling pathways (barth., 1997). moreover, extracellular matrix components, like laminin, can also regulate akt / gsk3 signalling (chen., 2009). in addition, p73 (a member of the p53 family) modulates jnk activity to regulate tau phosphorylation (wetzel., 2008), while cdk5 may increase its activity after an intracellular increase of calcium (patrick., 1999). all the previously indicated functions of tau can be regulated by phosphorylation, and since phosphorylation at different sites could have different consequences for each of these functions, a tau code could exist. for example, tau can interact with the neuronal plasma membrane through the amino terminal region of the protein and phosphorylation of that region could prevent such interactions (arrasate. the interaction of tau with microtubules is also regulated by phosphorylation whereby phosphorylation generally reduces the affinity of tau for microtubules (avila., 2004), although some exceptions have been reported (schneider., 1999). phosphorylation of the microtubule binding region (residues 244 - 368 of the tau molecule), mainly at serine 262 and 356, decreases the interaction of tau with microtubules (buee., 2000). moreover, phosphorylation in the regions adjacent to the microtubule binding domain may decrease the interaction of the tau protein with microtubules, for example phosphorylation at serine 214 or threonine 231. this phosphothreonine is the binding site for pin-1, a chaperone that facilitates dephosphorylation of tau at that site by the pp2a phosphatase (lu., 1999). 2008), as well as facilitating its interaction with other proteins that could regulate some of its activities, like 14 - 3 - 3 (sadik., 2009). in addition, the reported interaction of tau with muscarinic receptors appears to be regulated (decreased) by phosphorylation, probably at the c - terminal of tau (ser 396 - 404) (gmez - ramos, a.. finally, phosphorylation at residues 396/404 of the c - terminal region favours the formation of a heterotrimeric complex between phosphotau, -synuclein and gsk3 (duka., 2009). tau is also known to form self aggregates and this process is also modulated by phosphorylation (santa - maria., 2004). soluble hyperphosphorylated tau (p - tau), associated with ad, can sequester normal tau, map1 and map2, causing the inhibition of microtubule assembly and the disassembly of already formed microtubules (alonso., it was suggested that p - tau but not unmodified tau can interact with maps, although the nature of the phosphorylated residues involved in this interaction remains unclear. in ad, another post - translational modification of tau could also be regulated by phosphorylation, its truncation (mondragon - rodriguez., 2008 ; wischik., thus, phosphorylation of residues in different regions of tau produces different effects on tau function or dysfunction. up to 40 residues have been seen to be modified in tau protein isolated from the brain of ad patients (hanger., 2009), with gsk3 being identified as the kinase that could phosphorylate more sites in the tau molecule (figure 1). however, tau phosphorylation in ad could result from the phosphorylation of different tau isoforms modified at different sites, rather than the modification of the 40 sites in a single tau molecule (hernandez., 2003). (a) more than 40 residues of the different tau isoforms can be modified in alzheimer disease (ad). some of these sites are modified by gsk3, and the modified serine residues (184, 198, 199, 202, 210, 214, 235, 237, 258, 262, 289, 356, 396, 400, 404, 409, 412, 413, 416) or threonine residues (69, 175, 181, 212, 217, 231, 414) have been localized within the tau molecule (hanger., in addition, there are three possible tyrosine residues (18, 29, 394) that could be modified in ad, the main candidates each located in the amino terminal domain. fewer residues modified by gsk3 have been identified in tau from progressive supracellular palsy (psp). (b) these and other phosphorylation events can regulate the interaction of tau protein with the cell membrane, microtubules, other proteins or its self association. the circles indicate the localization of the phosphorylated residues that prevent interactions (ser 199 - 202) with the cell membrane, (ser 262) with microtubules, or (ser 396 - 404) with muscarine receptors. also, when the serines (396 - 404) are phosphorylated the interaction of phospho tau with -synuclein and gsk3 is favoured. three 3r and three 4r tau isoforms.) in other tauopathies, like progressive supranuclear palsy (psp), only eight phosphorylation sites have been reported (wray., 2008), although all of these sites (ser 46, thr181, ser202, thr217, thr231, ser235, ser396 or ser400, and thr403 or ser404) are also modified in ad and they might be modified by gsk3. it is not known whether these differences in the number of phosphorylated residues in tauopathies like ad or psp could provoke the appearance of different filamentous tau polymers in these conditions (arrasate., 1997), in addition to other reported factors that could also influence the morphology of these filaments (arrasate., 1997). in ad, paired helical (phf) and straight (sf) filaments can be detected in the patients brain (kidd, 1963), whereas mainly straight filaments are observed in psp (perez., 1998). alternatively, tau can be proteolyzed (truncated) in different ways in tauopathies like ad or psp, and truncated tau can be phosphorylated by kinases like gsk3 (rissman., 2004) and indeed, it has been suggested that tau cleavage may precede its hyperphosphorylation (cribbs., 2004). in ad, tau fragments of different sizes have been described that lack c - terminal residues (up to 17 kda) (binder., 2005 ; gamblin., 2003 ; however, in psp (wray., 2008) a 35 kda tau fragments lacking the n - terminal region has been identified. curiously, this tau fragment is less evident in the csf of psp patients (borroni., 2008), perhaps because it forms aggregates that are not secreted into the csf. on the other hand, tau truncated at asp 421 is toxic since it induces mitochondrial fragmentation and elevated oxidative stress, which could in turn result in neuron death (quintanilla., 2009). in summary, many tau protein isoforms can be generated from a single tau gene and some of these isoforms may undergo different modifications, such as phosphorylation. these differences in phosphorylation could reflect a tau code that could explain the different activities fulfilled by tau (figure 1b). indeed, phosphorylation of the n - terminal half of the tau molecule could affect its interaction with the cell membrane, while phosphorylation in the microtubule binding domain and its adjacent regions will impair the interaction of tau with microtubules, as well as influencing tau - tau self assembly. by contrast, phosphorylation at the c - terminal region affects the interaction of tau with other proteins, like muscarinic receptors, or the formation of the -synuclein - gsk3-tau complex. the author declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. on the other hand, | in this short review, i will focus on how a unique tau gene may produce many tau isoforms through alternative splicing and how the phosphorylation of these isoforms by different kinases may affect their activity and behaviour. indeed, each of the different tau isoforms may play a distinct role under both physiological and pathological conditions. thus, i will discuss whether a tau code exists that might explain the involvement of different tau isoforms in different cellular functions. |
accurate assessment of the central corneal thickness (cct) has become extremely important in ophthalmologic examinations. preoperatively, it helps the ophthalmologist to safely plan corneal refractive procedures and screen for refractive surgery candidates, in order to reduce the risk of postoperative complications. besides, cct measurements play a crucial role in the diagnosis and management of glaucoma because the value of intraocular pressure should be adjusted in accordance with cct. cct measurements also play an important role in the diagnosis of corneal diseases, such as fuchs ' corneal dystrophy and keratoconus [3, 4 ]. for many years, ultrasound pachymetry has been the most frequently used method to measure cct because it is relatively inexpensive and easy to use and has high intraoperator repeatability [2, 5 ]. nevertheless, ultrasound pachymetry has certain disadvantages, such as corneal - probe contact, the need for topical anesthesia, and the risk for transmission of infections and corneal epithelial lesions. besides, the reliability of ultrasound pachymetry results depends on the operator 's skill when placing the probe perpendicularly to the cornea. over the last decade, scheimpflug technology plays a major role, including pentacam (oculus, wetzlar, germany), sirius (costruzione strumenti oftalmici, florence, italy), galilei (ziemer, port, switzerland), and tms-5 (tomey, nagoya, japan). previous studies have shown that common used device pentacam has high intraoperator repeatability and interoperator reproducibility for cct measurements [79 ]. the corneal dynamic scheimpflug analyzer corvis st (oculus optikgerte, inc., wetzlar, germany) is relatively new, noncontact corneal biomechanics equipment, which is composed of an air puff indentation system and ultra - high - speed scheimpflug technology. the ultra - high - speed scheimpflug camera has a blue light led and acquires the deformation process at 4330 frames / s with an 8 mm horizontal coverage. because of the air impulse, the cornea experiences three stages : first applanation, highest concavity, and second applanation. furthermore, cct is obtained by the corneal initial state of the central horizontal cross - section diagram through the scheimpflug technology. few studies [1012 ] have evaluated the intraoperator repeatability of cct measurements obtained by this device in normal population. ali. evaluated the intersession reproducibility of the cct at different times of the day with this device. chen. assessed the intraoperator repeatability and interobserver reproducibility in virgin and post - prk eyes but only applied single value to evaluate interobserver reproducibility. however, to our knowledge, the interoperator reproducibility of corvis st for cct measurements using both single and average measurement value methods in normal eyes has not yet been evaluated. the purpose of the present study was to prospectively assess the intraoperator repeatability and interoperator reproducibility of cct measurements using both single and average methods acquired from the corvis st in normal eyes and compare the agreement with pentacam and ultrasound pachymetry. this prospective study was conducted on normal subjects recruited from the staff and students of the eye hospital of wenzhou medical university. the research protocol conformed to the tenets of the declaration of helsinki and was approved by the office of research ethics, eye hospital of wenzhou medical university. the exclusion criteria were active ocular pathology, any history of ocular surgery or trauma, recent contact lens wear (soft contact lens within two weeks and rigid contact lens within four weeks), systemic diseases with eye symptoms, and intraocular pressure > 21 mmhg. all subjects underwent a comprehensive ophthalmic examination, including uncorrected distance visual acuity, best - corrected visual acuity, slit - lamp microscopy, noncontact tonometer, and fundus examinations. subsequently, we applied pentacam, corvis st, and ultrasound pachymetry to measure cct. to avoid any effect of the ultrasound probe and topical anesthetic on the cornea measurements were acquired from the right eyes of subjects to avoid structural similarities between fellow eyes. in order to minimize the diurnal variation on cct readings, the subjects were required to completely blink twice before measurements, in order to form an optically smooth tear film on the cornea. corvis st examination applies four red alignment marks to position the center of the cornea on the computer screen. once positioned successfully, a puff of air with a pressure of 25 kpa is emitted automatically from the instrument aimed at the cornea at a distance of 11 mm. during the examination, the scheimpflug camera records corneal deformation process and cct. the three cct measurements obtained by each examiner were used to evaluate the intraoperator repeatability. the mean value of three successive measurements and the first measurement by each examiner were used to analyze the interoperator reproducibility. briefly, the subject was instructed to sit, open both eyes, and fixate on a target within the device. the real - time image of the subject 's eye on the computer screen was adjusted according to the pupil edge, center, and the corneal apex by moving the joystick. to avoid operator - dependent variables, the pentacam would automatically measure when correct alignment with the corneal apex and focus was achieved. only when the examination quality specification reading showed ok, it was recorded ; otherwise it was excluded and remeasured until three valid readings were obtained. after the cct measurements were obtained by pentacam and corvis st, an a - scan ultrasound pachymetry (sp-3000, tomey inc., nagoya, japan) was used. before the measurements, first, the cornea was anesthetized with topical 0.5% proparacaine hydrochloride (alcaine ; alcon laboratories, tx, usa). then, the subject in the supine position was asked to fixate on a target on the ceiling. then, five consecutive measurements were obtained, of which the highest and lowest were excluded, and the remaining three were recorded. statistical analysis. statistical analysis was performed using spss software for windows version 21 (ibm corporation, usa) and microsoft office excel (microsoft corp. the distributions were checked by kolmogorov - smirnov test, which showed that the data were normally distributed (p > 0.05). results were presented as means standard deviations. to assess the intraoperator repeatability of corvis st, within - subject standard deviation (sw), test - retest repeatability (trt), within - subject coefficient of variation (cov), and intraclass correlation coefficient (icc) were calculated for the three successive measurements obtained by the two operators. the trt is 2.77 times sw, which represents an interval within which 95% of the differences between measurements are expected to lie. the closer the icc is to 1, the better the consistency of measurement is. to evaluate the interoperator reproducibility of corvis st, the average method (the difference between the mean of the three successive measurements obtained by the two operators) and the single method (the first measurement of each operator) were used. then, the interoperator sw, trt, cov, and icc were also calculated. for multiple comparisons between cct measurements obtained by corvis st and pentacam or ultrasound pachymetry, the repeated - measures analysis of variance (anova) with bonferroni post hoc comparison test was used. furthermore, the 95% limits of agreement (loa) were calculated and bland - altman plots were produced to evaluate the agreement on the cct measurements between corvis st versus pentacam and corvis st versus ultrasound pachymetry. the 95% loa are defined as mean 1.96 sd, which represent an interval within which 95% of the differences between readings are expected to lie. this study enrolled 84 right eyes of 84 subjects (38 males and 46 females). the mean age of the subjects was 27.30 6.06 years (range 18 to 49 years). the mean spherical equivalent refraction was 4.12 2.66 d (range 10.50 to + 0.50 d). the cct measurements obtained using corvis st showed excellent intraoperator repeatability for both operators (table 1). the trt values were 13 m, the cov values were 0.97. the mean sd of cct, sw, trt, cov, and icc of corvis st are shown in table 2, which demonstrate high interoperator reproducibility. in the average method, the trt was 9.7 m, the cov was 0.65%, and the icc was 0.98. in the single method, the trt was 12.6 m, the cov was 0.85%, and the icc was 0.97. obviously, the error of the average method was smaller than the single method. the mean cct readings using corvis st, pentacam, and ultrasound pachymetry were 535.9 27.0 m, 539.0 25.70 m, and 543.7 27.52 m, respectively. the cct readings measured by corvis st were significantly thinner than pentacam (p 0.97, which represented high intraoperator repeatability in cct readings using corvis st in normal eyes. ali. obtained similar results in normal eyes, with trt, cov, and icc of 27 m, 1.83%, and 0.95, respectively. hon and lam reported trt, cov, and icc of 15.34 m, 1.01%, and 0.96, respectively, in normal subjects. nemeth. obtained similar icc of 0.97 and cov of 0.8% for cct in normal eyes. salvetat. reported icc of 0.99 in normal subjects and primary open - angle glaucoma patients. chen. obtained similar results in virgin eyes, with trt, cov, and icc of 12.56 m, 0.69%, and 0.99, respectively. however, they reported worse results in post - prk eyes, with trt and cov of 22.61 m and 2.29%, respectively. previous studies had assessed the intraoperator repeatability of cct values using other scheimpflug systems and specular microscopes, such as pentacam, sirius, galilei, orbscan ii (bausch & lomb, rochester, ny, usa), and sp-02 (costruzione strumenti oftalmici, italy). huang. evaluated the intraoperator repeatability of sirius and pentacam, a rotating scheimpflug camera combined with a placido disk corneal topographer and a rotating scheimpflug camera, respectively, in normal subjects. they indicated that the trt values were 8.79 m and 9.65 m, the cov values were 0.59% and 0.65%, and the icc values were 0.98 and 0.98, respectively. al - mohtaseb. assessed the intraoperator repeatability of the galilei, a dual rotating scheimpflug camera combined with a placido disk, in normal eyes. the cov was 0.36% and the icc was 0.99, which were also slightly better than our results with corvis st. maldonado. studied the intraoperator repeatability of the orbscan ii, a scanning - slit combined with placido disc topography, with trt and cov of 20.2 m and 1.5%, respectively, which were worse than our results. assessed the intraoperator repeatability of sp-02, a noncontact specular microscope, in normal eyes with trt, cov, and icc of 18.67 m, 1.23%, and 0.97, respectively, which were worse than our results with corvis st. these indirect comparisons indicate that the above - mentioned devices have high intraoperator repeatability, with galilei being the best. the galilei has two opposite scheimpflug cameras and can calculate the average value of the cct obtained by the two cameras. therefore, future studies should compare corvis st, galilei, and sirius. in the present study, we analyzed the interoperator reproducibility of cct measurements acquired with corvis st by applying the single and average methods. chen and lam [22, 23 ] demonstrated that the width of 95% loa was reduced by using an averaged result rather than the first result of each visit. ali. assessed the reproducibility of the cct with the device in normal eyes, which was intersession reproducibility at different times of the day. in their study, trt, cov, and icc were 11.0 m, 7.41%, and 0.995, respectively, while they were 9.70 m, 0.65%, and 0.984 in the current study, respectively. our results were obviously better than their results because our results were accomplished within 30 minutes, which eliminated the effects of different times on the cct. chen. measured the interoperator reproducibility of the cct by the single method in virgin and post - prk eyes, and the trt, cov, and icc were 13.24 m, 0.72%, and 0.98 in virgin eyes and 9.89 m, 0.58%, and 1.00 in post - prk eyes, respectively, which were similar to our results using the single method. salvetat. only applied icc to assess the interoperator reproducibility, which was 0.99 in normal subjects and primary open - angle glaucoma patients. we believe that ours is the first study to evaluate the interoperator reproducibility of corvis st using the average and single methods with trt, cov, and icc. in addition, we compared the cct readings between corvis st, pentacam, and ultrasound pachymetry in normal eyes. corvis st had a slightly lower cct measurement as compared to pentacam with a mean of 3.2 m. meanwhile, corvis st significantly underestimated cct as compared to ultrasound pachymetry with an average of 7.8 m. the 95% loa between corvis st and pentacam were narrow and comparable, with the cct diurnal pachymetric variation range of 11 to 11 m. therefore, corvis st and pentacam could be interchangeably used in normal eyes in most clinical applications. however, corvis st can not be interchangeably used with ultrasound pachymetry in normal eyes because of broad 95% loa between the two devices. our results were similar to or better than those previously reported when investigating agreement of cct measurements obtained from other scheimpflug systems, orbscan ii, and specular microscopes with respect to pentacam or ultrasound pachymetry (table 4). several reasons may explain the difference in cct readings between corvis st and ultrasound pachymetry. firstly, topical 0.5% proparacaine hydrochloride may cause corneal thickness to increase by 8.6 m in 80 seconds. secondly, the accuracy of ultrasound pachymetry depends on the operator 's proficiency and whether the corneal probe is perpendicularly placed on the center of the cornea. thirdly, if the posterior surface reflection point is closer to the anterior chamber, the cct measurement is thicker than the actual value. first, we only assessed the intraoperator repeatability and interoperator reproducibility in normal subjects and did not include keratoconus, glaucoma, or postrefractive surgery patients. further research is needed to assess the intraoperator repeatability and interoperator reproducibility in the above - mentioned patients. second, our study is restricted by the different algorithms each device uses for obtaining the cct. however, ultrasound pachymetry is performed over the pupil center, and its position depends on the operator 's experience. in conclusion, corvis st showed high intraoperator repeatability and interoperator reproducibility of cct measurements in normal eyes. corvis st and pentacam showed excellent agreement, which suggests that the two devices may be interchangeably used for cct measurements in the clinical setting. however, the cct readings between corvis st and ultrasound pachymetry are not directly interchangeable owing to the relatively wide 95% loa. | purpose. to assess the repeatability and reproducibility of central corneal thickness (cct) measurements by corneal dynamic scheimpflug analyzer corvis st in normal eyes and compare the agreement with pentacam rotating scheimpflug system and ultrasound pachymetry. methods. 84 right eyes underwent corvis st measurements performed by two operators. the test - retest repeatability (trt), within - subject coefficient of variation (cov), and intraclass correlation coefficient (icc) were used to evaluate the intraoperator repeatability and interoperator reproducibility. cct measurements also were obtained from pentacam and ultrasound pachymetry by the first operator. the agreement between the three devices was evaluated with 95% limits of agreement (loa) and bland - altman plots. results. corvis st showed high repeatability as indicated by trt 13.0 m, cov 0.97. the interoperator reproducibility was also excellent. the cov was 0.97. corvis st showed significantly lower values than pentacam and ultrasound pachymetry (p < 0.001). the 95% loa between corvis st and pentacam or ultrasound pachymetry were 15.8 to 9.5 m and 27.9 to 12.3 m, respectively. conclusions. corvis st showed excellent repeatability and interoperator reproducibility of cct measurements in normal eyes. corvis st is interchangeable with pentacam but not with ultrasound pachymetry. |
postoperative nausea and vomiting (ponv) can cause considerable distress and discomfort to participants undergoing surgery. several classes of antiemetic agents exist to combat these side - effects, though the 5-ht3 receptor antagonists have become the first line of treatment and are considered the gold standard in antiemetic therapy. compared with the older generation antiemetic drugs, 5-ht3 receptor antagonists are effective, well tolerated, and associated with few side - effects. most research on the 5-ht3 receptor antagonists has been performed with ondansetron, which has greater antivomiting than antinausea effects. not all surgical patients will benefit from antiemetic prophylaxis ; thus, identification of patients who are at increased risk is imperative. indeed, ondansetron is associated with cardiovascular effects, appears to cause qt interval corrected for heart rate (qtc) prolongation similar to that with droperidol ; droperidol and ondansetron induced similar clinically relevant qtc interval prolongations. when used in the treatment of ponv, a situation where prolongation of the qtc interval seems to occur, the safety of 5-hydroxytryptamine type 3 antagonists may not be superior to that of low - dose droperidol. thus ondansetron should be used with extreme caution in participants who suffer from or may develop prolongation of cardiac conduction intervals, thereby requiring dose adjustment. the qtc interval is a reflection of the action potential in the cardiac cells. homogenous or heterogeneous changes in the duration of action potential lead to alteration of qtc interval. the effect of a single intravenous dose of 1 mg, 8 mg, 16 mg ondansetron in preventing ponv was investigated in a randomized, double - blind, placebo - controlled, multicenter, international study and it showed that the maximum number of participants experienced no postoperative emesis in the 8 mg ondansetron recipient group than in the placebo group for the first 24-h postoperative period. the study showed that 2.7 - 3.5% qtc prolongation at 5 min after pre - induction injection of treatment medications, equal to 11.3 24.3 millisecond (ms) with 1.25 mg droperidol alone, and 9.9 34.7 ms with 4 mg ondansetron alone. a comparative study between droperidol (0.75 mg iv) and ondansetron (4 mg iv) in adults with ponv showed that the maximal qtc prolongation with droperidol was 179 msec, occurring at 2 min after administration and with ondansetron it was 2013 msec, occurring after 3 min. ondansetron is effective in the prevention and treatment of ponv associated with surgery. yet, qtc prolongation by 1 mg ondansetron has not yet been properly analyzed, though its prophylactic antiemetic effect was studied. we therefore aimed to evaluate the effect of 1 mg, 4 mg, and 8 mg bolus doses of intravenous ondansetron, relative to placebo, in prevention of ponv and the changes of qtc interval in healthy adult volunteers. this prospective randomized, placebo - controlled, double - blind study was carried out in various surgical operation theaters (ots) of a tertiary care postgraduate teaching hospital at kolkata between october 2008 and april 2009 after obtaining approval of the institutional ethics committee and informed consent of the participants. one hundred and thirty - six adult participants of both sexes, asa physical status i and ii, on no prescription of over - the - counter medication, with normal baseline electrocardiograms posted for breast surgery, vaginal hysterectomy, skin grafting surgery and reconstructive limb surgery were included in this study. participants were excluded from the study if they were known to have history of gastrointestinal disease, hormonal therapy, evidence of uncontrolled clinically important neurological, renal, hepatic, cardiovascular, metabolic or endocrine dysfunction or clinically important abnormalities in laboratory screening tests, vomiting during the 24-h period before surgery, vas > 4 at extubation, if the participants were not extubated after 30 min following study medication, were not responding to verbal commands after extubation, reaction to the study drug, had a nasogastric tube in situ postoperatively, weighed 90 kg, or were pregnant or lactating women. sample size estimation was done by a previously conducted pilot study that revealed an incidence of 60% ponv in untreated participants. thereafter a pre - study power analysis was computed which revealed that a minimum of 34 participants per group would be required to verify this difference at 95% confidence limit and 90% power of the test. following overnight fasting, all the participants were premedicated with oral lorazepam 0.04 mg / kg and tab omeprazole 40 mg 2 h prior to induction of anesthesia. on arrival in the operating room, an intravenous infusion was started with lactated ringer 's infusion. five - lead electrocardiogram (ecg), saturation of peripheral oxygen (spo2), non - invasive blood pressure (nibp), edn tidal carbon - di - oxide (etco2), airway pressures and temperature were monitored intra - operatively. anesthesia was maintained with 50% nitrous oxide and 0.5% sevoflurane in oxygen with top - up doses of inj. neuromuscular function was monitored in the right adductor pollicis muscle using tactile train of four (tof). approximately one hour before skin closure, the participants were randomly allocated into four groups with 34 participants in each group through a computer - generated random number, and were administered inj. ondansetron 1 mg (0.02 mg / kg) in group o1, 4 mg (0.08 mg / kg) in group o4 and 8 mg (0.16 mg / kg) in group o8 and normal saline in placebo group (group p). ondansetron 1 mg, 4 mg or 8 mg all diluted to 10 ml in normal saline and this was administered as an infusion over five minutes duration, 30 min before extubation on anticipation of ponv. inhalational agent was discontinued 30 min before skin closure while n2o was stopped 10 min before extubation. residual neuromuscular blocking effect was reversed with 50 g / kg neostigmine along with 10 g / kg glycopyrrolate after fulfilling the criteria of extubation. intra - operatively all the data were recorded by an independent observer blinded to the study drugs. data recorded were noninvasive arterial blood pressure, heart rate (before, during and after inj. of study drug) which was continuously monitored and noted at 5-min intervals, duration of anesthesia, intraoperative blood loss, intravenous fluid, total amount of fentanyl administered and visual analogue score (vas) at extubation. qtc interval changes were monitored in chest lead ii for each participant and changes from preoperative baseline value to 1, 3, 5, 10, 15, 20, 40, 60, 120, 240, and 360 min post dose period were noted for each participant. qt interval was accepted as prolonged, when qtc values exceeded 440 ms. treatment arrangements kept ready for any precipitated arrhythmias included blockade anti - bradycardia pacing, implantable automatic cardioverter - defibrillator (icd).[1113 ] all data were entered into an excel spreadsheet and were analyzed using standard statistical software like spss and statistica. parametric data were analyzed using one - way analysis of variance test (anova). all data were entered into an excel spreadsheet and were analyzed using standard statistical software like spss and statistica. parametric data were analyzed using one - way analysis of variance test (anova). one hundred and thirty - six adult participants of both sexes were comparable in terms of mean age, sex, body mass index (bmi), and duration of anesthesia. there were no significant differences among the groups with respect to type of surgery performed (chi square 0.5371, df 9, p=1.00) [table 1 ]. clinico - social cofactors of the study participants comparable operative data with respect to mean intra - operative intravenous fluid administration, mean estimated blood loss and mean intra - operative fentanyl use, in the different study groups showed that there were no marked differences among the groups in respect to the above - mentioned operative parameters [table 2 ]. distribution of patients groups as per operative data there were no significant differences among the groups with respect to vas score on extubation (p=0.857). but vas score was significantly different among the groups at 40 min (p<0.0001) and at 60 min (p=0.0077) [table 3 ]. distribution of patients groups as per visual analogue score (vas) at different time intervals the study revealed that first rescue antiemetic requirement was not administered before 60 min in any group receiving ondansetron. in the placebo group the percentage (%) of participants in the 1 mg (gr.o1), 4 mg (gr.o4), and 8 mg (gr.08) ondansetron groups who experienced first ponv and needed rescue antiemetic between 60 to 120 min were 28%, 24%, and 7% respectively. the percentage (%) of participants in the 1 mg (gr.o1), 4 mg (gr.o4) and 8 mg (gr.08) ondansetron groups who experienced first ponv and needed first rescue antiemetic between 120 to 240 min interval from initial dose, were 63%, 72%, and 57% respectively. in gr. o8, 36% of participants required their first antiemetic between 240 to 360 min [table 4 ]. percentage distribution of participants who required first rescue anti - emetic medication at different time intervals participants of gr. p and gr.o1 maintained an insignificant change in qtc (mm) throughout the study period with exception at 40 min, a slight increase due to extubation - related sympathetic effect. significant and maximal qt prolongation in the 8 mg (gr.o8) recipient participants was revealed within 3 min of administration and in the 4 mg (gr. 04) recipient participants within 5 min of administration, which was within the normal range, prior to administration of inj. distribution of patients groups as per changes in qtc (mm) interval at different time intervals many medications given to participants under anesthesia care are also known to cause qtc prolongation, such as inhaled anesthetics, propofol, thiopental, succinylcholine, and neuromuscular blocker antagonists. most currently available antiemetics also cause qtc prolongation, including phenothiazines, antihistamines, and 5ht3 antagonists such as ondansetron. in our study, qtc changes were monitored at different time intervals and it was observed that, among the three groups of participants receiving inj. ondansetron (1 mg, 4 mg, 8 mg), a maximal qtc prolongation was recorded in 8 mg recipient participants at 3 min post administration period (0.47 0.02). in one study it was observed that, a 2.7 - 3.5% qt prolongation recorded at 5 min after injection of pre - induction medications was equal to 11.3 24.3 ms with 1.25 mg droperidol alone, 9.9 34.7 ms with 4 mg ondansetron alone. another comparative study between droperidol (0.75 mg iv) and ondansetron (4 mg iv) on 85 adults with ponv showed that the maximal qt prolongation with droperidol was 179 ms, occurring at 2 min after administration. therefore, intra - operative management should continue to focus on prevention of excessive sympathetic activity and avoidance of factors that can prolong the qtc interval. non - invasive monitoring should commence before the induction of anesthesia and ideally should include ecg monitoring of more than one lead, as short bursts of torsades de pointes may be difficult to distinguish from monomorphic ventricular tachycardia, when only one lead is available for analysis. the effect of prolonging the qtc interval by a volatile agent may resemble the action of class iii antiarrhythmic agents that prolong the qtc interval. the concomitant presence of k+ and mg+ serum level abnormalities (increased level) or existant endocrine dysfunction may alter qt interval. other researchers opined that prophylactic ondansetron appeared to be more effective when administered at the end of surgery than prior to induction of anesthesia. in this study as neostigmine is never given in isolation to reverse neuromuscular block, its true effect is unknown, but one would predict that the inevitable resultant bradycardia may prolong qt up to the extent of torsades, which would be undesirable. most research on the 5-ht3 receptor antagonists has been performed with ondansetron, which has greater antivomiting than antinausea effects. ondansetron 4 mg has an number needed to treat (nnt) of approximately 7 in the prevention of nausea compared with placebo (024 h) ; the 8-mg dose has an nnt of approximately 6. for the prevention of vomiting (024 h), ondansetron 4 mg has an nnt of approximately 6 ; the 8-mg dose has an nnt of approximately 5. other researchers also concluded in their study that the treatment of ponv with ondansetron was more cost - effective than prevention in both a low - risk (30%) and a high - risk (60%) setting. the reason for this was the frequent success rate of treating established ponv, even with small doses of ondansetron (1 mg). reversible transient changes in the, pr, qrs and qtc intervals have been consistently observed in noncomparative and comparative trials with ondansetron. the present study showed stable and comparable hemodynamic parameters during the study period in all the groups, implicating a least association of contributory factors on qtc prolongation, other than the effects of the study drug in healthy adult participants. concurrent assessments of the qtc changes by the different anesthetic agents would have yielded better results for external validity. there are many factors both related and unrelated to anesthesia that may influence ponv, such as age, sex, body weight, type and duration of surgery, type of induction and maintenance of anesthesia and the neuromuscular blocking agent used. this study was conducted in a homogenous group and with a single molecule in terms of age, sex, body weight, duration of anesthesia, and type of surgery. emerging disparity among the 5-ht3 receptor antagonists suggests that the incidence and/or intensity of adverse events should not be regarded as a class effect. the side - effects of the supportive care are important particularly for those who are suffering from co - morbid conditions, such as cardiovascular disease and renal or hepatic impairment. therefore it is very important to know whether the variation in the incidence of nausea and vomiting is related to the difference in agents used or not. reversible transient changes in the, pr, qrs and qtc intervals have been consistently observed in noncomparative and comparative trials with ondansetron. the present study showed stable and comparable hemodynamic parameters during the study period in all the groups, implicating a least association of contributory factors on qtc prolongation, other than the effects of the study drug in healthy adult participants. concurrent assessments of the qtc changes by the different anesthetic agents would have yielded better results for external validity. there are many factors both related and unrelated to anesthesia that may influence ponv, such as age, sex, body weight, type and duration of surgery, type of induction and maintenance of anesthesia and the neuromuscular blocking agent used. this study was conducted in a homogenous group and with a single molecule in terms of age, sex, body weight, duration of anesthesia, and type of surgery. emerging disparity among the 5-ht3 receptor antagonists suggests that the incidence and/or intensity of adverse events should not be regarded as a class effect. the side - effects of the supportive care are important particularly for those who are suffering from co - morbid conditions, such as cardiovascular disease and renal or hepatic impairment. therefore it is very important to know whether the variation in the incidence of nausea and vomiting is related to the difference in agents used or not. participants receiving ondansetron in the doses of 4 mg and 8 mg were significantly better than those receiving placebo for prevention of emesis ; the 8-mg dose prevented emesis longer than 1 mg or 4 mg dose ; effect of 4 mg dose was not different from that of 1 mg. therefore, 1 mg rather than 4 mg or 8 mg may be chosen as the optimal dose in preventing postoperative nausea and vomiting, because of having less propensity for inducing cardiac effects by prolonging the qtc, which along with other precipitating factors of surgery may precipitate the life - threatening torsades, despite the excellent overall safety profiles of this 5-ht3 receptor antagonist. therefore it may be concluded that ondansetron in a dose of 1 mg in healthy adult participants can effectively prevent postoperative nausea and vomiting causing no or insignificant prolongation of qtc interval. | background : to avert nausea and vomiting the 5-hydroxytryptamine3 (5-ht3) antagonists have become the first line of treatment ifassociated with cardiovascular effects andappear to cause qt prolongation.objective:evaluate the effect of 1 mg, 4 mg, and 8 mg bolus doses of intravenous ondansetron, relative to placebo, in prevention of postoperative nausea and vomiting (ponv) and to find out the changes of qt interval corrected for heart rate (qtc).materials and methods : this prospective randomized, placebo - controlled, double - blind study was carried out among 136 adult participants of both sexes in a tertiary care postgraduate teaching institute at kolkata. mg, 4 mg or 8 mg inj. ondansetron was diluted to 10 ml with normal saline, was infused 30 min before extubation in relation with a control group. time to first rescue antiemetic medication and in qtc interval at different time intervals, in each group was noted in different in the various surgical operation theaters (ots).results : requirement of the first rescue antiemetic in the postoperative period between 60 to 120 min in the mg, 4 mg or 8 mg ondansetron groups was in 28%, 24% and 7% participants respectively ; between 120 to 240 min in 63%, 72% and 57% respectively ; and within 360 min in 9%, 4% and 36% respectively. significant and maximal qtc prolongation was observed in the participants with mg or 8 mg ondansetron 3 and 5 min of drug administration.conclusions:one mg ondansetron in healthy adult participants can effectively prevent ponv causing no or insignificant prolongation of qtc interval. |
grand multiparity has been considered an independent factor for increasing adverse outcome for both fetus and mother specially diabetes mellitus, antepartum hemorrhage, malpresentation, cesarean section rate, postpartum hemorrhage, iron deficiency anemia, and a high perinatal mortality rate al jf (1). more recent reports, however, have demonstrated that in the presence of good perinatal care, grand multiparity no longer need to be considered an obstetrical risk in the presence of satisfactory health care conditions (2, 3). the majority of the studies argued that grand multiparas are more likely to be of old age which might be the reasons for increased morbidity and mortality. in our clinical practice, such factor is difficult to be removed because women s age is the most important biological variable that influences the reproductive events which we study. in saudi arabia, large family is desirable for cultural reasons ; consequently, a high incidence of grand multiparity is expected. the fertility rate in saudi arabia was last reported at 2.81 in 2010, according to a world bank report published in 2012 (4). in addition, early age of marriage might be one of the reasons for this high incidence of grand multiparas. the current study was conducted in a tertiary hospital where medical care is given free of charge for all mothers. the aims of the current study were to determine the prevalence and to investigate the feto - maternal outcomes related to grand multiparity. in this retrospective study, the data were gathered from patient s case notes over a period of a 1-year from january 1, 2012 through december 31, 2012 at the maternity and children hospital (mch), buraidah, saudi arabia in an attempt to determine the prevalence of grand multiparity and its associated risks. uncomplicated cases received antenatal care at the level of primary health care centers, whereas complicated and referred cases are managed at the hospital. all deliveries took place in the hospital, and no home confinements were allowed. in this study, a grand multiparas woman was defined as a woman who gave birth to 5 and more deliveries after 24 weeks gestations (5). a total of 8040 deliveries was performed during the year, of these 430 were grand multiparas which were the actual number of grand multiparity during the whole year. they were matched to 657 multiparas (parity 1 - 4) women who delivered during the same time scale. sociodemographic factors, obstetric complications, and neonatal morbidity for both groups were recorded from the case note. maternal variables we assessed included diabetes mellitus, hypertensive disorders of pregnancy, premature rupture membrane, placental abruption, placenta previa, medical problems (such as asthma, epilepsy and hypothyroidism), postpartum hemorrhage, tears, cesarean hysterectomy, preterm labor, mode of delivery and post term labor(diabetes was assessed separately because it is important variable for pregnancy outcomes). each of these variables was analyzed against each group. for clarity, medical problems included (asthma, epilepsy and hypothyroidism) and diabetes included both pre - existing and gestational diabetes. fetal variables we assessed were admission to nursery, small for gestational age, fetal death, apgar score, fetal weight, gestational age at delivery, fetal distress and macrosomia. this study was approved by the ethics committee of the college of medicine of qassim university. the statistical package for the social sciences (spss 17 for windows) was used for recording and statistical analyses of data. the descriptive statistics used included the mean, the frequency distribution and the standard deviation. a chi - square test was used to compare the means of qualitative data, whereas a student s t - test was used to compare the means of quantitative data. in multivariate analysis the results of the analysis are presented as odds ratio (or) and 95% confidant interval (95% ci). the statistical package for the social sciences (spss 17 for windows) was used for recording and statistical analyses of data. the descriptive statistics used included the mean, the frequency distribution and the standard deviation. a chi - square test was used to compare the means of qualitative data, whereas a student s t - test was used to compare the means of quantitative data. in multivariate analysis the results of the analysis are presented as odds ratio (or) and 95% confidant interval (95% ci). the total number of deliveries during the study period was 8040, of these 430 were grand multiparas. of 430, grand multiparas, 28.6% (123) were below 35 years of age (younger grand multiparas), in this group the csr was 27.2% (72). there was no significant differences in the cs rate when they were compared with those above 35 years of age 72.8 % (307) p=0.666 as show in table 1. values were presented as number (percentage) table 2 shows the frequency of the individual parity and the associated percentage. values are presented as number (percentage) in this study, the distribution of age according to parity showed a linear relationship with good agreement with p - p plot distribution. there were significant differences in maternal age (28.88285.26145 vs. 36.84884.40522 ; p0.05). values are presented as number (percentage) chi - square test and fisher s exact test (cell count less than 5) were used to examine the differences between some post partum obstetrical complications between multiparas and grand multiparas. grand multiparas when compared to multiparous women they were at an increased risk of cesarean delivery (p0.05) (table 4). values are presented as number (percentage) binary logistic regression was used to explore the association of some selected antenatal and postnatal variables between multiparas and grand multiparas as presented in table 5. grand multiparas were significantly associated with increased incidence of cesarean section (or=2.699, ci=2.072 - 3.515, p0.05. nassar and colleagues observed no significant differences in antepartum hemorrhage, intrauterine growth restriction and stillbirth rates (8, 19). however, rayamajhi., reported stronger association of hypertensive disorders in pregnancy, preterm birth, anemia, postpartum hemorrhage in the grand multiparity (20). in his study, 3 (.7%) cases of grand multiparity underwent hysterectomies, one for complete placenta previa and two for uncontrolled postpartum hemorrhage, giving a hospital incidence of one in 2680 deliveries, a comparable incidence of one in 2581 was reported from tunisia (21)., there was no significant association between grand multipara and admission to icu, intrauterine fetal death and low birth weight babies. the shortcomings of this study are its retrospective nature and the gathering of data from a single center rather than multicenter (the latter of which could be more reprehensive of the population). in view of the results obtained in this study, we feel that grand multiparity continue to pose additional risk for pregnancy outcomes even in modern obstetrics care. in a community where large family is desirable, again, conduction of good antenatal and intrapartum care will result in much reduction of these adverse outcomes for both fetus and mother. | aim : the aim of the current study was to determine the prevalence of grand multiparity and the associated risks factors.methods:four hundred thirty grandmutliparas (parity 5 or more) were compared with multiparous population (parity 2 - 4) with regard to maternal age, gestational age, mode of delivery, fetal and maternal outcomes and inter - current medical and obstetrical problems.results:there were significant association between grand multiparity and adverse pregnancy outcomes such as cesarean delivery (or=2.699, ci=2.072 - 3.515, p<0.001), fetal macrosomia (or=1.675 ; 95% ci=1.004- 2.796, p=.048), diabetes mellitus (or=1.634, 95%ci=1.076 - 2.481, p=0.021), and pregnancy induced hypertension (or=1.838, 95% ci=1.054 - 3.204, p=.032). no significant associations were seen in placenta abruption, placenta previa, preterm labor, postpartum hemorrhage and the frequency of admission to neonatal intensive care unit. no prenatal or maternal mortality was reported in this study.conclusion:grand multiparty remains a major obstetrics problem. it is associated with many medical and obstetrical complications. in communities where large family is desirable it is important to address the value of family planning and conduction of meticulous antenatal care. |
addictions are chronic relapsing health conditions associated with many negative consequences at individual and population levels. these include, but are not limited to, higher morbidity and mortality rates for the addicted person, health and financial damages for family or community members, and increased economic and social costs for society as a whole (effertz & mann, 2013 ; mcginnis & foege, 1999 ; single, robson, xie, & rehm, 1998). addictions are among the most prevalent mental disorders, especially when behavioral addictions are considered (sussman, lisha, & griffiths, 2011). although conceptualization, criteria, and categories of behavioral addictions have been vigorously debated, there is emerging consensus that they are similar to substance - related addiction problems insofar as they generate short - term rewards that promote behavioral persistence, despite knowledge of adverse consequences (demetrovics & griffiths, 2012 ; grant, potenza, weinstein, & gorelick, 2010 ; karim & chaudhri, 2012 ; mudry., 2011). these range from behaviors that are now widely viewed as legitimate addictions [e.g., gambling and online gaming addiction (hellman, schoenmakers, nordstrom, & van holst, 2013 ; wong & hodgins, 2014) ] through controversial behaviors [e.g., television, sex, and pornography addictions (clarkson & kopaczewski, 2013 ; garcia & thibaut, 2010 ; sussman & moran, 2013) ], to highly speculative addictions [e.g., love, tanning, or shoplifting addiction (kourosh, harrington, & adinoff, 2010 ; shulman, 2003 ; sussman, 2010) ]. from a clinical perspective, loss of control over these behaviors can lead to neglect of role obligations and health - protective behaviors as well as interpersonal conflict and/or direct physical harm. the phenomenon of reduced self - control despite negative consequences is one of the key characteristics uniting this broader conception, making it plausible to view these different activities as behavioral (or process) addictions (mudry., 2011). clinicians have noted that addictions frequently co - occur in the same individual and that there might be a systematic progression from having difficulties with one excessive behavior to struggling with another (gossop, 2001 ; haylett, stephenson, & lefever, 2004). moreover, compared with people experiencing only a single problematic addictive behavior, individuals with co - occurring addictions are at increased risk for negative outcomes including victimization, poorer physical health status, or even suicide (rush, urbanoski, bassani, castel, & wild, 2010). furthermore, when addictions co - occur, they can interact with each other, complicating both accurate assessment and effective treatment ; for instance, one excessive behavior might mask another addiction or addictions may alternate with each other (freimuth., 2008). despite these considerations, addiction treatment providers and programs often do not explore comorbidity issues (especially comorbid substance - related and behavioral addictions) and as a result do not provide integrated interventions despite their clear advantage over services offered in parallel or successively (rush., 2010). moreover, although high rates of co - occurring addictions have been discussed in the empirical literature, most of this work emphasizes co - occurring problems with substances and often excludes behavioral addictions. only a small body of research has addressed co - occurring substance - related and behavioral addictions. using a variable - centered (i.e., factor - analytic) approach, stephenson, maggi, lefever, and morojele (1995) examined co - occurrences among 16 excessive behaviors in a clinical sample. the authors identified a nurturance factor (e.g., excessive eating, shopping, exercise, work, or caffeine use) and a hedonism factor (e.g., use of alcohol, nicotine, recreational drugs, or gambling and excessive sexual behavior). (2004) attempted to replicate these findings using the same set of addictive behaviors and reported four groupings : a self - regarding nurturance (e.g., excessive eating, shopping, or caffeine use), an other - regarding nurturance (e.g., excessive work and compulsive helping), a sensation - seeking hedonism (e.g., use of recreational drugs, prescription drugs, and nicotine), and a dominance - related hedonism (2005) investigated another set of behaviors / disorders in a clinical sample and identified three groups characterized by reward deficiency (e.g., trichotillomania, pathological gambling, and hypersexual disorder), impulsivity (e.g., compulsive shopping, kleptomania, and excessive eating), and others identified only two groups in a non - clinical sample of youth when analyzing the co - occurrence of 11 excessive behaviors : a generally non - addicted and a work hard, play hard group (e.g., excessive sexual behavior, exercise, or internet use) (sussman., 2014). extant research in this area is limited because of the use of small and/or age - specific samples (sussman., 2014 ; villella., 2011 ; willoughby, chalmers, & busseri, 2004), restricted coverage of substance - related and behavioral addiction problems (freimuth., 2008 ; sussman., 2011), and variable - centered (i.e., factor - analytic) as opposed to person - centered (e.g., cluster analytic) approaches. to address these limitations, the first aim of this study was to describe the prevalence of single versus multiple addiction problems using a large, representative sample and a broad range of behaviors and substances. in doing so, we adopted a lay epidemiology approach to co - occurring addictive behaviors (konkol thege., 2015). lay epidemiology proposes that fields of symptomatology, nosology, aetiology, and epidemiology have identifiable counterparts in the thoughts and activities of people outside the formal medical community from this perspective, systematic investigations of inferences made by the lay public about health conditions can provide important insights into how they construe risks and how to craft intervention strategies (lawlor, frankel, shaw, ebrahim, & smith, 2003). the second aim of this study was to identify distinct subgroups of people experiencing one or more substance - related and behavioral addiction problems and to explore whether and how members of separate addiction clusters differ in relation to sociodemographic characteristics and psychological well - being. first, an online survey of 4,000 alberta (canada) adult members (18 + years of age) of an established research panel (ipsos canadian online panel) were recruited. target quotas, based on 2006 canadian census data, were set for age, gender and region, and a random, representative sample of panel members were sent invitations to take part in the survey. to widen the repertoire of methodology used, and balancing the possible bias resulted from online data collection (granello & wheaton, 2004), a computerized assisted telephone survey of an additional population - based sample of 2,000 alberta adults was also conducted in 2010. further details of the survey methodology have been described elsewhere (konkol thege., 2015). to address our first research aim, the combined online and telephone survey dataset (n = 6,000) was used. both original data bases were independently weighted to ensure that regional, age, and gender composition reflected that of the actual alberta population aged 18 years or older according to 2006 census data. although sociodemographic characteristics (with the exception of sex and income) and the occurrence of problem behaviors (with the exception of excessive shopping and work) differed across the two data sets, the effect sizes of these differences fell in the negligible range ; the only exception was excessive sexual behavior where cramer s v (0.11) was just above the border of negligible and small effect size. a more detailed analysis of survey mode differences in these samples has been reported elsewhere (konkol thege., 2015). to address our second research aim, we created an analytic subsample consisting of respondents who reported one or more addiction problems in the past year. the total sample (n = 6,000) and the subsample used for clustering (n = 2,728 ; 45.5%) differed across almost all sociodemographic characteristics ; however, the effect sizes of the differences fell again in the negligible or small range (table 1). furthermore, in the analytic sub - subsample, 1,850 individuals (67.8% of the subsample) were recruited as part of the online survey and 878 individuals (32.2% of the subsample) were participants of the telephone survey, which considering the original sample sizes of the telephone (n = 2,000, 33.3%) and online (n = 4,000, 66.6%) samples also indicates that the telephone and online samples were generally comparable in terms of addiction prevalence. sociodemographic characteristics of the total sample and the analytic subsample used for the cluster analysis are summarized in table 1. sociodemographic characteristics of the samples the survey included items assessing participants sex, age, educational level, marital status, employment, and income (table 1 describes response options for each of these sociodemographic items). in some of the analyses, sociodemographic variables were recoded into fewer categories to improve clarity (table 3). the survey also included questions regarding four substances (alcohol, tobacco, marijuana, and cocaine use) and six behaviors (problematic gambling, eating, shopping, sexual behavior, video gaming, and work) which were presented in random order for each respondent, regardless of survey mode. consistent with our emphasis on lay epidemiology, that is, views of the public about addiction problems rather than expert - derived signs and symptoms (konkol thege., 2015), a definition was provided for each problem behavior (table 2), which was intended to broadly define self - attributed problems for the substances and behaviors without using the term addiction to avoid respondent reactivity. to assess the occurrence of the excessive behaviors included, a single question (thinking back over your life, have you ever personally had a problem with [problem behavior ] ?) was used with three available response categories (no ; yes, but not in the past 12 months ; and yes, in the past 12 months). since, in this study we focused on the co - occurrence of past - year behaviors only, the first two response options were collapsed. definition of the problem behaviors provided to respondents sociodemographic characteristics in relation to number of self - reported addiction problems in the previous year (n = 6,000) to assess general well - being of the respondents, the eight - item personal wellbeing index (international wellbeing group, 2006) was administered. the scale contains eight areas of satisfaction, each rated on an 11-point scale (0 = completely dissatisfied, 5 = neutral, 10 = completely satisfied) : standard of living, health, achieving in life, relationships, safety, community - connectedness, future security, and spirituality. internal consistency of the scale was very good in the present sample (cronbach s = 0.88). statistical analyses were executed using spss 23.0 (spss, chicago, il, usa). chi - square tests were employed to compare respondents reporting no, one, two, and three or more past - year problem behaviors across categorical sociodemographic variables (e.g., sex and marital status) using cramer s v to quantify effect size. ordinal and non - normally distributed continuous sociodemographic characteristics (e.g., age and income) and well - being scores of the groups were compared using the non - parametric kruskal a multinomial logistic regression analysis was also run to model associations between sociodemographic variables and well - being scores and cluster membership. hierarchical cluster analysis using ward s method with squared euclidean distance as the distancing metric was employed to explore the patterns of co - occurring addictions. input for this analysis were the 10 variables indicating the past - year presence vs. absence of each of the problem behaviors investigated. number of clusters to retain was based on the approach of seeking for the largest change in agglomeration schedule coefficients [cf. stopping rules (clatworthy, buick, hankins, weinman, & horne, 2005) ]. cluster members were also compared across sociodemographic and well - being characteristics using the chi - square and kruskal wallis tests. first, an online survey of 4,000 alberta (canada) adult members (18 + years of age) of an established research panel (ipsos canadian online panel) were recruited. target quotas, based on 2006 canadian census data, were set for age, gender and region, and a random, representative sample of panel members were sent invitations to take part in the survey. to widen the repertoire of methodology used, and balancing the possible bias resulted from online data collection (granello & wheaton, 2004), a computerized assisted telephone survey of an additional population - based sample of 2,000 alberta adults was also conducted in 2010. further details of the survey methodology have been described elsewhere (konkol thege., 2015). to address our first research aim, the combined online and telephone survey dataset (n = 6,000) was used. both original data bases were independently weighted to ensure that regional, age, and gender composition reflected that of the actual alberta population aged 18 years or older according to 2006 census data. although sociodemographic characteristics (with the exception of sex and income) and the occurrence of problem behaviors (with the exception of excessive shopping and work) differed across the two data sets, the effect sizes of these differences fell in the negligible range ; the only exception was excessive sexual behavior where cramer s v (0.11) was just above the border of negligible and small effect size. a more detailed analysis of survey mode differences in these samples has been reported elsewhere (konkol thege., 2015). to address our second research aim, we created an analytic subsample consisting of respondents who reported one or more addiction problems in the past year. the total sample (n = 6,000) and the subsample used for clustering (n = 2,728 ; 45.5%) differed across almost all sociodemographic characteristics ; however, the effect sizes of the differences fell again in the negligible or small range (table 1). furthermore, in the analytic sub - subsample, 1,850 individuals (67.8% of the subsample) were recruited as part of the online survey and 878 individuals (32.2% of the subsample) were participants of the telephone survey, which considering the original sample sizes of the telephone (n = 2,000, 33.3%) and online (n = 4,000, 66.6%) samples also indicates that the telephone and online samples were generally comparable in terms of addiction prevalence. sociodemographic characteristics of the total sample and the analytic subsample used for the cluster analysis are summarized in table 1. the survey included items assessing participants sex, age, educational level, marital status, employment, and income (table 1 describes response options for each of these sociodemographic items). in some of the analyses, sociodemographic variables were recoded into fewer categories to improve clarity (table 3). the survey also included questions regarding four substances (alcohol, tobacco, marijuana, and cocaine use) and six behaviors (problematic gambling, eating, shopping, sexual behavior, video gaming, and work) which were presented in random order for each respondent, regardless of survey mode. consistent with our emphasis on lay epidemiology, that is, views of the public about addiction problems rather than expert - derived signs and symptoms (konkol thege., 2015), a definition was provided for each problem behavior (table 2), which was intended to broadly define self - attributed problems for the substances and behaviors without using the term addiction to avoid respondent reactivity. to assess the occurrence of the excessive behaviors included, a single question (thinking back over your life, have you ever personally had a problem with [problem behavior ] ?) was used with three available response categories (no ; yes, but not in the past 12 months ; and yes, in the past 12 months). since, in this study we focused on the co - occurrence of past - year behaviors only, the first two response options were collapsed. definition of the problem behaviors provided to respondents sociodemographic characteristics in relation to number of self - reported addiction problems in the previous year (n = 6,000) to assess general well - being of the respondents, the eight - item personal wellbeing index (international wellbeing group, 2006) was administered. the scale contains eight areas of satisfaction, each rated on an 11-point scale (0 = completely dissatisfied, 5 = neutral, 10 = completely satisfied) : standard of living, health, achieving in life, relationships, safety, community - connectedness, future security, and spirituality. internal consistency of the scale was very good in the present sample (cronbach s = 0.88). statistical analyses were executed using spss 23.0 (spss, chicago, il, usa). chi - square tests were employed to compare respondents reporting no, one, two, and three or more past - year problem behaviors across categorical sociodemographic variables (e.g., sex and marital status) using cramer s v to quantify effect size. ordinal and non - normally distributed continuous sociodemographic characteristics (e.g., age and income) and well - being scores of the groups were compared using the non - parametric kruskal a multinomial logistic regression analysis was also run to model associations between sociodemographic variables and well - being scores and cluster membership. hierarchical cluster analysis using ward s method with squared euclidean distance as the distancing metric was employed to explore the patterns of co - occurring addictions. input for this analysis were the 10 variables indicating the past - year presence vs. absence of each of the problem behaviors investigated. number of clusters to retain was based on the approach of seeking for the largest change in agglomeration schedule coefficients [cf. stopping rules (clatworthy, buick, hankins, weinman, & horne, 2005) ]. cluster members were also compared across sociodemographic and well - being characteristics using the chi - square and kruskal wallis tests. more than half (50.8%) of the participants in the total sample of 6,000 respondents reported experiencing a problem with one or more of the substances and behaviors examined in the 12 months preceding the study (prevalence rates for the individual addictive behaviors in this sample have been described elsewhere, see konkol thege., 2015). about one - third (29.8%) reported a problem with only one substance or behavior in the past year, while 13.1% reported two problems, and 7.9% reported problems with three or more substances and behaviors in the year before the study. members of these groups differed significantly across all sociodemographic characteristics as well as well - being scores (table 3). when entering the sociodemographic variables and well - being into a multinomial logistic regression model predicting whether respondents reported one, two, or three or more problems (reference group = respondents reporting no addiction problems in the past year), age and well - being were the only consistently significant predictors (each associated with decreased probability of excessive behaviors) ; sex, educational attainment, and marital status were associated with group membership only occasionally, while income and employment status did not seem to have a role in distinguishing between the groups of individuals with no, one, two, and three or more addiction problems (table 4). results of the multinomial logistic regression investigating correlates of reporting no versus one, two, or three or more addiction problems (odds ratios with 95% confidence intervals) p <.05, p <.01, p <.001. the results of the cluster analysis suggested a seven - cluster solution. as shown in table 5, the first cluster (26.0% of the sample used when conducting the clustering) represented individuals with smoking as their shared problem behavior. the second cluster (21.8%) consisted of participants reporting excessive eating as their only problem behavior. the third cluster (16.2%) represented individuals with work problems, while the fourth cluster (13.0%) consisted of participants characterized by a large number of different addiction problems without a clearly dominant behavior. the fifth cluster (9.5%) represented mainly individuals reporting excessive sexual behavior, while the sixth (8.9%) and seventh (4.7%) clusters consisted of participants with shopping and video gaming as their shared behavioral problem, respectively. highest average number of past - year addictive behaviors was observed among excessive video game players (cluster vii), while the lowest was found among excessive eaters (cluster ii). detailed information on the addiction characteristics of each cluster are described in table 5. prevalence (%) of each problem behavior in the addiction clusters (n = 2,728) note. alc : problematic alcohol use, tob : tobacco use problems, mar : problems with marijuana use, coc : problematic cocaine use, gamble : gambling problems, shop : excessive shopping, video : problematic video gaming, eat : problematic eating, sex : excessive sexual behavior, and work : excessive work. cluster membership was significantly associated with sex : the proportion of males was 34.9%, 27.7%, 40.6%, 47.7%, 64.1%, 20.6%, and 44.1% in the seven clusters, respectively. all of the ordinal level (educational attainment) or non - normally distributed continuous sociodemographic variables (age and income) were also associated with cluster membership. finally, cluster members significantly differed in terms of well - being as well : members of the excessive buyer, smoker, sex addict, and polyaddict clusters showed clearly (indicated by non - overlapping confidence intervals) lower well - being scores than workaholics and excessive eaters (figure 1). sociodemographic characteristics in relation to cluster membership (n = 2,728) details on the measurement of income can be found in table 1. means and 95% confidence intervals of the personal wellbeing index in the seven clusters more than half (50.8%) of the participants in the total sample of 6,000 respondents reported experiencing a problem with one or more of the substances and behaviors examined in the 12 months preceding the study (prevalence rates for the individual addictive behaviors in this sample have been described elsewhere, see konkol thege., 2015). about one - third (29.8%) reported a problem with only one substance or behavior in the past year, while 13.1% reported two problems, and 7.9% reported problems with three or more substances and behaviors in the year before the study. members of these groups differed significantly across all sociodemographic characteristics as well as well - being scores (table 3). when entering the sociodemographic variables and well - being into a multinomial logistic regression model predicting whether respondents reported one, two, or three or more problems (reference group = respondents reporting no addiction problems in the past year), age and well - being were the only consistently significant predictors (each associated with decreased probability of excessive behaviors) ; sex, educational attainment, and marital status were associated with group membership only occasionally, while income and employment status did not seem to have a role in distinguishing between the groups of individuals with no, one, two, and three or more addiction problems (table 4). results of the multinomial logistic regression investigating correlates of reporting no versus one, two, or three or more addiction problems (odds ratios with 95% confidence intervals) p <.05, p <.01, p <.001. as shown in table 5, the first cluster (26.0% of the sample used when conducting the clustering) represented individuals with smoking as their shared problem behavior. the second cluster (21.8%) consisted of participants reporting excessive eating as their only problem behavior. the third cluster (16.2%) represented individuals with work problems, while the fourth cluster (13.0%) consisted of participants characterized by a large number of different addiction problems without a clearly dominant behavior. the fifth cluster (9.5%) represented mainly individuals reporting excessive sexual behavior, while the sixth (8.9%) and seventh (4.7%) clusters consisted of participants with shopping and video gaming as their shared behavioral problem, respectively. highest average number of past - year addictive behaviors was observed among excessive video game players (cluster vii), while the lowest was found among excessive eaters (cluster ii). detailed information on the addiction characteristics of each cluster are described in table 5. prevalence (%) of each problem behavior in the addiction clusters (n = 2,728) note. alc : problematic alcohol use, tob : tobacco use problems, mar : problems with marijuana use, coc : problematic cocaine use, gamble : gambling problems, shop : excessive shopping, video : problematic video gaming, eat : problematic eating, sex : excessive sexual behavior, and work : excessive work. cluster membership was significantly associated with sex : the proportion of males was 34.9%, 27.7%, 40.6%, 47.7%, 64.1%, 20.6%, and 44.1% in the seven clusters, respectively. all of the ordinal level (educational attainment) or non - normally distributed continuous sociodemographic variables (age and income) were also associated with cluster membership. finally, cluster members significantly differed in terms of well - being as well : members of the excessive buyer, smoker, sex addict, and polyaddict clusters showed clearly (indicated by non - overlapping confidence intervals) lower well - being scores than workaholics and excessive eaters (figure 1). sociodemographic characteristics in relation to cluster membership (n = 2,728) details on the measurement of income can be found in table 1. integrated treatment for multiple addictive disorders is important because failure to identify and treat comorbid addiction problems is associated with poorer outcomes (substance abuse and mental health services administration, 2009). unfortunately, treatment providers are most prone to recognize disorders that fit the focus of their training, which rarely includes behavioral addictions (freimuth., 2008). therefore, the aim of the present study was to provide further information on how a relatively large number of substance and behavioral addictions co - occur to help inform treatment providers and service planners about typical combinations of addictions. our findings regarding the overall prevalence of addictive problems were consistent with us data indicating that about half of the adult population struggles with at least one excessive behavior in a given year (sussman., 2011). results of the present study also demonstrated that about 30 percent of the adult population had difficulties with one addictive behavior, while an additional 21% reported problems with two or more behaviors and/or substances. while many argue that the frequent co - occurrence of some forms of addiction suggests the presence of an underlying non - addiction specific proneness responsible for dependence (shaffer., 2004), the present data showing that almost 60% of those reporting addictive problems have difficulties only with one behavior does not provide a clear support for this syndrome model of addictions. however, it is also possible that many individuals reporting only one excessive behavior in the past year have had or will have problems in other areas but successively instead of simultaneously (cf. in addition, our data revealed that the patterning of the excessive behaviors studied fitted best with a seven - cluster solution, which is greater than the number of groups or dimensions (24) typically reported in previous studies (haylett., 2004 ; lochner., 2005 ; maclaren & best, 2010 ; stephenson., 1995 worthy of note, however, that direct comparison of the results across the investigations is problematic because of the large variability in study methodology including the number and type of addictions examined and the statistical methods employed (factor analysis, cluster analysis, latent class analysis, and correlations among scales measuring addictions). one strength of this study is the use of two independent, relatively large canadian samples representative of the adult alberta population across sex, age group, and region. a further strength is the simultaneous assessment of a relatively large number of both substance - related and behavioral addictions, providing the opportunity of taking a broader look at the whole addiction field. on the other hand, there were several limitations of the present research that deserve to be highlighted. first, response rates were relatively low in both survey modes, which weakens the generalizability of our findings (konkol thege., 2015). in addition, although the single question method for assessing problematic addictive behaviors is often employed in epidemiological surveys (bowling, 2005 ; cook, 1987), the reliability of single - item scales is generally weaker than that of multi - item scales. also, although previous research indicated that self - identification through a single question for an excessive behavior is a reliable and clinically meaningful tool in identifying persons with addictive disorders, these studies concentrated on substance addictions, pathological gambling, and video gaming (cook, 1987 ; king, delfabbro, & griffiths, 2013 ; widyanto, griffiths, & brunsden, 2011). the generalizability of the methodological appropriateness of this assessment method to all behavioral addictions is, therefore, questionable. our aim was to provide a brief behavioral description of each behavior that emphasized impairment and to avoid the use of terms such as addiction to minimize respondent reactivity. how exactly participants interpreted these items was not examined in this study, and it is possible that impairment was defined broadly in some instances. for example, problematic eating as defined for the respondents, can incorporate not only excessive eating of food addicts but also the restrictive behavioral patterns of anorexics, which despite its destructive nature is not classified as an addiction in the current nosological systems. despite these limitations, this study calls our attention to the considerable large number of individuals experiencing several addictive disorders simultaneously who thus need special consideration and the use of integrated treatment approaches when receiving mental health services. we hope that this work will assist in the accurate assessment and treatment of patients presenting addiction symptoms, encouraging professionals to consider the possibility of likely co - occurring substance and behavioral addictions above those emphasized initially by their clients. bkt conducted the literature searches and provided summaries of previous research studies, conducted the statistical analysis, and wrote the first draft of the manuscript. all authors had full access to all data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. | background and aimsthe aims of this study were (a) to describe the prevalence of single versus multiple addiction problems in a large representative sample and (b) to identify distinct subgroups of people experiencing substance - related and behavioral addiction problems.methodsa random sample of 6,000 respondents from alberta, canada, completed survey items assessing self - attributed problems experienced in the past year with four substances (alcohol, tobacco, marijuana, and cocaine) and six behaviors (gambling, eating, shopping, sex, video gaming, and work). hierarchical cluster analyses were used to classify patterns of co - occurring addiction problems on an analytic subsample of 2,728 respondents (1,696 women and 1032 men ; mage = 45.1 years, sdage = 13.5 years) who reported problems with one or more of the addictive behaviors in the previous year.resultsin the total sample, 49.2% of the respondents reported zero, 29.8% reported one, 13.1% reported two, and 7.9% reported three or more addiction problems in the previous year. cluster - analytic results suggested a 7-group solution. members of most clusters were characterized by multiple addiction problems ; the average number of past year addictive behaviors in cluster members ranged between 1 (cluster ii : excessive eating only) and 2.5 (cluster vii : excessive video game playing with the frequent co - occurrence of smoking, excessive eating and work).discussion and conclusionsour findings replicate previous results indicating that about half of the adult population struggles with at least one excessive behavior in a given year ; however, our analyses revealed a higher number of co - occurring addiction clusters than typically found in previous studies. |
gmbs solution was flowed through the peg functionalised go chip at a 20 l / min flow rate using a syringe pump (harvard apparatus). after 30 minutes of incubation, the device was washed with ethanol at 100 l / min. 50 g / ml neutravidin was prepared and flowed through the device at 20 l / min. after 1 hour incubation, the device was flushed with phosphate buffered saline (pbs) at 100 l / min to remove the excessive neutravidin. finally, biotinylated epcam antibody at a concentration of 20 g / ml in pbs with 1% (w / v) bsa was flowed through the device for 10 minutes at 20 l / min. after 1 hour incubation, pbs was flowed to wash then, 1% or 3% bsa solution in pbs was flowed at 100 l / min for 5 minutes. after flowing bsa solution, the device was allowed to incubate for 30 minutes. tissue culture reagents were purchased from gibco invitrogen corporation / life technologies life sciences unless otherwise specified. mcf-7/hs-578 t and pc-3 cells were cultured in dmem and dmem / f12 medium containing 10% fetal bovine serum and 1% penicillin - streptomycin solution. when cells reached more than 7080% confluence, they were harvested and labeled with a green cell tracking dye (invitrogen, celltracker green cmfda, c7025). subsequently, these fluorescence tracked cells were used to perform the capture efficiency experiments. for a low number cell spiking (320), cells were diluted in serum - free medium starting at an initial concentration of 1 10 cells / ml. 1 l of the concentrated cell suspension was transferred to a low - attachment 96-well plate. the transferred cells were counted under the microscope, then immediately pipetted into a 1 ml of whole blood. after removing the cells from the 96-well plate, we counted the remaining cells at the same position. by subtracting these cells left behind from the original spot, after flowing blood samples with low number of non - labeled cells, the captured cells were washed with pbs, fixed with 4% paraformaldehyde (pfa), permeabilized with 0.2% triton - x and incubated for 30 minutes followed by pbs wash. the device was incubated for 30 minutes with 1 ml of blocking buffer containing 2% normal goat serum and 3% bsa. anti - cytokeratin 7/8 (bd biosciences) and anti - cd45 (bd biosciences) were diluted to 5 g / ml in 1% bsa. these antibodies were flowed through the go chip for 20 minutes at 50 l / min and incubated for 1 hour. anti - cytokeratin was probed with alexa fluor 488 igg2a fitc (invitrogen) and the anti - cd45 was probed with alexa fluor 546 igg1 (invitrogen). the secondary antibodies were diluted in 1% bsa at a 1:200 ratio, flowed through the go chip for 20 minutes at 50 l / min, incubated for 1 hour and followed by washing with pbs. to stain nuclei of the captured cells, dapi (1:1000 dilution in pbs) was flowed for 20 minutes at 50 l / min and the device was incubated for 15 minutes and washed with pbs. blood samples were drawn from patients with tumours and healthy donors after obtaining informed consent under an irb - approved protocol. all specimens were collected into edta tubes and were processed within 3 hours. to measure cells ability to proliferate, click - it edu imaging kit (invitrogen, c10340) was used. after capturing cells, the go chip was washed with pbs, and 10 m edu solution was added to the chip. the chip was incubated overnight, washed with pbs and followed by cell fixation with 4% pfa. after 15 minutes of incubation, the chip was washed with 3% bsa twice, followed by cell permeabilization with 0.5% triton x-100 in pbs and incubated for 20 minutes. the chip was washed with 3% bsa twice and 0.5 ml of click - it reaction cocktail was added, followed by 30 minutes incubation and washing once with 3% bsa. for nucleus staining, 1 ml of 1x hoechst 33342 solution was added and cells in the chip were incubated for 30 minutes and washed with 1 ml of pbs. the rna from captured ctcs either from spike samples or from patient s blood samples was extracted from the go chip using arcturus picopure rna isolation kit according to the manufacturer s instruction (abi, life technologies). then concentrated preparation of total rna for each sample was used in rt reaction followed by pre - amplification of cdnas using the pooled taqman gene expression assays of target genes and cell - to - ct kit according to the manufacturer s instruction (ambion, life technologies) on the eppendorf mastercycler pro s instrument. finally, gene expression experiments for each pre - amplified sample were performed using taqman gene expression assays for gapdh and her2 (life technologies) in a multiplex qpcr setting on the abi 7900ht instrument. gmbs solution was flowed through the peg functionalised go chip at a 20 l / min flow rate using a syringe pump (harvard apparatus). after 30 minutes of incubation, the device was washed with ethanol at 100 l / min. 50 g / ml neutravidin was prepared and flowed through the device at 20 l / min. after 1 hour incubation, the device was flushed with phosphate buffered saline (pbs) at 100 l / min to remove the excessive neutravidin. finally, biotinylated epcam antibody at a concentration of 20 g / ml in pbs with 1% (w / v) bsa was flowed through the device for 10 minutes at 20 l / min. after 1 hour incubation, pbs was flowed to wash then, 1% or 3% bsa solution in pbs was flowed at 100 l / min for 5 minutes. after flowing bsa solution, the device was allowed to incubate for 30 minutes. tissue culture reagents were purchased from gibco invitrogen corporation / life technologies life sciences unless otherwise specified. mcf-7/hs-578 t and pc-3 cells were cultured in dmem and dmem / f12 medium containing 10% fetal bovine serum and 1% penicillin - streptomycin solution. when cells reached more than 7080% confluence, they were harvested and labeled with a green cell tracking dye (invitrogen, celltracker green cmfda, c7025). subsequently, these fluorescence tracked cells were used to perform the capture efficiency experiments. for a low number cell spiking (320), cells were diluted in serum - free medium starting at an initial concentration of 1 10 cells / ml. 1 l of the concentrated cell suspension was transferred to a low - attachment 96-well plate. the transferred cells were counted under the microscope, then immediately pipetted into a 1 ml of whole blood. after removing the cells from the 96-well plate, we counted the remaining cells at the same position. by subtracting these cells left behind from the original spot, after flowing blood samples with low number of non - labeled cells, the captured cells were washed with pbs, fixed with 4% paraformaldehyde (pfa), permeabilized with 0.2% triton - x and incubated for 30 minutes followed by pbs wash. the device was incubated for 30 minutes with 1 ml of blocking buffer containing 2% normal goat serum and 3% bsa. anti - cytokeratin 7/8 (bd biosciences) and anti - cd45 (bd biosciences) were diluted to 5 g / ml in 1% bsa. these antibodies were flowed through the go chip for 20 minutes at 50 l / min and incubated for 1 hour. anti - cytokeratin was probed with alexa fluor 488 igg2a fitc (invitrogen) and the anti - cd45 was probed with alexa fluor 546 igg1 (invitrogen). the secondary antibodies were diluted in 1% bsa at a 1:200 ratio, flowed through the go chip for 20 minutes at 50 l / min, incubated for 1 hour and followed by washing with pbs. to stain nuclei of the captured cells, dapi (1:1000 dilution in pbs) was flowed for 20 minutes at 50 l / min and the device was incubated for 15 minutes and washed with pbs. blood samples were drawn from patients with tumours and healthy donors after obtaining informed consent under an irb - approved protocol. to measure cells ability to proliferate, click - it edu imaging kit (invitrogen, c10340) was used. after capturing cells, the go chip was washed with pbs, and 10 m edu solution was added to the chip. the chip was incubated overnight, washed with pbs and followed by cell fixation with 4% pfa. after 15 minutes of incubation, the chip was washed with 3% bsa twice, followed by cell permeabilization with 0.5% triton x-100 in pbs and incubated for 20 minutes. the chip was washed with 3% bsa twice and 0.5 ml of click - it reaction cocktail was added, followed by 30 minutes incubation and washing once with 3% bsa. for nucleus staining, 1 ml of 1x hoechst 33342 solution was added and cells in the chip were incubated for 30 minutes and washed with 1 ml of pbs. the rna from captured ctcs either from spike samples or from patient s blood samples was extracted from the go chip using arcturus picopure rna isolation kit according to the manufacturer s instruction (abi, life technologies). then concentrated preparation of total rna for each sample was used in rt reaction followed by pre - amplification of cdnas using the pooled taqman gene expression assays of target genes and cell - to - ct kit according to the manufacturer s instruction (ambion, life technologies) on the eppendorf mastercycler pro s instrument. finally, gene expression experiments for each pre - amplified sample were performed using taqman gene expression assays for gapdh and her2 (life technologies) in a multiplex qpcr setting on the abi 7900ht instrument. | the spread of cancer throughout the body is driven by circulating tumour cells (ctcs)1. these cells detach from the primary tumour and move from the blood stream to a new site of subsequent tumour growth. they also carry information about the primary tumour and have the potential to be valuable biomarkers for disease diagnosis and progression, and for the molecular characterization of certain biological properties of the tumour. however, the limited sensitivity and specificity of current methods to measure and study these cells in patient blood samples prevent the realization of their full clinical potential. the use of microfluidic devices is a promising method for isolating ctcs2, 3 ; however, the devices are reliant on three - dimensional structures, which limit further characterization and expansion of cells on the chip. here we demonstrate an effective approach to isolate ctcs from blood samples of pancreatic, breast and lung cancer patients, by using functionalised graphene oxide nanosheets on a patterned gold surface. ctcs were captured with high sensitivity at low concentration of target cells (73% 32.4 at 35 cells / ml blood). |
constipation is a common complaint encountered by physicians, and women are two times as likely to report constipation than men. constipation is commonly multifactorial and can result from metabolic, endocrine, or neurological disorders, or from the adverse effect of medications. in functional defecation disorders, patients perceived constipation as difficulties in defecating or the presence of hard stools despite a normal evacuation rate. the prevalence rate (usually on the higher side) of constipation varies considerably due to different methods applied and differences in study populations. the prevalence of constipation in the iranian population is reported to be 1.437%, and the prevalence of functional constipation is reported to be 2.411.2%. chronic constipation impairs health - related quality of life, and thus, appropriate management is very important. a recent population - based study estimated the economic burden of functional bowel disorders (including functional constipation) in iran and reported a heavy financial burden on the iranian national health system. constipation, especially when chronic and severe, can result in complications such as fecal impactions, hemorrhoid and rectal bleeding, colorectal obstruction, colorectal ulcers, rectal prolapse, anal fissures, and pelvic wall insufficiency, thereby increasing the burden of the disorder on the patient 's health. a long - lasting management of chronic constipation usually begins with lifestyle modifications and increased intake of dietary fibers and fluids alone or in combination with an osmotic laxative. polyethylene glycol, sodium picosulfate, bisacodyl, prucalopride, lubiprostone, and linaclotide are considered to be more effective than placebo in treating chronic functional constipation. although there are also other preparations used for treating chronic functional constipation, only limited evidence is available to support their efficacy. thus, their use may be restricted to patients who fail to respond to conventional treatments. many patients are still treated empirically as there is a lack of an evidence - based algorithm so far. the high costs and widespread complications associated with chemical drugs force patients to consume alternative medication, which sometimes have higher efficacy and lower side effects and cost. in western european countries and the united states, approximately 850% of individuals use alternative medication ; in fact, this rate is even higher in developing countries. patients consider herbal medicines appropriate because they are easy to obtain, affordable, and have fewer side effects. we designed this study to evaluate a herbal preparation (lax - asab), which is traditionally used for the treatment of chronic constipation in some regions of iran and is well - known for higher efficacy, lower side effects, and lower cost in comparison with other currently available chemical and herbal remedies. this double - blind, randomized control clinical trial was carried out at the outpatient clinic of tabriz university of medical sciences, iran, during 20112012. the study protocol was in accordance with the declaration of helsinki and was approved by the regional ethic committee on august 20, 2010 (reference no. the study is also registered as a clinical trial in the iranian registry of clinical trials (www.irct.ir ; registration no. all patients diagnosed by a gastroenterologist to have functional constipation according to the rome iii criteria (for at least 3 months) were included if they agreed to avoid using any laxative medication for 1 week before beginning the study. a thorough physical examination was performed, and patients ' medical history and biochemical parameters (including liver, renal, and thyroid function tests) were reviewed and collected before the trial. imaging or additional evaluations were carried out based on the clinical judgment of the attending gastroenterologist. participants with a history of organic gastrointestinal (gi) disorders (including cancer and celiac disease) ; renal, hepatic, cardiac, pulmonary, neurologic, and hematologic diseases ; malignancy ; inflammatory bowel disease ; drugs or alcohol - use problems ; hypersensitivity to herbal preparations ; pregnancy ; lactation ; history of gi bleeding ; emergence of intolerable, serious drug side effects ; and those noncompliant to the study protocol were excluded from the study. all of the patients were allowed to leave the study at any time and would subsequently receive standard care. medications that may affect gi motility were not allowed during the study period (e.g., antidepressants, narcotic analgesics, and medications with an anticholinergic effect). selected patients received either lax - asab (lax - asab group) or placebo (control or placebo group) for 4 weeks (fig. 1). lax - asab is a traditional herbal preparation (dried and powdered) that contains equal proportions of cassia angustifolia vahl. (xi y fn xi y), mentha piperita l. (h jio b h), zingiber officinale rosc. (shng jing), and glycyrrhiza glabra l. (gn co). because c. angustifolia is the only component with laxative properties (the active component), the mixture of the other three was used as the placebo. thus, the placebo powder contained the same intergradient as that of lax - asab, except c. angustifolia leaves. lax - asab and placebo were supplied in identical containers by saeb - darou pharmaceutical plant company (mashhad, iran). all study personnel involved in the clinical evaluation and patients were blinded to the study treatments. patients ingested 1 g of either lax - asab or placebo powder, which was provided in identical packages numbered by a study member blinded to clinical evaluations. patients dissolved the powder in one glass of boiled water and ingested the medication before breakfast on alternative days. noncompliance for 3 consecutive days or for a total of 7 days (self - reported by study population) resulted in exclusion. the dietary (food frequency questionnaire) and physical activities [international physical activity questionnaire, last 7 days (self - administered format) ] of patients were controlled weekly according to the standard questionnaire to maintain a constant dietary composition and physical activity during the study. side effects were recorded each week, beginning with an open question about any complaints and using a checklist asking about cramps, abdominal pain, nausea, vomiting, diarrhea, gastric discomfort, and excess bloating. the severity of constipation (defined as number of defecation per week) and defecation difficulties (1 = dry stool ; 2 = straining force applied during defecation ; and 3 = obstruction during defecation) were recorded before and after the trial. data were collected using checklists and the efficacy of lax - asab, determined by a symptom scale, was compared with that of placebo during and at the end of the study. chi - square, fisher exact, and independent t tests, and repeated measurements were used to estimate differences wherever appropriate. the patients were randomly enrolled to receive treatment with either lax - asab (n = 24) or placebo (n = 24). of these 48 patients, 40 completed the trial [24 men (60%), mean age, 21.0 4.2 years ; 16 women (40%), mean age, 20.1 4.3 years ]. the mean weekly defecation time was 1.8 and 1.7 times, respectively, in patients receiving lax - asab and placebo (p = 0.729). most patients (n = 32, 80%) had previously used at least one medication for constipation in the past 6 months. the primary complaint of patients was incomplete defecation followed by abdominal bloating / distension, abdominal discomfort, and symptoms due to hemorrhoids. the mean of weekly defecation times increased in both groups (p = 0.001) ; from 1.8 0.41 to 4.8 1.12 times in patients receiving lax - asab and from 1.7 0.44 to 2.2 0.61 times in patients receiving placebo. a time treatment interaction showed that this increase was significantly higher in the intervention group (p < 0.0001). all the other measured outcomes concerning defecation difficulty were significantly improved in patients receiving lax - asab compared with placebo. no statistically significant difference between the two groups with regard to side effects was observed. side effects were reported by three patients who received placebo and by seven who received lax - asab [odds ratio = 3.05 ; 95% confidence interval (0.6514.13) ; p = 0.27 ]. major side effects reported were watery diarrhea followed by abdominal cramp, nausea, and distension in the intervention group, and abdominal pain followed by distension in the placebo group (table 2). other side effects such as allergic reaction or drug - induced hepatitis were not observed / reported. this randomized, double - blind, placebo - controlled study suggested the benefits of lax - asab, a traditional herbal remedy containing equal proportions of cassia angustifolia vahl. (xi y fn xi y), mentha piperita l. (h jio b h), zingiber officinale rosc. (shng jing), and glycyrrhiza glabra l. (gn co), which is used for the treatment of functional constipation. in addition, the formulation increased the frequency of defecation and decreased complaints as early as 2 weeks after beginning the treatment. patients mostly define constipation by these symptoms : infrequent stools (typically <3 times / wk), hard stools, the need for excessive straining force during defecation, a sense of incomplete bowel evacuation, and excessive time spent in the toilet or unsuccessful defecation. leaves of senna (c. angustifolia vahl.) have been used as an alternative medicine and as an aid to treat constipation.. not all uses of senna are approved by the food and drug administration, and senna preparations are often sold as herbal supplements. response relationship but it has been reported that a combination of laxative and senna is more efficient in treating constipation. senna leaves are well - known for their laxative effects in iran but their use is limited due to the side effects, which can be very severe. common side effects are electrolyte disturbances, potassium deficiency, dehydration, and diarrhea accompanied by cramps. however, in this study, lax - asab, an herbal formulation that contains senna leaves along with other herbal ingredients in equal proportions, effectively decreased defecation problems and the patients did not experience any serious side effects. ginger (z. officinale rosc.) is a well - known flavoring agent in the east and has been traditionally used for gi upsets. ginger preparations have been reported to be effective against nausea associated with motion sickness and chemotherapy. human studies and clinical trials of peppermint leaf (m. piperita l.) are limited. however, peppermint has a relaxing effect on gi tissue, as well as analgesic and anesthetic effects on the central and peripheral nervous systems in animal models. the final ingredient of lax - asab, g. glabra l., has various pharmacological properties, including antitumor, antiparasitic, antiulcer, and antioxidative effects. despite the fact that it is has been traditionally used in the preparation, none of these properties seemed efficient at reducing the side effects or increasing the efficacy of the preparation. although we did not use a satisfaction questionnaire, none of participants stopped using lax - asab because of bad taste or intolerable side effects. the herbal combination seems to limit the side effects of senna while giving a tolerable taste. however, this is only a preliminary study and further studies for determining the dosage are necessary. speculations about complementary and alternative medicines producing only a placebo effect still exist. however, similar to other studies that evaluated the efficacy of herbal preparations in treating functional constipation, safety analysis revealed that there were no statistically significant differences in side effects between the two groups. adverse gi events included abdominal distension, pain, diarrhea, flatulence, and nausea ; their incidence was not different between the two groups. in addition, the side effects were not considered to be related to the study medication. although the main significance of these findings could be providing evidence about the efficacy of this preparation for clinicians, so they can use this formulation in their practice with functional constipation patients, these results could be a base for further research to optimize preparations based on senna, involving identifying the exact dosage required and the effects of other ingredients. in addition, although this study did not evaluate the financial benefits of using lax - asab, this preparation will obviously cost less, given the fact that patients face a chronic condition lasting for several months, which requires expensive medications otherwise. a possible limitation of this study is that although we evaluated the changes in bowel movements (determined based on frequency of defecations), other potentially related variables such as stool consistency, stool weight, and ease of defecation were not evaluated. in other words, measurements were limited to subjective reports, and objective evaluations, which would be time consuming and costly, were not feasible. however, these results would have clinical relevance as subjective complaints have a very important impact on the everyday life of patients, especially in those with functional disorders. another limitation was the short duration of follow up, which was a result of the preliminary nature of this study and should be improved in further studies that could target cost effectiveness as well. this study provides evidence for the safe and effective use of lax - asab in treating chronic functional constipation. | functional constipation is a common clinical complaint of patients with unsatisfactory treatment outcome. we designed this study to evaluate the efficiency of a traditional herbal preparation (lax - asab) in treating chronic constipation. in this double - blind, randomized, placebo - controlled clinical trial, participants with chronic constipation (n = 48) were randomly selected to receive either the lax - asab powder (n = 24) or placebo (n = 24) on alternative days for 4 weeks. the lax - asab powder contains equal amounts of cassia angustifolia vahl. (xi y fn xi y), mentha piperita l. (h jio b h), zingiber officinale rosc. (shng jing), glycyrrhiza glabra l. (gn co). a total of 40 patients completed the study. we determined the severity of constipation based on defecation frequency (per week) and defecation difficulties. of the total of 48 patients who participated, 40 completed the trial [24 men (60%), mean age, 21.0 4.2 years ; 16 women (40%), mean age, 20.1 4.3 years ]. the mean of weekly defecation frequency increased in both groups ; from 1.8 0.41 to 4.8 1.12 times in patients who received lax - asab and from 1.7 0.44 to 2.2 0.61 times in patients who received placebo. a time treatment interaction showed that this increase was significantly higher in the intervention group. defecation difficulties improved significantly more in patients who received lax - asab than patients who received placebo. there was no statistically significant difference between the two groups with regard to the side effects observed. this study confirms the efficacy and tolerability of an iranian herbal preparation, lax - asab, in treating patients with chronic functional constipation. |
cancer screening programmes internationally often experience challenges in maximizing uptake. in scotland, breast screening (via three - yearly mammography) and colorectal cancer screening (involving biennial faecal occult blood testing) have uptake rates of 74.5% and 54.9% respectively. improving screening uptake for these two programmes has been prioritized by the scottish government s detect cancer early campaign. the many reasons why people do not attend screening include fear of results and not liking the test. feedback from clinicians locally suggested that some invited participants have a specific medical reason for not being screened. this could mean that screening would be inappropriate (eg. if the patient is being treated palliatively for a terminal condition), or that greater surveillance is already being undertaken and so screening programme participation is not necessary. this study aims to quantify the percentage of patients in two scottish gp practices with medical reasons for non - participation in breast and bowel screening programmes. case notes were analyzed from 700 patients who had not participated in the previous round of breast or bowel screening. these patients were registered to one of two gp practices : practice one, located in an affluent area, with a scottish index of multiple deprivation score in the least deprived quintile with around 11,000 patients, and practice two, located in the middle quintile for deprivation, but where over half of the around 12,500 patients are in the two most deprived quintiles. the patients were randomly selected from non - participants in the 2012 bowel screening programme and from the most recent (20092012) breast screening programme. there is no clear guidance on what is considered a medical reason not to be invited for screening in scotland, so criteria were defined by local experts before data collection. for breast screening medical reasons were considered to be : bilateral mastectomy, recent mammogram (within the last 6 months), pregnancy, familial screening, terminal illness, lack of capacity to undergo screening, and cancer follow - up. for bowel screening, medical reasons were : no functioning bowel, recent colonoscopy, familial screening, terminal illness, lack of capacity to undergo screening (ie. patients who had moved or died were excluded because the electronic notes available were not adequate to determine if there was a medical reason for non - participation. of the 700 non - respondents, 69 patients had a medical reason for not participating (table 1). of those with a medical reason, the largest two groups for both breast and bowel screening were current follow - up for breast cancer with 8.86% (95% ci : 6.10, 12.3) and recent investigation (mammogram) with 6.57% (95% ci : 4.21, 9.70). other reasons accounted for a much smaller percentage (from 0.29% to 1.14%). the largest group in each screening programme was no medical reason, with 82.6% (95% ci : 78.2, 86.4) breast screening non - participants and 97.7% (95% ci : 95.6, 99.0) of bowel screening non - participants. table 1.medical reasons for non - participation in the scottish 20092012 breast screening and 2012 bowel screening in a random sample of non - participants in two gp practices in the edinburgh and the surrounding area. total n = 700.breastboweln%n%no medical reason28982.6 (95% ci : 78.2, 86.4)34297.7 (95% ci : 95.6, 99.0)hospital follow up318.86 (95% ci : 6.10, 12.3)41.14 (95% ci : 0.31, 2.90)recent investigation236.57 (95% ci : 4.21, 9.70)20.57 (95% ci : 0.07, 2.05)familial screening41.14 (95% ci : 0.31, 2.90)00previous surgery20.57 (95% ci : 0.07, 2.05)10.29 (95% ci : 0.01, 1.58)lack of capacity to undergo screening10.29 (95% ci : 0.01, 1.58)10.29 (95% ci : 0.01, 1.58)terminal illness0000total350350a = either colonoscopy or mammogram within 6 months. c = patients without capacity to decide to participate in screening, whose healthcare team concluded not being screened is in their best interest. medical reasons for non - participation in the scottish 20092012 breast screening and 2012 bowel screening in a random sample of non - participants in two gp practices in the edinburgh and the surrounding area. n = 700. a = either colonoscopy or mammogram within 6 months c = patients without capacity to decide to participate in screening, whose healthcare team concluded not being screened is in their best interest. this study demonstrated that there are patients who have medical reasons that mean it may not be appropriate for them to be invited for either breast or bowel screening. there was a much greater number of patients who were potentially inappropriately invited for screening amongst breast cancer screening patients (17.4%) compared with bowel cancer screening patients (2.3%). these figures are less disparate when considering that 26.5% of those invited do not attend breast screening compared with 45.1% for bowel screening. patients with a medical reason for not participating represent 4.6% of the total number of people invited for breast screening and 1.04% for bowel screening. this is the first study to quantify the number of patients with a medical reason for not participating in either breast or bowel cancer screening. a previous study in birmingham, aiming to increase breast cancer screening uptake, identified 548 persistent non - attenders and invited them to attend for screening, of whom 482 gave a response. it was found that eight women (1.66%) had recently had a mammogram and three (0.62%) were under care for other conditions. these figures are not directly comparable with the estimated 8.86% of non - participants in our study who were still in hospital follow - up and 6.57% had recently had a mammogram, most likely reflecting differences in study design. our study was in a small population and would need to be replicated using a larger sample of representative practices across scotland to gain more meaningful results. however, it establishes the principle that non - participation for medical reasons could be important and require further investigation. despite this study s limitations, the results are of high policy relevance. identifying patients who should not be invited for breast and bowel screening would allow more accurate monitoring of the effect of interventions to increase attendance. it is also important to consider the psychological impact invitation for screening could have on patients with medical reasons for non - participation, and to reduce the risk of this occurring as much as possible. clear guidelines on medical reasons that mean a patient should not be invited for screening are necessary. currently only patients fitting very strict exclusion criteria (ie. have had a bilateral mastectomy or are terminally ill) are not invited for breast screening in scotland. if the details of patients being followed - up for breast cancer, or patients who have recently had a mammogram could be sent to the breast - screening centres, 78% of patients deemed to have been inappropriately invited for breast screening in this study would not have been invited. this study demonstrates that patients with medical reasons for not being screened are present in both the breast and bowel screening programmes in two gp practices in edinburgh and the surrounding area. potential to prevent inappropriate offers for screening exists, but further work is needed to calculate the extent to which medical reasons account for screening non - participation in a larger representative population. svk is funded by the chief scientist office at the scottish health directorates as part of the evaluating social interventions programme at the mrc / cso social and public health sciences unit (mc_u130059812 and mc_uu_12017/4). | objectiveincreasing uptake of cancer screening is a priority for health systems internationally, however, some patients may not attend because they are undergoing active treatment for the cancer of interest or have other medical reasons that mean participation would be inappropriate. this study aims to quantify the proportion of non - participants who have a medical reason for not attending cancer screening.methodsmedical reasons for not participating in breast and bowel screening were defined a priori on the basis of a literature review and expert opinion. the notes of 700 patients at two gp practices in scotland were reviewed, to ascertain the prevalence of medical reasons amongst non - participants. simple proportions and confidence intervals were calculated.results17.4% of breast and 2.3% of bowel screening non - participants had a medical reason to not participate. the two most common reasons were previous breast cancer follow up (8.86%) and recent mammogram (6.57%).conclusionthese patients may not benefit from screening while also being distressed by receiving an invitation. this issue also makes accurate monitoring and target - setting for improving uptake difficult. further work is needed to estimate robustly the extent to which medical reasons account for screening non - participation in a larger population. |
rectal cancer accounts for approximately 30% of colorectal cancers and is a leading cause of cancer death worldwide. the introduction of total mesorectal excision (tme) and preoperative chemoradiotherapy (crt) has dramatically decreased the local recurrence rates and improved survival in rectal cancer patients. however, these improvements have been relatively modest for patients with very low rectal cancer, compared with those with mid - to - upper rectal cancer. this poor prognosis of low rectal cancer is mainly ascribed to the insufficient resection plane of the current abdominoperineal resection (arp) technique, which is still regarded as the standard treatment for low rectal cancer.indeed, many studies have shown that apr is associated with a high frequency of circumferential resection margin (crm) involvement and greater tumor perforation. however, it is still unknown whether the worse outcome in patients undergoing apr rather than restorative resection is related to tumor biology, surgical, and/or surgeon - specific technique, or patient factors. with these variable confounding factors, the lack of standardization of surgery causes a wide variation in apr rates depending on the geographical area or period over which the study was conducted. for example, in the dutch trial that included 27% of tumors located within 5 cm of the anal verge, apr was performed after preoperative crt in 28% of cases, suggesting that most low rectal cancers were treated by apr. the heterogeneous surgical treatment of low rectal cancer is also apparent in a united states - based study in which the rate of apr varied between 6% and 100%. despite these wide variations, the use of apr has been consistently declining worldwide. this may be accounted for in part by the growing use of intersphincteric resection (isr) as an alternative to apr. isr is usually combined with preoperative crt and is used in cases where a stapling technique can not be applied. the invasion to the external sphincter is currently considered an absolute oncological indication for apr. in this regard, the assessment of tumor depth is very important and influences the selection of the surgical method in low rectal cancer. although magnetic resonance imaging (mri) has shown the best accuracy for preoperative staging, restaging mri (postchemoradiation mri) is not as accurate as preoperative mri due to the difficulty in assessing diffuse fibrotic changes in the initial tumor area. in this clinical setting, an important, but unanswered, question is whether apr of a low rectal cancer after crt has different oncological outcomes to isr, especially in tumors for which it is virtually impossible to define a safe resection plane. hence, we conducted this study to compare the clinicopathologic factors and oncologic outcome in patients with locally advanced low rectal cancers who were treated using preoperative crt, followed by either apr or isr. in addition, in order to minimize the selection bias described above, the propensity score matching (psm) was used. between 2006 and 2011, 202 consecutive patients who underwent apr or isr after receiving neoadjuvant crt for locally advanced (radiological t3 - 4 and/or n+) rectal cancer were enrolled in this study. cancer was staged using pelvic mri (n = 144) and erus (n = 58) to determine the extent of local disease at presentation. the median time between the end of neoadjuvant crt and the restaging mri was 32 days (range, 2237 days) and was performed in 198 patients. abdominopelvic computed tomography (ct), chest ct or x - ray imaging, and positron emission tomography (pet)-ct were used to determine the extent of extrapelvic disease. the location of the tumor was defined as the distance from its lowest margin to the anal verge as measured by rigid sigmoidoscopy. briefly, a total dose of 5040 gy in 25 fractions of 180 cgy / d over 5 weeks was delivered. chemotherapy consisted of continuous intravenous infusion of 5-fluorouracil (5-fu ; 425 mg / m / d) and leucovorin (20 mg / m / d) during the first and fifth weeks of radiotherapy. one hundred ninety - three patients (95.5%) received postoperative chemotherapy, the majority (81.8%) of whom were administered 5-fu / leucovorin, while the others (18.2%) received 5-fu / leucovorin with either oxaliplatin or irinotecan, or oral capecitabine. experienced surgeons performed radical oncological surgery, including total mesorectal excision, high vascular ligation (inferior mesenteric artery and vein), and en bloc resection of adjacent involved organs, 6 to 8 weeks following the completion of crt. although the decision to perform sphincter - preserving surgery was based on a variety of clinical factors (distance from the anal verge, response to neoadjuvant treatment, preoperative anal sphincter function, and the patient 's preference), this procedure was not used when the tumor had invaded the external sphincter. for apr, transabdominalmesorectal excision was performed in the same manner as isr down to the pelvic floor, and the perineal resection was then started with dissection in the ischiorectal space, and completed with en bloc resection of the internal and external sphincter complex and rectum altogether. rectal tumors were staged by 2 gastrointestinal pathologists on the basis of the final pathological features, and in accordance with the seventh uicc tumor - node - metastasis (tnm) staging system. the circumferential resection margin (crm) was considered positive if microscopic tumor was identified within 1 mm of the surgical resection margin. tumor regression grade (trg) was defined on the basis of the ratio of fibrosis to residual cancer and was scored as follows ; grade 0, no regression ; grade 1, minor regression (dominant tumor mass with obvious fibrosis in 25% or less of the tumor mass) ; grade 2, moderate regression (26% fibrosis < 50%) ; grade 3 (fibrosis 50%) ; and grade 4, total regression (no viable tumor cells, fibrotic mass only). patients were followed up at 3-month intervals for 2 years, then at 6-month intervals for the next 3 years, and once annually thereafter. follow - up examinations were conducted on a semiannual basis or when disease recurrence was suspected, and included physical examination, serum carcinoembryonic antigen (cea) assay, chest x - ray or ct, abdomino - pelvic ct or mri, and colonoscopy. pet - ct was selectively used when recurrence was strongly suspected based on ct or mri findings. the main pattern of recurrence was recorded as the first site of detectable lesion during the follow - up period. to identify the factors associated with each surgical method in the entire cohort, univariate analysis was performed, consisting of the test for comparing proportions and the t test for comparing continuous variables. after comparing the demographic data between theses 2 groups, we performed propensity analysis using logistic regression analysis to compensate for potential baseline confounding variables. subsequently, these groups were matched on a 1:1 basis using the nearest neighbor matching method. the kaplan meier method was used to calculate cumulative recurrence rates and the log - rank test was used to compare survival between groups. variables with statistical significance (p < 0.1) in univariate analysis were further analyzed using the multivariate cox forward stepwise logistic regression model. statistical analyses were carried out using the spss statistical package (version 21.0 ; spss inc, chicago, il) and r 2.14.1. to identify the factors associated with each surgical method in the entire cohort, univariate analysis was performed, consisting of the test for comparing proportions and the t test for comparing continuous variables. after comparing the demographic data between theses 2 groups, we performed propensity analysis using logistic regression analysis to compensate for potential baseline confounding variables. subsequently, these groups were matched on a 1:1 basis using the nearest neighbor matching method. the kaplan meier method was used to calculate cumulative recurrence rates and the log - rank test was used to compare survival between groups. variables with statistical significance (p < 0.1) in univariate analysis were further analyzed using the multivariate cox forward stepwise logistic regression model. statistical analyses were carried out using the spss statistical package (version 21.0 ; spss inc, chicago, il) and r 2.14.1. a total of 202 patients who were followed up for a median period of 45.3 months (range, 585.2 months) were eligible for the present analysis, and of these, 40 (19.8%) underwent apr and 162 (80.2%) required isr. before psm, there were considerable imbalances in the patients demographical characteristics between the apr and isr groups : patients selected for apr tended to be older than those in the isr group, and patients in the apr group had tumors that were closer to the anal verge and larger than those in the isr group, which was reflected in more extensive circumferential lumen involvement. in addition to these clinical parameters, patients undergoing apr tended to have significantly more adverse pathologic predictors, such as poorly differentiated tumor and perineural involvement. positive crms were also more frequently identified in patients undergoing apr compared with isr, although this difference was not significant (17.5% vs 6.2%, p = 0.172) (table 1). tumor perforation was observed in a total of 8 patients : 5 (3.1%) in the isr group and 3 (7.5%) in the apr group. multivariate logistic regression analysis of the preoperatively determined variables (age, sex, asa score, yct and n stage, distance from the anal verge, tumor size, and circumferential extent) revealed that surgeons more frequently performed apr in patients who were older and had a tumor with extensive luminal involvement and/or was closer to the anal verge (table 2). in addition to these 3 variables, pathologic t and n categories, crm, and the number of lymph nodes retrieved were all assessed by constructing 1:1 matched - pairs and, after matching, the 2 groups were balanced with the exception of maximal tumor size (p = 0.01). comparison of patients characteristics before and after propensity score matching logistic regression of treatment selection (propensity) for apr as opposed to isr a total of 202 patients who were followed up for a median period of 45.3 months (range, 585.2 months) were eligible for the present analysis, and of these, 40 (19.8%) underwent apr and 162 (80.2%) required isr. before psm, there were considerable imbalances in the patients demographical characteristics between the apr and isr groups : patients selected for apr tended to be older than those in the isr group, and patients in the apr group had tumors that were closer to the anal verge and larger than those in the isr group, which was reflected in more extensive circumferential lumen involvement. in addition to these clinical parameters, patients undergoing apr tended to have significantly more adverse pathologic predictors, such as poorly differentiated tumor and perineural involvement. positive crms were also more frequently identified in patients undergoing apr compared with isr, although this difference was not significant (17.5% vs 6.2%, p = 0.172) (table 1). tumor perforation was observed in a total of 8 patients : 5 (3.1%) in the isr group and 3 (7.5%) in the apr group. multivariate logistic regression analysis of the preoperatively determined variables (age, sex, asa score, yct and n stage, distance from the anal verge, tumor size, and circumferential extent) revealed that surgeons more frequently performed apr in patients who were older and had a tumor with extensive luminal involvement and/or was closer to the anal verge (table 2). in addition to these 3 variables, pathologic t and n categories, crm, and the number of lymph nodes retrieved were all assessed by constructing 1:1 matched - pairs and, after matching, the 2 groups were balanced with the exception of maximal tumor size (p = 0.01). comparison of patients characteristics before and after propensity score matching logistic regression of treatment selection (propensity) for apr as opposed to isr the actuarial 5-year local recurrence - free survival and distant metastases - free survival rates for the whole cohort were 85.3% and 71.1%, respectively (table 3). patients undergoing apr had a higher 5-year local recurrence (p < 0.001) and distant metastasis rate (p = 0.01) (figure 1). it was particularly noteworthy that the higher local recurrence rate for apr persisted even after psm (table 3), and these findings were verified in the multivariate analyses not only for the entire cohort but also after psm (table 4). in addition, patients with advanced tumors, as assessed by restaging mri and luminal circumferential involvement, suffered local recurrence significantly more frequently if they had been treated using apr rather than isr (figure 2). impact of different clinical and pathologic factors on oncologic outcome before and after propensity score matching kaplan meier curves depicting distant metastasis - free survival and local recurrence - free survival according to the surgery type. multivariate analysis for metastases - free and local recurrence - free survival after chemoradiotherapy and rectal cancer resection kaplan meier curves depicting local recurrence - free survival of patients who underwent abdominoperineal resection (apr) or intersphincteric resection (isr). local recurrence rates with respect to luminal circumferential involvement ; a < 50%, b 50%. local recurrence rates with respect to restaging magnetic resonance image findings this study in patients with low rectal cancer treated with either apr or isr after preoperative crt shows that local failure is more common among the former, even after correcting for significant biases between these 2 groups. furthermore, this difference in local recurrence was more pronounced among patients with advanced rectal cancers, as assessed preoperatively by their depth and degree of luminal invasion. as a result, in this subgroup, a more radical operation rather than conventional apr should be considered, if it is not possible to preserve the sphincter. our findings generally agree with previous studies, in which patients undergoing apr more frequently suffered local recurrence compared with patients undergoing restorative surgery. it has also been suggested that inadequate excision, resulting in a greater crm involvement and a less intact tme plane, is a major determinate of outcome. nagtegaal reported that local recurrence rates were higher among patients with positive crms, regardless of the surgical technique used, and a more positive margin was present in patients undergoing apr (30.4%) compared with anterior resection (ar) (10.7%, p = 0.01). on the basis of these findings, they attributed the poor oncologic outcome of the patients who underwent apr to an insufficient resection plane, and concluded that the current form of apr is a nonradical surgery. however, patients who had a positive crm had a 5-year local recurrence rate of 30.4% after apr and 17.1% after ar. it is possible, therefore, that other factors may lead surgeons to select apr and are responsible to some extent for the higher local recurrence rates. in a similar setting, reshef demonstrated that patients had a worse outcome after apr rather than ar even in the absence of crm. taken together, although technical factors such as crm involvement are important and obtaining a clear crm is critical in reducing the risk of local recurrence, these findings suggest that there are also other factors influencing local control. indeed, reshef expressed their opinion that matching the apr and restorative surgery groups for tumor - specific factors, patient factors, and technical factors would be necessary, and other previous studies were also not free from this type of selection bias. patients undergoing apr were on average older, had a higher asa score, and a lower mean tumor distance from the anal verge. in addition, even adverse histologic features such as worse tumor differentiation, higher pathologic t stage, and a greater frequency of lymph node involvement were more common in patients undergoing apr. this is supported by a study comparing the use of apr and isr following preoperative crt, in which the former was performed more frequently for patients showing a lesser degree of tumor regression (p = 0.01). to our knowledge, our study is the first report applying psm to assess the oncologic outcome of apr compared with isr following the neoadjuvant crt. we selected variables that were considered to be important factors based on the findings of previous studies, or our own multivariate analysis for this matching. patients factors (age and sex), technical factors (crm and the number of lymph nodes retrieved), and tumor factors (pathologic t and n categories, distance from anal verge, extent of luminal circumference, tumor differentiation, and perineural invasion) were selected and optimally adjusted. after compensating for bias in this way, patients who underwent apr still had significantly worse local control. we therefore suggest that the current apr method is insufficient to achieve local disease eradication when compared with isr that does achieve an acceptable local recurrence rate, even for advanced stage disease. apr may thus ultimately be modified to more extensive surgery, especially for advanced disease. theoretically, the isr technique or a modified version of it could allow a sphincter - preserving operation for the majority of patients who underwent apr for low rectal cancer, even if the tumor invades the anal canal. in agreement with these reports, 305 (88.4%) patients underwent sphincter preserving surgery (143 ; lar, 162 ; isr) after preoperative crt in our study. furthermore, the local recurrence rate remains within a favorable range regardless of tumor stage in patients who undergo isr, despite the current lack of consensus on the best type of surgery for patients with very low rectal cancer. surgeons would prefer a stapled anastomosis if they could avoid dissecting some, or all of the anal sphincter. however, as stated above, it was even more difficult to decide whether to use apr and isr for patients who have undergone radiotherapy. currently, the invasion of the sphincter muscle or an undifferentiated tumor with aggressive characteristics is regarded as an absolute indication for apr. base on this guideline, relatively few patients actually undergo apr, especially as sphincter invasion is rare above the anal canal. holzer reported that the external anal sphincter was infiltrated by cancer in 2 (5%) of 40 patients with histologically verified adenocarcinoma of the lower rectum but without evidence of distant metastases. similarly, demonstrated that only 2 (2.7%) of 75 patients in whom the lowest edge of the tumor was located more than 2 cm above the dentate line showed external sphincter invasion. in this present study, we also found that only 2 (5%) of 40 patients treated with apr showed pathological sphincter invasion. therefore, when the tumor is located more than 2 cm from the dentate line, the decision as to which surgical method should be used is based not on the likely oncologic outcomes but is instead a functional problem. as a result, the decision as to which surgical technique will provide the best oncological outcome is difficult when the tumor is located in the anal canal or within 2 cm of the dentate line. in this situation, when the tumor is assessed as ymrt3 or involves a luminal circumference greater than 50%, more radical surgery, such as cylindrical apr will be needed. in this present study, we demonstrated that the ymrt category and the extent of luminal circumference could be used to help decide whether more radical surgery is required. mri has been shown to be the best modality for assessing rectal tumor invasion, and for selecting the optimal surgical method. some authors have drawn attention to the limited accuracy of restaging mri due to post - therapeutic inflammation and fibrosis. in fact, the discrimination is more prominent when analyzed using the ypt rather than the ymrt category ; the 5-year local recurrence - free survival rates in patients who underwent apr and isr, and who were confirmed to have tumor stage ypt3 - 4, were 81.0% and 39.5%, respectively (p = 0.01), but they were 83.3% and 50.2%, respectively, when analyzed in patients with ymrt3 - 4 tumors (p = 0.02). the number of cancer cells in post - therapeutic inflammation and fibrosis is usually overestimated. in a recently published meta - analysis evaluating the accuracy of restaging mri it was shown that most t0 rectal cancers could not be accurately identified, although only a few were misdiagnosed (sensitivity ; 15.3%, specificity ; 94.6%). in contrast, t3 - 4 lesions were correctly diagnosed, although t2 lesions were apt to be over - staged to the t3 - 4 category. however, it is still a valuable diagnostic method as it can prevent under - treatment. moreover, mr sensitivity and specificity for crm involvement were 85.4% and 80.0%, respectively. this means that restaging mri is a critical tool for the treatment of locally advanced rectal cancer. finally, as pointed out by rullier, careful digital rectal examination when the anal sphincter is fully relaxed would be more helpful than any other preoperative diagnostic tool. we demonstrated that the local recurrence rate in patients with a ypt3 - 4 tumor was similar to that of the whole patient population (86.1% vs 91.0%). as discussed above, invasion of the external sphincter was rare, and some of the factors that may have influenced the choice of surgical method could not always be determined. second, it can be argued this study includes too few patients, and in fact, although tumor perforation is regarded as an important predictive factor for local recurrence following resection of low rectal cancer, we could not fully assess its impact in our study due to its rarity. finally, more refined mri predictors, such as crm involvement, were not used.in a future study, a more detailed and informative analysis based on preoperative mri may provide more individualized treatment guidelines. in conclusion, apr is still essential for the treatment of low rectal cancer following preoperative crt. however, we found that there was a significantly worse outcome after apr compared with isr in terms of local disease eradication, even after risk adjustment in propensity score analyses. a more detailed preoperative diagnostic guideline is needed for the individualized treatment of patients with very low rectal cancer after preoperative crt. | abstractdue to selection bias, the oncologic outcomes of apr and isr have not been compared in an interpretable manner, especially in patients treated with preoperative crt. to assess factors influencing oncologic outcomes in patients with locally advanced low rectal cancer treated with preoperative chemoradiotherapy (crt) followed by abdominoperineal resection (apr) or intersphincteric resection (isr).between 2006 and 2011, 202 consecutive patients who underwent apr or isr after preoperative crt for locally advanced rectal cancer were enrolled in this study. the median follow - up period was 45.3 months (range : 585.2 months). multivariate and propensity score matching (psm) analyses were performed to reduce selection bias.of the 202 patients, 40 patients (19.8%) underwent apr and 162 (80.2%) required isr. in unadjusted analysis, patients undergoing apr had a higher 5-year local recurrence (p < 0.001) and distant metastasis rate (p = 0.01), respectively. however, the higher local recurrence rate for apr persisted even after psm, and these findings were verified in the multivariate analyses. moreover, patients with advanced tumors, as assessed by restaging magnetic resonance imaging and luminal circumferential involvement, had a significantly higher local recurrence rate after apr compared with isr.this is the first psm based analysis providing evidence of a worse oncologic outcome after apr compared with isr. in addition, the results of the subgroup analysis suggest that a more radical modification of the current apr is required in cases of advanced cancer. |
the micronucleus (mn) test as a short and easy assay for the evaluation of the effect of mutagenic action (schmid 1975) is a suitable substitution for the time - consuming analysis chromosome aberrations (countryman and heddle 1976). recently, the mn test with fluorescent stains proved to be more sensitive in detecting small micronuclei compared to the mn test using traditional methods, such as feulgen s reaction (dias. 2005). therefore, the use of fluorescence has been proposed as an accurate method for the detection of micronuclei. interphase cytogenetics using fluorescence in situ hybridization (fish) with specific dna probes not only provides a sensitive tool to detect small chromosome rearrangements, but also offers the possibility of better understanding the origin of the micronuclei (maluszynska. the micronucleus test applied with fish employing chromosome or chromosome region specific dna probes is widely applied in human cytogenetics, toxicological and radiation studies. in plant cytogenetics the repetitive dna sequences recognizing a specific chromosome region, such as centromeres and telomeres, as well as rdna are used most extensively as probes for fish for plant chromosomes (bolzan and bianchi 2006). there is only one morphological type of micronuclei which differs in size, however they can originate from different chromosomes or chromosome fragments. it was proved that the mn assay using fish with telomeric and/or centromeric dna sequences is able to detect the clastogenic or aneugenic effect (acar.. studies concerning the evaluation of the origin of chemically induced micronuclei by maleic hydrazide (mh) and n - nitroso - n - methylurea (mnu) in barley cells were previously done by our group (juchimiuk. the cytogenetic effects of a gamma ray in the root tips of many plant species were measured previously (evans and hof 1975). the specific localization of the radiation induced chromosome aberrations using traditional chromosome staining has been the subject of studies in barley and other plant species (natarajan and ahnstrom 1970 ; kunzel. a gamma ray, causing breaks in one or two chains of dna, is routinely used in plant mutagenesis (hagberg and persson 1968) and most barley mutant varieties were developed by applying this type of radiation. here, we quantitatively analyze the gamma ray - induced micronuclei in order to examine the involvement of specific chromosomes or chromosome fragments in their formation. fish with different dna probes (5s and 25s rdna, telomere- and centromere - specific dna sequences) was applied in the analysis of the micronuclei. the next aim was a comparison of the possible origin of the micronuclei induced by physical and chemical treatment (mh and mnu) in hordeum vulgare cells. vulgare, a model plant, was chosen as the majority of large chromosomes can be distinguished because of the specific localization of rdna. barley (hordeum vulgare l., 2n = 14) seeds of the cv. the irradiation was carried out in the international atomic energy agency, seibersdorf laboratory, austria. after irradiation the seeds were presoaked in distilled water for 8 h and germinated in petri dishes at 21c in the dark. roots of m1 seedlings were used as the source of meristems for the investigations of aberrations. the material was fixed in ethanol : glacial acetic acid (3:1) at 60 h of germination. fluorescence in situ hybridization was applied according to the method described by maluszynska and heslop - harrison (1991) with some minor modifications. four dna probes were used in two fish experiments : clone ht100.3 containing 30 copies of arabidopsis - type telomeric repeats ((tttaggg)n) labelled with rhodamine-5-dutp by pcr (roche), clone ccs1 containing a part of the centromeric retrotranspozons isolated from brachypodium sylvaticum (aragon - alcaide. 2000) labelled with digoxigenin-11-dutp using pcr (roche), clone pta-794 containing 5s rdna from triticum aestivum (gerlach and dyer 1980 ; hasterok. 2002) labelled with rhodamine-5-dutp using a pcr labeling kit (amersham life sciences), and clone pclai containing 25s rdna isolated from arabidopsis thaliana (unfriend and gruendler 1990) labelled with digoxigenin-11-dutp by nick translation (roche). prior to fish, pretreatment with rnase, washing, dehydration of the chromosome preparations were applied as in a previous work of our group (juchimiuk. the hybridization mixture containing 2.5 g / ml of labelled dna, 50% formamide, 10% dextran sulphate and 0.1 mg/l salmon testes dna in 2 ssc was denaturated at 75c for 10 min and immediately placed on ice for a few minutes. the hybridization mixture (38 l) was added to the chromosome preparations and covered with a plastic coverslip. the chromosomes and dna probes were denatured for 5 min at 70c on a hot plate (hybaid thermal cycler pcr in situ). hybridization was carried out at 37c in a moist chamber for 20 h. after hybridization, slides were subsequently washed for 4 min in 2 ssc at 42c, 2 4 min in 0.1 ssc at 42c, 3 3 min in 2 ssc at 42c, 3 3 min in 2 ssc at room temperature and for 5 min in 0.2% tween in 4 ssc at room temperature. the digoxigenin - labelled probe was detected using fitc - conjugated anti - digoxigenin antibodies (roche) and then the signal was amplified by a fitc - conjugated secondary antibody (dako). after three washes for 8 min in 0.2% tween in 4 ssc at 37c and dehydration in the ethanol series, the slides were mounted in a vectashield medium (vector laboratories) containing 6 g/ ml dapi (sigma). images were captured using a hamamatsu c5810 ccd camera and processed using adobe photoshop 4.0. the frequency of micronuclei with specific dna signals and without signals was calculated. for each experimental group 100 cells with micronuclei analysed on three slides, each made from three meristems, were evaluated. the total frequencies of dapi - stained micronuclei in 3000 interphase cells were analyzed on the same slides before the fish experiments. barley (hordeum vulgare l., 2n = 14) seeds of the cv. the irradiation was carried out in the international atomic energy agency, seibersdorf laboratory, austria. after irradiation the seeds were presoaked in distilled water for 8 h and germinated in petri dishes at 21c in the dark. roots of m1 seedlings were used as the source of meristems for the investigations of aberrations. the material was fixed in ethanol : glacial acetic acid (3:1) at 60 h of germination. fluorescence in situ hybridization was applied according to the method described by maluszynska and heslop - harrison (1991) with some minor modifications. four dna probes were used in two fish experiments : clone ht100.3 containing 30 copies of arabidopsis - type telomeric repeats ((tttaggg)n) labelled with rhodamine-5-dutp by pcr (roche), clone ccs1 containing a part of the centromeric retrotranspozons isolated from brachypodium sylvaticum (aragon - alcaide. 2000) labelled with digoxigenin-11-dutp using pcr (roche), clone pta-794 containing 5s rdna from triticum aestivum (gerlach and dyer 1980 ; hasterok. 2002) labelled with rhodamine-5-dutp using a pcr labeling kit (amersham life sciences), and clone pclai containing 25s rdna isolated from arabidopsis thaliana (unfriend and gruendler 1990) labelled with digoxigenin-11-dutp by nick translation (roche). prior to fish, pretreatment with rnase, washing, dehydration of the chromosome preparations were applied as in a previous work of our group (juchimiuk. the hybridization mixture containing 2.5 g / ml of labelled dna, 50% formamide, 10% dextran sulphate and 0.1 mg/l salmon testes dna in 2 ssc was denaturated at 75c for 10 min and immediately placed on ice for a few minutes. the hybridization mixture (38 l) was added to the chromosome preparations and covered with a plastic coverslip. the chromosomes and dna probes were denatured for 5 min at 70c on a hot plate (hybaid thermal cycler pcr in situ). hybridization was carried out at 37c in a moist chamber for 20 h. after hybridization, slides were subsequently washed for 4 min in 2 ssc at 42c, 2 4 min in 0.1 ssc at 42c, 3 3 min in 2 ssc at 42c, 3 3 min in 2 ssc at room temperature and for 5 min in 0.2% tween in 4 ssc at room temperature. the digoxigenin - labelled probe was detected using fitc - conjugated anti - digoxigenin antibodies (roche) and then the signal was amplified by a fitc - conjugated secondary antibody (dako). after three washes for 8 min in 0.2% tween in 4 ssc at 37c and dehydration in the ethanol series, the slides were mounted in a vectashield medium (vector laboratories) containing 6 g/ ml dapi (sigma). images were captured using a hamamatsu c5810 ccd camera and processed using adobe photoshop 4.0. the frequency of micronuclei with specific dna signals and without signals was calculated. for each experimental group 100 cells with micronuclei analysed on three slides, each made from three meristems, were evaluated. the total frequencies of dapi - stained micronuclei in 3000 interphase cells were analyzed on the same slides before the fish experiments. dapi staining applied before fish experiment allowed analysis of the total frequency of gamma ray - induced micronuclei in barley root meristematic cells (fig. 1). the frequencies of micronuclei are shown for 60 h of postincubation as they were the highest during this time. the frequency of micronuclei after gamma irradiation varied from 4.5% to 18.2%, depending on dose and postincubation time (data not presented). 225 gy of gamma ray was a stronger inducer of micronuclei than 175 gy. no published data on gamma ray - induced micronuclei are known for barley (only chromosome aberrations). in the present study, the frequencies of gamma ray - induced micronuclei in barley cells showed that they were higher than the frequencies of chemically (mh- and mnu-) induced micronuclei in applied and estimated doses earlier (juchimiuk. 2007). 1frequencies of gamma ray - induced micronuclei in meristematic cells of barley roots after 60 h of postincubation frequencies of gamma ray - induced micronuclei in meristematic cells of barley roots after 60 h of postincubation fish was used for a detailed characterization of the micronuclei induced by gamma ray in barley cells. ht100.3 and ccs1 were used as probes in the first fish experiments, whereas in the second one 25s and 5s rdna were used. the analysis of the number of rdna signals in micronuclei makes the identification of individual chromosome / chromatid engaged in their formation possible (juchimiuk. this was possible due to the large number of rdna sites in this species : only one chromosome pair is characterized by a lack of rdna signals, two chromosome pairs with 25s rdna signals (nor chromosomes), four chromosomes pairs with 5s rdna (fig. 2). the application of fish with centromeric and telomeric probes allowed the question of whether interstitial or distal chromosome / chromatid fragments or whole chromosomes are involved in micronuclei formation to be answered. in this study micronuclei with different fish signals were observed. examples of interphase control and irradiated cells with signals of different dna sequences with the examples of probable chromosome origin of gamma ray - induced micronuclei are presented in figs. 3 and 4. it is well known that a gamma ray can produce chromosome and chromatid aberrations ; however, it is difficult to distinguish between them on the basis of the size of the signals of probes applied in interphase nuclei due to their three - dimensional structure. after the first fish experiment with ht100.3 and ccs1 as probes, the micronuclei with telomeric signals or both telomeric and centromeric signals were observed. 3b does not contain signals of the probes applied, and the chromosome origin of this micronuclei is unknown ; however, the involvement of the interstitial chromosome region in its formation might be confirmed. 3c f, because they possess only a telomere - specific dna, are examples of the involvement of a different number of acentric and distal chromosome fragments in their formation. g contains two telomeric and two centromeric signals and thus indicates its origin from one incomplete chromosome and one centric fragment, or from three chromosome fragments : two centric and one acentric terminal fragment. 3h has four telomeric signals and one centric signal and thus there are few possible origins of its complex origin : from one complete, laggard chromosome, chromosome without telomeres, acentric terminal fragment, or from 3 fragments including the centric one. surprisingly, the presence of micronuclei with telomeric and centromeric sequences proved that a gamma ray can cause spindle fibre defects. telomeric sequences - red, centromeric sequences - green, dapi staining - blue. control cell, without micronucleus (a) ; the cell with : micronucleus without specific signals (b), micronucleus with one telomere specific signal (c), micronucleus with two telomere specific signals (d), micronucleus with three telomere specific signals (e), micronucleus with four telomere specific signals (f), micronucleus with two telomere and two centromere specific signals (g), micronucleus with four telomere and one centromere specific signals (h). 5s rdna sequences - red, 25s rdna sequences - green, dapi staining - blue. control cell, without micronucleus (a) ; the cell with : micronucleus without specific signals (b), micronucleus with one 5s rdna specific signal (c), micronucleus with two 5s rdna specific signals (d), micronucleus with four 5s rdna specific signals (e), micronucleus with one 25s rdna specific signal (f), micronucleus with two 25s rdna specific signals (g), micronucleus with two 5s rdna and two 25s rdna specific signals (h). bars represent 10 m hordeum vulgare cv. start idiogram with the distribution of the rrna genes and chromosome numbering. 5s rdna red, 25s rdna green hordeum vulgare interphase cells and probable origin of micronuclei after gamma irradiation of seeds. telomeric sequences - red, centromeric sequences - green, dapi staining - blue. control cell, without micronucleus (a) ; the cell with : micronucleus without specific signals (b), micronucleus with one telomere specific signal (c), micronucleus with two telomere specific signals (d), micronucleus with three telomere specific signals (e), micronucleus with four telomere specific signals (f), micronucleus with two telomere and two centromere specific signals (g), micronucleus with four telomere and one centromere specific signals (h). bars represent 10 m hordeum vulgare interphase cells and probable origin of micronuclei after gamma irradiation of seeds. 5s rdna sequences - red, 25s rdna sequences - green, dapi staining - blue. control cell, without micronucleus (a) ; the cell with : micronucleus without specific signals (b), micronucleus with one 5s rdna specific signal (c), micronucleus with two 5s rdna specific signals (d), micronucleus with four 5s rdna specific signals (e), micronucleus with one 25s rdna specific signal (f), micronucleus with two 25s rdna specific signals (g), micronucleus with two 5s rdna and two 25s rdna specific signals (h). bars represent 10 m the use of rdna makes the analysis of the involvement of specific chromosomes and/or specific chromosome fragments in the micronuclei formation possible. the origin of the micronucleus in fig. 4b is unknown due to the lack of rdna signals : it could originate from an interstitial fragment (without rrna genes) of any chromosome or from whole chromosome no. 4c contains one signal of 5s rdna and it probably originated from the chromatid fragment with 5s rrna genes of chromosome no. 1, 2, 3 or 4. 4d that possesses two signals of 5s rdna is an example of an aberration formed after a double - chromatid break of one of the chromosome no. 1, 2, 3 or 4 or from single - chromatid breaks of two of these chromosomes. the presence of four signals of 5s rdna in the micronucleus indicates that two chromosomes from among chromosomes no. 1, 2, 3 or 4 might be involved in the formation of this aberration (fig. 4f clearly proved its origin from a whole chromatid or a chromatid fragment of chromosome no. 6 or 7. the total number of 25s rdna sites in this cell is five, indicating the probability of a duplication event of chromosome fragment including 25s rdna region. chromosome / chromosome fragments no. 6 or 7 and chromosome / chromosome fragments no. 1, 2, 3, or 4 may have created the micronucleus in fig. the applied doses of gamma ray did not influence the frequencies of micronuclei with particular signals (data not presented). figure 5 shows a lack of correlation between the postincubation times used in the study and the frequency of micronuclei with specific signals, thus all of the data obtained were pooled (diagrams). fish with telomeric and centromeric dna as probes showed that the micronuclei with telomeric dna were observed most frequently (81%), and rarely were micronuclei without signals (14%). only 5% of the gamma - induced micronuclei are characterized by the presence of both : telomeric and centromeric dna sequences. calculating the centromere signal - positive and centromere signal - negative micronuclei is well known in plant cells ; however, it is used more often in human cells (schuler. 1997 ; jovtchev. although, individual chromosome painting of plants is difficult, the rdna has become a useful chromosome marker for a few plant species, including arabidopsis and barley (weiss and maluszynska 2000). in this work applying rdna - fish to barley chromosomes enabled an analysis of the engaging of rrna - bearing chromosomes in micronuclei formation. the relatively high frequency (50%) of micronuclei including 5s rdna signals after hybridization and micronuclei without signals (34%) was found. 12% of micronuclei are characterized by the presence of 25s rdna sites, and only 4% with both rdna signals. therefore, even taking into consideration the higher number of 5s rdna - bearing chromosomes (5 pairs) than 25s rdna bearing chromosomes (2 pairs) in the barley diploid genome, it can be concluded that chromosomes no. 1, 2, 3 or 4 were involved in formation of gamma ray - induced chromosome aberrations more often than chromosomes no. 5, 6 or 7. theoretically, we can state that the mean percent of micronuclei per one pair of 5s rdna - bearing chromosomes is 12.5%, whereas per one pair of 25s rdna - bearing chromosomes only 6%. fish applied in a similar way in relation to the involvement of chromosomes with known markers was done in humans and has shown that different human chromosomes are not randomly involved in mn (guttenbach and schmidt 1994). 5the frequency of micronuclei with signals of specific dna sequences in barley cells after gamma irradiation in 36 h, 48 h, 60 h of postincubation. (a) fish with telomeric and centromeric dna as probes (b) fish with rdna as probes. the pie charts show data, which were pooled for all postincubation times the frequency of micronuclei with signals of specific dna sequences in barley cells after gamma irradiation in 36 h, 48 h, 60 h of postincubation. (a) fish with telomeric and centromeric dna as probes (b) fish with rdna as probes. the pie charts show data, which were pooled for all postincubation times a similar study concerning a comparison of the frequency of micronuclei with specific dna sequences was performed for mh- and nmu - treated barley cells (juchimiuk. a comparison of the possible origin of the micronuclei induced by physical (done in this work) and chemical treatment (mh and mnu) in cells of hordeum vulgare was possible. the comparison of the effect of mutagenic treatment showed that a gamma ray in applied doses induced micronuclei with almost a 2 times higher frequency than mh and mnu. however, gamma ray, mh and mnu caused terminal deletions most often and with a similar frequency in barley cells. (2002) reported that most mnu - induced micronuclei showed telomere - specific signals. surprisingly, no micronuclei with only centromere - specific signal were observed in the case of treatment with gamma ray, which indicates that large interstitial fragments including only centromeric dna are not generated by physical treatment, but formed by chemical treatment. the comparison showed that micronuclei without centromeric- and telomeric - specific fish signals, probably arising form interstitial fragments, were observed with similar frequencies (about 15%) after gamma ray treatment and after chemical treatment. it should be underlined that not all initially radiation - induced chromatin breaks are observed as they can remain open and rejoin with another break or restitute. our studies confirm earlier observations that some chromosomes and chromosome regions are more prone to the formation of aberrations than others (kumar and natarajan 1965 ; natarajan and ahnstrom 1970). it was confirmed that small chromosome regions in the median and distal arm positions were identified as preferred sites for translocation breakpoints induced by gamma radiation in barley (kunzel. many factors could be responsible for distributions of chromosome aberrations that are not random : interphase organization, the transcriptional activity of different chromosome regions and chromosome size (natarajan 2002). fish with rdna and ht100.3/ccs1 sequences as probes confirmed its usefulness in the characterization of micronuclei content, as well as in understanding and comparing the mechanisms of the actions of mutagens applied in genetic toxicology. based on the number of centromeric and telomeric signals in micronuclei the possible mechanism of gamma - ray induced micronuclei formation was stated : the micronuclei originated from the acentric fragments as a result of chromosome breakage or whole lagging chromosomes as a result of the dysfunction of the mitotic apparatus. an application of rdna as probes allowed it to be stated that 5s rdna - bearing chromosomes are involved in micronuclei formation more often than nor - bearing chromosomes. the recent work allowed the origin of physically- and chemically - induced micronuclei in barley cells to be compared. even though the mode of action of chemical and physical mutagens is different, the origin of micronuclei was most often from terminal fragments. molecular cytogenetics techniques gives us insight into the mechanisms of the formation of chromosome aberrations during genotoxicity studies. in future studies, the combination of multiple dna probes labelled with various fluorochromes can make fish a more powerful tool in plant mutagenesis. | a micronucleus test in combination with fluorescent in situ hybridization (fish) using telomere-, centromere - specific probes and 5s and 25s rdna was used for a detailed analysis of the effects of gamma ray irradiation on the root tip meristem cells of barley, hordeum vulgare (2n = 14). fish with four dna probes was used to examine the involvement of specific chromosomes or chromosome fragments in gamma ray - induced micronuclei formation and then to explain their origin. additionally, a comparison of the possible origin of the micronuclei induced by physical and chemical treatment : maleic hydrazide (mh) and n - nitroso - n - methylurea (mnu) was done. the micronuclei induced by gamma ray could originate from acentric fragments after chromosome breakage or from whole lagging chromosomes as a result of a dysfunction of the mitotic apparatus. no micronuclei containing only centromeric signals were found. an application of rdna as probes allowed it to be stated that 5s rdna bearing chromosomes are involved in micronuclei formation more often than nor chromosomes. this work allowed the origin of physically- and chemically - induced micronuclei in barley cells to be compared : the origin of micronuclei was most often from terminal fragments. fish confirmed its usefulness in the characterization of micronuclei content, as well as in understanding and comparing the mechanisms of the actions of mutagens applied in plant genotoxicity. |
tryptophan is essential for all mammalian systems, and tryptophan catabolism is regulated through the l - kynurenine pathway. the first, rate - limiting step of the kynurenine pathway is the oxidation of l - tryptophan to n - formylkynurenine (nfk, scheme 1a). this is an o2-dependent process and is catalyzed by one of two heme enzymes : tryptophan 2,3-dioxygenase (tdo) or indoleamine 2,3 dioxygenase (ido).(1) the mechanism of tryptophan oxidation is of essential interest from a clinical perspective because tryptophan catabolism generates a number of secondary metabolites such as 3-hydroxyanthranilic acid, anthranilic acid, 3-hydroxykynurenine, and quinolic acid that are implicated in a wide range of neurological disorders, cataract formation, and suppression of t cell proliferation. in this sense ido and tdo are emerging as important drug targets because they have the power to regulate the supply of tryptophan (trp) and other kynurenine pathway metabolites.(2) in consequence, the development of dioxygenase inhibitors is ongoing(3) (with some compounds, such as 1-methyl - tryptophan (me - trp)), in clinical trials.(4) (a) the reaction catalyzed by the heme dioxygenases. (b) left : previous proposal for the first step of the reaction mechanism, involving base - catalyzed abstraction. (c) proposed(1) criegee (top, solid line) and dioxetane (bottom, dashed line) reaction mechanisms for formation of n - formylkynurenine. despite years of investigation going back as far as the 1930s,(5) the reaction mechanism of the dioxygenases is still not clarified. early studies(6) concluded that the mechanism involved base - catalyzed deprotonation of the indole nh group as the initiating step (scheme 1b), and were based on an assignment of me - trp as an inhibitor. structural data are not consistent with the proposal since not all dioxygenases human indoleamine 2,3-dioxygenase being one example(7)contain an active site base (presumed to be a histidine). and, in cases where histidine is present, mutagenesis studies do not support an essential role for it. this led to an alternative proposal that did not require an obligate base (scheme 1b). however, both proposals (scheme 1b) are problematic because neither is consistent with the chemistry of indoles (which do not react by loss of the indole proton(11)). that deprotonation of the indole proton might be a red herring was first suggested(12) from computational work but the experimental evidence has so far been from mass spectrometry(13) which has shown that me - trp is not an inhibitor (as previously suggested(6)) but a slow substrate. since the indole proton is missing in me - trp (scheme 1a), the logical conclusion is that the mechanism can not involve its loss. the later parts of the mechanism leading to formation of nfk are, as yet, unclarified. the literature(1) describes either criegee or dioxetane rearrangements (scheme 1c), but there is no experimental evidence for either mechanism. recent resonance raman work(16) has suggested that the mechanism may proceed by sequential insertion of oxygen. in the present paper, we have examined product formation in a number of different heme dioxygenases under turnover conditions. we find evidence, from mass spectrometry data, for insertion of a single oxygen atom into the substrate. this is not consistent with widely cited(1) proposals for the concerted addition of both oxygen atoms. human tdo (htdo), human ido (hido) and x. campestris tdo (xtdo) were prepared as previously described. for lc ms experiments, enzyme (15 m), o2 and l - trp (1 mm, sigma) or 1-methyl - l - tryptophan (me - trp, 0.5 mm, aldrich) were incubated in a sealed vessel for varying amounts of time (10120 min), with conditions as previously described.(13) the reducing system in all cases was ascorbate / methylene blue, as for normal dioxygenase assays,(8) or in some cases with dithionite - reduced htdo with o2 and l - trp (1 mm) in 50 mm tris - hcl buffer, ph 8.0 (25.0 c). steady - state formation of nfk was monitored at 321 nm using a varian cary 50 probe uv reactions were terminated by the addition of 30% (v / v) trichloroacetic acid although control reactions were also carried out in which tca was not used. in all cases samples were centrifuged (13,000 rpm for 5 min in a microcentrifuge) to collect degraded protein and the supernatant was then filtered using a millipore 0.45 m filter at 25 c. for o2 experiments, all buffers and solutions were purged with nitrogen and then left to equilibrate in the glovebox for at least 2 h. samples for the o2-experiments were prepared by bubbling o2 (98%, ck gas products ltd.) into the assay mixture and the reaction initiated by the addition of enzyme. for reactions carried out in o - labeled water, all solutions were prepared using h2o (98%, ck gas products ltd.) and the assays carried out as outlined above. reactions were terminated by the addition of 30% (v / v) trichloroacetic acid as described above. lc ms analyses used a micromass quatro lc spectrometer and samples were run with acetonitrile / h2o (0.1% formic acid) as the solvent (in a ratio of 10:90 or 20:80) on a c18 column. the 3a - hydroxypyrroloindole-2-carboxylic acid compound, referred to as the cyclic amino acetal in this paper (see scheme 2, species y), was synthesized (> 95% purity, assessed by nmr) via dye - sensitized photooxygenation of l - tryptophan.(20) nmr and mass spectrometry data are given in the supporting information (si). the species labeled x and y have also been suggested(40) as intermediates in the prnb mechanism (see discussion). in our lc ms analyses looking for nfk (m / z = 237) formation in a number of dioxygenases, a peak eluting with m / z = 221 has been routinely observed in addition to the expected peak at m / z = 237. a representative example is shown in figure 1 for htdo, but similar data have been obtained for other bacterial (xtdo) and mammalian (hido) enzymes under a range of conditions. here, the products from the reaction of htdo with o2 and l - trp were examined using lc ms (figure 1a - d). using selected ion monitoring (figure 1a), the elution profile revealed a peak eluting with a mass at 221 (figure 1b). this is consistent with insertion of a single oxygen atom into the substrate (mr = 204 ; table s1, si). the observation of a peak at m / z = 221 did not depend on the reaction time (varied between 10 and 120 min), and is not a feature of the tca precipitation treatment since the equivalent peaks at m / z = 221 (and m / z = 237) are also observed when tca is not used. nor is it a contaminant from the adventitious reaction of trp with o2 under aerobic conditions, because no such peaks (either at m / z = 221 or m / z = 237) are observed for steady state assays obtained in the absence of enzyme. this is expected since trp is only reactive with o2 in the presence of a sensitizer ; in fact, this is used for the chemical synthesis of 3a - hydroxypyrroloindole-2-carboxylic acid below. samples of l - trp alone (i.e., l - trp in buffer under aerobic conditions) similarly showed no peaks at m / z = 221 or m / z = 237 (and nmr analysis of l - trp itself showed no peaks from impurities, data not shown). ms / ms analyses of the products obtained from the o2-dependent reaction of htdo with l - trp in the steady state. (a) elution profile for selected ion chromatogram with m / z = 221. corresponding positive esi mass spectrum for the product eluted at 5.72 min, showing a peak at m / z = 221. (c) elution profile for the same peak (at 5.60 min in this elution profile) and (d) corresponding ms / ms spectrum, showing the peak at 221 and a fragmentation pattern corresponding to the structure proposed in scheme 2. (e) positive esi mass spectrum for the product eluted at 7.02 min, showing a peak at m / z = 237 corresponding to nfk formation. for all enzymes examined, the intensity of the 221 peak is invariably lower than that for the 237 peak, but we have been unable to extract quantitative time profiles from the mass spectrometric analyses. we speculated that this peak may originate from initial conversion of the substrate to the corresponding 2,3-epoxide, which had been initially identified(12) by computational studies (but considered energetically unfavorable in the gas phase) and also implicated from resonance raman work.(16) the proposed 2,3-epoxide is a known compound but is unstable and undergoes facile conversion to the more stable cyclic aminoacetal (scheme 2). mass spectrometry data for this derivative are published,(21) including the expected fragmentation patterns in ms / ms experiments, which provides a useful control compound for the enzymatic turnover experiments. in lc ms / ms analyses of product formation as above, a peak eluting at the same position is observed (figure 1c) and with m / z = 221, and the fragmentation pattern, figure 1d, matches that predicted for the cyclic aminoacetal shown in scheme 2 (peak at 175 : loss of cooh ; peak at 203 : loss of h2o ; peak at 157 : loss of h2o and hcooh).(21) we thus assign the 221 peak detected in our mass spectrometry experiments as arising from initial formation of an epoxide intermediate, in which one atom of oxygen is inserted into the substrate during turnover, followed by conversion to the more stable amino acetal compound under the conditions of our experiments.(22) in these experiments, we can also isolate an ion with m / z = 237 (figure 1e), which is assigned as arising from nfk formation (mr= 236, table s1, si) by comparison with previous work.(13) to compare the mass spectrometry data above with the authentic 3a - hydroxypyrroloindole-2-carboxylic acid compound (scheme 2), we synthesized the compound (> 95% purity, assessed by nmr) via dye - sensitized photooxygenation of l - tryptophan.(20) the compound was isolated as a mixture of two diastereoisomers in a ratio of 2:1, and the structure confirmed by nmr analysis (see si). both isomers have a cis-5,5-ring junction with the major isomer having a cis - relationship between the hydroxyl and carboxylic acid groups (these assignments were confirmed by noe experiments). lc ms analysis of this synthesized sample indicated that it had the same retention time and the fragmentation pattern (see supporting information) as the 221 peak observed in the htdo reactions. in control experiments, we found that the purified cyclic aminoacetal (as a mixture of isomers) was not turned over to product by any of the dioxygenases examined in this work (no identifiable product peaks in mass spectrometry). this would be consistent with observations from mass spectrometry that the 221 species is a minor product (with most of the substrate turned over to nfk, as judged by comparing intensities of peaks eluting from lc since the epoxide itself is not an isolatable compound, it was not possible to spike the reaction with epoxide in the same way. to confirm that the 221 peak was generated from o2-dependent turnover, the reactions with l - trp were repeated under the same conditions but using o2 to assess whether the oxygen atom in the 221 species originated from o2 (or o2). the results of these lc ms / ms experiments are shown in figure 2 for htdo with l - trp and for hido with me - trp, but similar data have been observed for xtdo with l - trp. for htdo, a peak is observed with m / z = 223 (figure 2a), which corresponds to insertion of a single atom of o into l - trp (table s1, (si)). in this spectrum there is also a peak at m / z = 177, which corresponds to the o - equivalent of the m / z = 175 fragmentation peak reported above (loss of cooh, as above). an ion with m / z = 241 was also detected, figure 2b, which corresponds to the mass of the nfk product carrying two atoms of o (m / z = 241, table s1 (si)). in the corresponding reactions of hido with me - trp and o2, we also observe peaks eluting with m / z = 237 and m / z = 255 (figures 2c, d), which are as expected for the o - labeled mono - oxygenated intermediate species and o - labeled n - formylmethylkynurenine product (mr = 254, table s1(si)).(23) formation of nfk during dioxygenase - catalyzed turnover using o2. ms / ms analyses of the products obtained on reaction of htdo with l - trp and o2 ((a) fragmentation pattern for ion with m / z = 223, (b) fragmentation pattern for ion with m / z = 241) and on reaction of hido with me - trp and o2 ((c) fragmentation pattern for ion with m / z = 237, (d) fragmentation pattern for ion with m / z = 255). for the reaction of htdo and o2, we also observed a product peak with m / z = 239 for the reaction with l - trp (figure 3a), and in parallel experiments a peak with m / z = 253 for the reaction with me - trp (figure 3b). in both instances, this corresponds to the mass of nfk carrying one atom each of o and o (i.e., oo - nfk and oo - me - nfk, instead of the expected product carrying two atoms of o2). it is known from other work that both carbonyl oxygen atoms in nfk are exchangeable with solvent. in recent analyses, ronsein and co - workers(26) mixed species (oo - nfk, m / z = 239) for nfk formed using singlet o2 and have used the fragmentation patterns to show that the two carbonyl oxygen atoms in nfk have different rates of exchange with solvent, with the carbonyl oxygen group of nfk (blue in figure 4) exchanging faster than the carbonyl oxygen of the amide group (red in figure 4).(26) in our experiments for htdo, xtdo, and hido with o2 (figures 4a c), the m / z = 239 peak for the original mixed oo species fragments in all cases to give m / z = 222 (loss of the nh3 group of the side chain), 204 (loss of nh3 then h2o), and m / z = 192 (loss of nh3 then co from the carbonyl of the amide group(26)). this is consistent with initial formation of oo - nfk, with subsequent exchange of the carbonyl oxygen to give oo - nfk (m / z = 239) followed by fragmentation leading to loss of o (from loss of co, red) and leaving o in the product (blue).(26) to verify this, we carried out a control reaction with o2 but with the protein exchanged into h2o - containing buffer. in these experiments, oo - nfk (m / z = 237) formed initially, followed by exchange (as evidenced by a peak at m / z = 239, figure 4d), but the fragmentation pattern is different (peaks at m / z = 222 as above, 202 (loss of nh3 then h2o), and 194 (loss of nh3 then co), figure 4d)) this is again consistent with faster exchange of the carbonyl oxygen with o (from h2o water), because the fragmentation (figure 4d) now leads to loss of co (red) from the carbonyl of the amide group (not o, as above in figure 4a c). together, these experiments provide assurance that the peaks for the mixed oo - nfk species are secondary products derived from subsequent exchange reactions with water (during handling). ms / ms analyses of nfk (m / z = 239) carrying one atom of o and one atom of o from reaction of (a) htdo with l - trp and o2 (oo - nfk) and (b) hido with me - trp and o2 (oo - me - nfk). ms / ms analyses of the nfk product peak (m / z = 239) in reactions of (a) htdo, (b) hido, and (c) xtdo with l - trp and o2 in h2o buffer ; and lc ms / ms analyses of the nfk product peak (m / z = 239) in reactions of htdo (d) with l - trp and o2 in h2o buffer. for clarity of discussion in the results, the activation of atmospheric oxygen is a fundamental requirement in biology that all aerobic organisms have to overcome. heme iron is often used to overcome these difficulties, and nature uses heme enzymes for a large number of quite different, and sometimes difficult, biological oxidations. the mechanism of o2 activation and nfk formation in the heme dioxygenases has yet to be clarified. the mass spectrometry data presented in this paper reveal the presence of a stable amino acetal species, assigned as originating from initial formation of an unstable 2,3-epoxide, and consistent with insertion of a single atom of oxygen into the substrate during o2-driven reactions. this is in contrast to the originally proposed mechanisms(1) for conversion of l - trp to nfk, scheme 1c, but would agree with more recent evidence(16) from resonance raman work in which sequential insertion of oxygen into the substrate has been suggested. we envisage two possible mechanisms (scheme 3, steps 1a and 1b(27)), both of which fit with our data and with the supposed formation of ferryl heme. one route is through electrophilic addition (steps 1b and 2b), which is well - known in indole chemistry. however, in this mechanism the bound o2 is denoted as an electrophile which could be problematic since o2 bound to ferrous heme is often formulated as a ferric - superoxide species.(30) an alternative suggested route is through a radical - mediated reaction and formation of ferryl heme (scheme 3, steps 1a and 2a). this would be attractive if the ferrous oxy species exists as ferric - superoxide (for which there is evidence(16)) and would at the same time overcome the thermodynamic barrier to the required activation of o2 (by one - electron reduction). recent computational work favors the radical addition mechanism.(33) it is perhaps also worth remembering that heterolytic cleavage typically requires considerable charge separation ; hence, there is a requirement for electrostatic stabilization by residues in the distal pocket (arg for example in the peroxidases), and the dioxygenase structures solved so far do not reveal large numbers of charged residues close to the heme iron (although there is an active - site arg involved in substrate binding(18)). regardless of which mechanism is used (scheme 3, steps 1a or 1b), neither requires abstraction of the indole proton and would thus be in agreement with most of the recent data. epoxide formation is envisaged by two possible routes, an electrophilic mechanism (step 1a) or by radical addition (step 1b) : as we highlight in the discussion, both routes can lead to formation of an epoxide, but neither mechanism has been definitely proven. ring - opening is indicated in step 3 (leading to the species labeled as x in scheme 2), but could be synchronous with step 4. cleavage of the c c bond and formation of nfk is subsequently straightforward. for further details the amine group of the side chain could potentially form a hydrogen bond to the o of the singly oxygenated intermediate. the later stages of the mechanism leading to formation of nfk have been proposed as occurring through either criegee or dioxetane pathways (scheme 1c). a singularly misleading aspect of the literature is that these proposals are based on early speculation(34) rather than on experimental fact (formation of a peroxyindole species, usually generated photolytically, is an implicit requirement, and we presume this would be difficult to generate under enzymatic conditions). the conditions of our turnover experiments and mass spectrometry analyses allow us to identify a cyclic aminoacetal derivative, which we assign as a byproduct of the reaction that originates from ring - opening of the initially formed (and unstable) 2,3-epoxide (scheme 2). it is well - known that epoxides can undergo facile c o bond cleavage (due to the adjacent nitrogen lone pair(36)), and for this reason we propose that the amino acetal species arises from ring - opening of the initial epoxide (scheme 2).(37) by analogy, this indicates to us that a heterolytic (two - electron) mechanism, again involving ring - opening of the initial epoxide, may be the initiating step in the dioxygenase enzymes (scheme 3, step 3). this would allow for reactivity of both l - trp and me - trp, both of which are substrates.(13) direct attack of the ferryl heme on the substrate can then be envisaged (step 4), after which a heterocyclic (two - electron) mechanism for the required cleavage of the c c bond is feasible (step 5).(39) we have found that the amino acetal is not, under the conditions of our experiments, turned over by the enzyme to nfk, but we note that the very same species (labeled x and y in scheme 2) have been proposed(40) as intermediates in the prnb enzyme (species y being referred to as the tricylic intermediate by naismith.(40)), which catalyzes cleavage of the c n bond of 7-cl - trp but without insertion of o2 (scheme 4a). in scheme 4b, we suggest how the two enzyme mechanisms may align : both proceed through intermediate x but then branch, in the dioxygenases leading to further oxygen insertion and nfk formation, while in prnb a different product emerges via cyclization and protonation.(40) the proposed lack of protons in the dioxygenase active site(14) might be a controlling factor in discriminating one pathway against the other. (a) the reaction catalyzed by prnb.(40) (b) comparison of possible reaction mechanisms in prnb (right) and the dioxygenases (left). in both, formation of the species labeled x is implicated, but after that the mechanisms branch (the species labeled x and y are the same as also shown in scheme 2). in prnb this leads to formation of the tricyclic (amino acetal) intermediate, without insertion of oxygen, whereas in the dioxygenases c c bond cleavage and further o atom insertion is the preferred route. epoxidation of substrates is, of course, well - known in the p450s in this case through direct reaction with the more powerfully oxidizing compound i intermediate but drawing on this, we considered other mechanisms for nfk formation involving h atom abstraction from different positions on the substrate. direct h atom abstraction from the c h group of the epoxide by compound ii was considered (scheme s1a, si) but subsequent conversion to nfk is not easily visualized. the most plausible alternative was considered to involve abstraction of the indole nh (which has precedent in p450 chemistry) and is attractive because it leads to nfk formation through a simple and chemically convincing radical mechanism, scheme s1b (si). however, this mechanism has the disadvantage that it does not accommodate the known reactivity of me - trp,(13) and we thus considered it less likely because me - trp and l - trp are, at this stage, presumed to react through similar mechanisms. there is logic in the idea that the shared heme structure used across all heme enzymes, together with the similarity of their reactions with o2 or its derivatives, means that there may be common patterns of reactivity across the entire family. typically, other o2-dependent oxidative enzymes (e.g., p450s, no synthase) react with o2 or derivatives of it (h2o2 in the peroxidases) to give an oxidized compound i intermediate. irreversible formation of compound i is a reaction that the dioxygenases apparently need to avoid because formation of compound i and recycling of the heme demands both protons (for evolution of water) and a continuous supply of electrons, neither of which is known to be required for dioxygenase turnover. quite how such discrimination is achieved in the dioxygenases has yet to be established, but regulation of the reactivity of the ferrous - oxy species by limiting the supply of protons / electrons to the heme would be one solution to the problem. in this sense, the dioxygenases are unique because, rather than using the same oxidizing intermediate, they appear to use different heme intermediates for insertion of the two atoms of oxygen. this may be one of the key controlling features that helps the dioxygenases to specialize in nfk formation. | heme dioxygenases catalyze the oxidation of l - tryptophan to n - formylkynurenine (nfk), the first and rate - limiting step in tryptophan catabolism. although recent progress has been made on early stages in the mechanism, there is currently no experimental data on the mechanism of product (nfk) formation. in this work, we have used mass spectrometry to examine product formation in a number of dioxygenases. in addition to nfk formation (m / z = 237), the data identify a species (m / z = 221) that is consistent with insertion of a single atom of oxygen into the substrate during o2-driven turnover. the fragmentation pattern for this m / z = 221 species is consistent with a cyclic amino acetal structure ; independent chemical synthesis of the 3a - hydroxypyrroloindole-2-carboxylic acid compound is in agreement with this assignment. labeling experiments with 18o2 confirm the origin of the oxygen atom as arising from o2-dependent turnover. these data suggest that the dioxygenases use a ring - opening mechanism during nfk formation, rather than criegee or dioxetane mechanisms as previously proposed. |
ischaemic colitis is a reversible and transient ischemic intestinal disease without occlusion of the main trunk of the mesenteric artery. although both the large and small intestines are susceptible to ischemia, the latter rarely presents with ischaemic enteritis because of its rich collateral arteries. because of the length of the small intestine, its inspection using endoscopy or gastrofluorography is difficult however, with the recent advent of capsule endoscopy and balloon enteroscopy, an increasing number of small intestinal diseases have been detected. most ischaemic enteritis cases are accompanied with hypertension, diabetes mellitus, ischaemic heart disease and cerebral infarction ; thus, ischaemic enteritis is considered to be associated with atherosclerosis. a 69-year - old male was admitted to our institution because of a sudden onset of vomiting and abdominal distention in april 2012. his general history did not include smoking or alcohol consumption ; however, his medical history included a femoral head fracture, which was treated with an artificial femoral head when he was 60 years old, and congestive heart failure, which improved with diuretic administration in november 2011. his past history also included an episode of ischaemic colitis which presented with severe left - sided abdominal pain. abdominal radiography and computed tomography revealed no abnormal findings, although colonoscopy revealed longitudinal ulcers in the descending colon (fig. 1b) and single - balloon enteroscopy confirmed the same in the distal ileum (fig. ischaemic enteritis was suspected and the patient was prescribed a treatment that included fasting and blood transfusion. single - balloon enteroscopy revealed circumferential ulceration of the distal ileum (c) colonoscopy revealed circumferential ulceration of the descending colon (a). single - balloon enteroscopy revealed circumferential ulceration of the distal ileum (c) on admission, the patient s blood pressure was 103/54 mmhg, heart rate was 72 beats / min, body temperature was 36.3 c and oxygen saturation was 97 % in room air. on clinical examination, his weight was 51 kg, height was 166 cm and body mass index was 18.5 kg / m. inspection of the palpebral conjunctiva revealed evidence of mild anaemia, whereas a chest auscultation revealed no abnormal findings and physical examination revealed no oedema or cyanosis. the patient s abdomen was markedly distended and reduced peristalsis was evident with mild tenderness over abdominal region. blood chemistry revealed a normal white blood cell count (7000 cells/l), mild anaemia (red blood cell count, 321 10/l ; haemoglobin, 9.2 g / dl), elevated c - reactive protein level (4.8 mg / dl), mild hypoproteinaemia (5.5 g / dl), mild hypoalbuminaemia (2.4 g / dl), mildly elevated serum creatine level (1.36 mg / dl), mildly elevated blood urea nitrogen level (25.6 mg / dl), hyponatraemia (128 meq / l) and an elevated glucose level (144 mg / dl ; table 1). plain abdominal computed tomography revealed extensively dilated small intestinal loops, a calibre change around the ileal end and an absence of ascites (fig. therefore, small intestinal obstruction was diagnosed and a transnasal ileus tube was placed (fig. 3a). during decompression of the small intestinal obstruction using the ileus tube, the patient was fasting and was treated with total parenteral nutrition. the ileus tube was progressively moved along the small intestine until it reached the distal ileum on post - admission day 4. transanal single - balloon enteroscopy performed to inspect the stricture revealed a circumferential and afferent tubular ulcer in the distal ileum, 5 cm from the ileocecal valve (fig. 4b), which was dilated using balloon catheters on several occasions but could not be improved.table 1laboratory data on admission hematology wbc7000/l rbc321 10/l hb9.2 g / dl ht28.3 % mcv88.0 fl plt27.2 10/lbiochemistry tp5.5 g / dl alb2.4 g / dl t - bil1.1 mg / dl gtp179 iu / i alp434 iu / i ast19 iu / i alt17 iu / i s - amy58 iu / i ldh168 iu / i bun25.6 mg / dl cr1.36 mg / dl ck19 iu / i na128 meq / l k4.0 meq / l ci86 meq / l glu144 mg / dl crp4.8 mg / dlfig. plain abdominal computed tomography revealed extensively dilated small intestinal loops and calibre change (white arrow) around the ileal end (b axial view, c coronal view)fig. 3a transnasal ileus tube was placed on post - admission day 1 (a). on post - admission day 4, contrast medium from the ileus tube revealed a distal ileal stricture (white arrow) (b)fig. 4transnasal single - balloon enteroscopy revealed a circumferential and afferent tubular ulceration of the distal ileum, 5 cm from the ileocecal valve to the oral side (a). gastrofluorography during the balloon enteroscopy revealed a stricture (white arrow) (b) laboratory data on admission abdominal plain radiography on admission revealed dilated small intestinal loops (a). plain abdominal computed tomography revealed extensively dilated small intestinal loops and calibre change (white arrow) around the ileal end (b axial view, c coronal view) a transnasal ileus tube was placed on post - admission day 1 (a). on post - admission day 4, contrast medium from the ileus tube revealed a distal ileal stricture (white arrow) (b) transnasal single - balloon enteroscopy revealed a circumferential and afferent tubular ulceration of the distal ileum, 5 cm from the ileocecal valve to the oral side (a). gastrofluorography during the balloon enteroscopy revealed a stricture (white arrow) (b) despite treatment, the patient s nutritional status deteriorated and his anaemia progressed, with the latter necessitating a blood transfusion., 42 cm of the ileum, including the known stricture, was resected. several circumferential ulcers with clear margins were detected in the ileum at 528 cm from the ileocecal valve (fig. 6a c) : stenotic portions of the ileum formed ulcers of grade ul - ii with an intact muscularis mucosa, the ileal lumen was covered with fibrin and fibrous connective tissue, an inflammatory cell infiltrate was present in all layers, particularly lymphocytes and eosinophils, dilatation and congestion of capillary vessels was observed in the submucosa and haemosiderin staining revealed sideroferous cells in the submucosal layers. considering these findings, ischaemic enteritis was confirmed in the resected specimen.fig. in total, 42 cm of the ileum, including the known stricture, was resected. several circumferential ulcers with clear margins were detected 528 cm from the ileocecal valve towards the oral side (b)fig. 6stenotic portions of the ileum formed an ulcer ul - ii excluding the muscularis mucosa. inflammatory cell infiltrate, particularly of lymphocytes and eosinophils, was observed throughout all layers. dilatation and congestion of the submucosal capillary vessels were observed (h&e staining : a low - power field, b high - power field). haemosiderin staining revealed sideroferous cells in the submucosal layers (c) perioperative findings revealed several strictures in the distal ileum (a). in total, 42 cm of the ileum, including the known stricture, was resected. several circumferential ulcers with clear margins were detected 528 cm from the ileocecal valve towards the oral side (b) stenotic portions of the ileum formed an ulcer ul - ii excluding the muscularis mucosa. inflammatory cell infiltrate, particularly of lymphocytes and eosinophils, was observed throughout all layers. dilatation and congestion of the submucosal capillary vessels were observed (h&e staining : a low - power field, b high - power field). haemosiderin staining revealed sideroferous cells in the submucosal layers (c) the patient s post - operative course was uneventful. oral intake was initiated and the patient experienced no recurrence of small intestinal obstruction or symptom of abdominal pain. ischaemic colitis was first reported by boley. ; and marston. defined it as an irreversible mucosal injury induced by intestinal ischaemia without organic occlusion of the mesenteric artery. on the basis of its severity, ischaemic colitis is classified as transient and reversible ischaemia, ischaemic ulcers with strictures and gangrenous ischaemic colitis. subsequently, gangrenous ischaemic colitis was removed from the classification because the progress of intestinal necrosis is irreversible at this stage. in the small intestine, the same condition as ischaemic colitis is termed ischaemic enteritis, which is fairly rare and shares several similarities with ischaemic colitis. both ischaemic colitis and enteritis first, the small intestine rarely presents with ischaemic changes because of its rich collateral flow. third, the small intestine is not easy to examine ; therefore, although small bowel pathology is rare, examination of acute small intestinal disease is liable to be delayed and its detection unsuccessful. patients with ischaemic enteritis commonly present with abdominal pain, nausea, fever and vomiting, while melena is rare. stenotic ischaemic enteritis occasionally presents with small intestinal obstruction after resolution of the initial symptoms. ischaemic enteritis is classified as transient type, when it resolves completely within a few days without sequelae, or as stenotic type, when it presents with tubular stricture after the course of chronic inflammation [6, 7 ]. some case reports [8, 9 ] demonstrated a small bowel obstruction due to multiple ulcerations of ischaemic enteritis with circumferential strictures which required surgical resection. before the advent of capsule endoscopy and balloon enteroscopy, it was difficult to observe small intestinal lesions ; therefore, most transient ischaemic enteritis cases could have been missed. ischaemic enteritis is characterized by the formation of strictures, circumferential segmental ulcers and afferent tubular stenosis, but seldom presents with longitudinal ulcers. in contrast, ischaemic colitis is characterized by longitudinal ulcers with eccentric deformation and sacculation. strictures in ischaemic colitis tend to spontaneously resolve over time, whereas those in ischaemic enteritis sometimes tend to progressively worsen and ultimately result in total occlusion. therefore, because the strictures of ischaemic enteritis may fail to resolve even after prolonged periods of conservative management, surgical intervention should be considered sooner rather than later. in our case, the stricture on which balloon dilatation was performed was refractory to several attempts at balloon dilatation ; therefore, the stricture was surgically resected. the macroscopic characteristics of ischaemic enteritis are : afferent tubular stenosis, circumferential stenosis with clear boundaries and severe thickening of the intestinal wall. the histological characteristics of ischaemic enteritis include the following [6, 9]:variable ulcer depth with most at ul - ii or ul - iiiulcer bases lined with vessel - rich granulation tissuesevere fibrosis seen mainly within the submucosal layerssevere inflammatory cell invasion (primarily lymphocytes and plasma cells)haemosiderin - laden macrophages scattered throughout the entire thickness of the intestine. variable ulcer depth with most at ul - ii or ul - iii ulcer bases lined with vessel - rich granulation tissue severe fibrosis seen mainly within the submucosal layers severe inflammatory cell invasion (primarily lymphocytes and plasma cells) haemosiderin - laden macrophages scattered throughout the entire thickness of the intestine. endoscopic analysis revealed circumferential, afferent, tubular and segmental ulcers with clear boundaries, whereas microscopic analysis revealed inflammatory cell infiltration throughout all layers, with dilatation and congestion of submucosal capillary vessels. the differential diagnoses in our case included small bowel tuberculosis, crohn s disease and non - specific multiple ulcers of the small intestine. these diagnoses were excluded because of the absence of cryptogenic abscesses and non - caseating granulomas. we reported a rare case of small intestinal obstruction resulting from stenotic ischaemic enteritis. when encountering small intestinal ulcerated lesions with tubular afferent stenosis ischaemic enteritis may result in small intestinal obstruction due to intestinal stenosis in its chronic phase. therefore, when a diagnosis of ischaemic enteritis is confirmed using enteroscopy or gastrofluorography, surgical treatment should be the intervention of choice. | a 69-year - old male was admitted to our institution because of a sudden onset of vomiting and abdominal distention. his past history of illness included femoral head fracture, congestive heart failure and ischaemic colitis. plain abdominal computed tomography revealed extensively dilated small intestinal loops with a calibre change around the end of the ileum. small intestinal obstruction was diagnosed and a transnasal ileus tube was placed. the ileus tube was constantly moved towards small intestine until it reached the distal ileum. contrast medium from the ileus tube revealed a distal ileal stricture. subsequently, transanal single balloon enteroscopy was performed to inspect the stricture, revealing a circumferential and afferent tubular ulcer in the distal ileum, 5 cm from the ileocecal valve ; gastrofluorography confirmed the stricture. although the stricture was dilated on several occasions using balloon catheters, the stricture could not be improved. however, during the treatment, his general condition worsened over time ; thus, surgical treatment was decided. operative findings revealed several circumferential ulcers with a clear margin 528 cm from the ileocecal valve : all lesions were successfully resected. pathological findings were consistent with ischaemic enteritis. we report a case of small intestinal obstruction resulting from stenotic ischaemic enteritis. |
parasitic helminths infect up to one - third of the global population due to having evolved numerous strategies to balance their survival with that of the host. one mechanism employs secretion of molecules that subtly modulate the host immune response (review ref (2)) to prevent clearance of the parasite without leaving the host vulnerable to opportunistic infections. an understanding of the molecular aspects of this mechanism has been approached through characterization of molecules such as es-62, a protein discovered in the secretions of the rodent filarial nematode acanthocheilonema viteae.(3) es-62 has a range of immunomodulatory effects, many of which involve subversion of tlr4 signaling to induce an anti - inflammatory immunological phenotype. the molecule has therefore been studied for its therapeutic potential in human diseases associated with aberrant inflammation such as rheumatoid arthritis (ra) and has been found to be protective in the mouse model of ra, collagen - induced arthritis (cia), via targeting of pathogenic pro - inflammatory cytokines, in particular il-17 and ifn. as a tetrameric protein of 240 kda (review ref (10)), es-62 is immunogenic and hence unsuitable for use as a drug. however, key anti - inflammatory activities are associated with its post - translational glycosylation and subsequent esterification by phosphorylcholine (pc ; review ref (11)). thus, the development of low molecular weight, nonimmunogenic, pc - based derivatives demonstrating es-62-like biological properties might offer a better approach to drug discovery. indeed, we have previously found that short synthetic peptides containing pc - esters of tyrosine as well as pc - containing glycosphingolipids replicate some of the immunomodulatory properties of es-62 in vitro (and unpublished results). here, we describe the design and synthesis of a library of novel small molecule analogues (smas) related to pc and provide proof of concept in the in vivo cia model that such compounds can be active against inflammatory diseases like ra for which improved drugs are sought. initially, the smas were screened in vitro to determine their effect on pro - inflammatory cytokines, promoting th1 (ifn)/th17 (il-17) responses that are secreted by macrophages in response to pathogen - associated molecular patterns (pamps). the screening strategy was chosen, not only because of the pathogenic roles ascribed to th1/th17 polarizing cytokines in ra but also because tlrs are highly expressed by fibroblasts and macrophages in the synovium, and synovial expression of tlr2, tlr4, and tlr9 is further upregulated by il-17 (in an il-1 and il-6-dependent manner) in cia. indeed, recent studies suggest that elevated tlr2 levels contribute to the spontaneous release of pro - inflammatory cytokines by synovial tissues. moreover, production of pro - inflammatory chemokines, cytokines, and matrix metalloproteinases, as well as promotion of angiogenesis and cellular invasion and also decreases in matrix biosynthesis, can be stimulated by triggering of tlrs by pamps or endogeneous damage - associated molecular patterns (damps) that are present in synovial tissue of patients (e.g., tlr2, gp96, snapin ; tlr4, hsp22, tenascin - c). it has thus been proposed that such aberrant tlr signaling drives the chronic inflammation characteristic of ra (reviews refs (2022)). collectively, these data have contributed to the identification of tlrs as therapeutic targets in inflammatory disease. hence our previous findings that es-62 exerts its anti - inflammatory effects at least in part by subverting tlr4 signaling to suppress tlr2, tlr4, and tlr9 responses suggests this screening approach for novel ra - targeted drugs is appropriate. thus, following the in vitro screen, we selected one of the molecules with properties most similar to es-62, a sulfone (11a) (termed s3 in uk patent application no. ra has been proposed to exhibit a th1/th17 phenotype of autoimmune inflammation, and we have recently shown that es-62 suppresses both ifn and il-17 production in cia. while we previously showed that the parasite product modulates th1 responses by suppression of their priming by dendritic cells (dc), we found that its protective effects against pathogenic il-17 responses reflect suppression of a cellular network involving dc, th17, and t cells. therefore, to address the therapeutic potential of pc - based smas of es-62 in arthritis, we first determined whether a pc - conjugated protein, pc - bsa, could suppress th1/th17 responses in cia. analysis of its effects (relative to bsa) confirmed and extended our previous findings using pc - ovalbumin (ova) in that pc - bsa suppressed the severity of disease in terms of articular score (figure 1a) and hind paw width (figure 1b) as well as reducing incidence of pathology (figure 1c), especially that pertaining to high articular score (figure 1d ; score 4). also, as with es-62, pc - bsa reduced the serum levels of il-17 (figure 1e), and this was reflected by reduced percentages and numbers of il-17-producing cd4 and t cells stimulated with pma / ionomycin ex vivo (figure 1f, h, i). similarly, and also as observed with es-62, pc - bsa suppressed ifn production by cd4, cd8, and t cells (figure 1g, j collectively, these data suggested that pc - based smas could be a suitable starting point for the development of novel anti - inflammatory drugs for ra. n = 6 (a)) and hind paw width (b), expressed as mean scores sem for bsa- or pc - bsa - treatment groups where n = number of individual mice exposed to collagen and disease incidence (c, d), indicated by the % of mice developing a severity score 2 (c) or 4 (d). serum il-17 levels are plotted as mean values of triplicate il-17 analyses of serum from individual mice (nave, n = 3 ; bsa, n = 6 ; pc - bsa, n = 6 (e)). (f, g) exemplar plots of gating strategy of intracellular il-17 and ifn expression by dln (draining lymph node) cells pooled from bsa- and pc - bsa - treated mice with cia show cd4 or expression on the x - axis versus cytokine expression on the y - axis, with the relevant % cytokine positive cells annotated. the numbers of cytokine - expressing cd4 t cells (h, j), t cells (i, l), and cd8 t cells (k) present in the pooled dln cells from the nave (not exposed to collagen), bsa, and pc - bsa groups are shown. for statistical analysis, p < 0.05. our previous work had shown that pc alone and pc esters of short peptides (unpublished results) and lipids could reproduce some of the actions of es-62. however both the size and/or the lability of these compounds suggested that they would not be appropriate as drugs. moreover, pc esters are known to have a wide range of biological actions, and selectivity would be important for any new drug. a potential solution to these problems is provided by the use of isoesters of the naturally occurring phosphate ester, in which the alkylamino chain and a tetrahedral analogue of the phosphate are included. on the basis of the structure of one of the short peptides, which contained pc - tyrosine, a simple structure was adopted that removed the labile phosphate esters and replaced them with phosphonates, sulfones, sulfonamides, and carboxamides. choline phosphonates, like phosphates, are zwitterionic and likely to have limited cellular penetration. to obtain monocationic small molecule analogues, therefore, of the peptide backbone, small substituents of differing electronic and steric properties were included in the benzene ring, leading to a generic structure (1) in which this substituent, the methylene chain length, and the substituted amino group could be varied (figure 2). design of small molecule analogues (smas) of es-62 based upon a tyrosyl - phosphoryl choline peptide. sulfones have been used in medicinal chemistry principally in peptidomimetics where they link amino acid - like components to give transition state analogues and activate alkenes to michael addition in irreversible inhibitors. cathepsin c is an anti - inflammatory target for which vinyl sulfone containing inhibitors have been described. away from the peptide field, sulfones have featured in inhibitors of terpenoid biosynthesis in farnesyl diphosphate mimetics. it has also been shown that sulfone and sulfonamide analogues of fosmidomycin are inactive compared with the parent compound ; in that case, the loss of the negative charge was considered to be significant. the closest structural relatives to the compounds described in this paper can be found in the patent literature, but most are arylsulfones in which the sulfone group is directly attached to the aromatic ring, unlike the new compounds described here in which there is a methylene group between aromatic ring and sulfonyl group. sulfonamides have featured in anti - inflammatory compounds as benzensulfonamides in cox-2 inhibitors, and some recent studies have included n - alkyl or aryl substituents. thus the indole - based pla2 inhibitors contain n - alkyl sulfonamides within highly hydrophobic structures. a similar substructure is found in some n - dialkylsulfonamide - containing h4 receptor antagonists. phosphonic acid precursors were prepared from the corresponding benzyl bromides (2a2c) by the arbuzov reaction, hydrolysis of the phosphonate esters (3a3d) to give the corresponding phosphonic acids (3a3d), activation using thionyl chloride, and finally esterification with choline or an appropriate analogue to give the target compound (scheme 1, 5a5e). a phosphate was prepared from the corresponding phenol by phosphorylation with phosphoryl chloride and coupling with choline iodide (scheme 1, 6). sulfones were prepared from the relevant benzylic halide by alkylation of either 2-thioethanol or 3-thiopropanol followed by oxidation of the thioethers (7a7d, 13a, b, schemes 2 and 3, respectively) to give the sulfones (8a8d, 14a, b). mesylation in the case of the ethyl derivatives afforded a mixture of the vinyl sulfones (9) and the mesylates (10), which was used without separation to react with the appropriate secondary amine to give a series of the required smas (11a11p, 17a17f). the exact proportions of the vinyl sulfone and mesylate were typically 7080% vinyl sulfone depending upon substituent and batch. if the reaction time was 48 h or longer, only vinyl sulfone was obtained. this synthetic step was not optimized because both vinyl sulfone and mesylate afforded the same product under equivalent conditions in the following steps. the corresponding quaternary salts (12b12d) were obtained by alkylation with methyl iodide. for the propyl derivatives in which elimination does not occur under mild conditions, substitution of the mesylate directly afforded the required smas. a third series of phenylsulfones (scheme 3, 18a18l) with shorter length than the foregoing compounds was prepared by addition of the appropriate amine to phenylvinylsulfone. benzyl sulfonamides were simply prepared from the relevant sulfonyl chloride and amine under standard conditions (scheme 4, 19a19aa). arylamino sulfonamides (21a21p) were prepared from the vinyl sulfonamide (20a20e) of the appropriate aniline or benzylamine. (19) is referred to as the csn type, (21) as the cns type, and (23) as the nsc type in the text. carboxamides (24a24d, 25a25d) were prepared by acylation of the appropriate amine with the acid chloride in dichloromethane solution (scheme 5). the isoquinolylmethylamide (24e) was prepared by hbtu coupling of the amine and sodium 3-(4-morpholinyl)propanoate. a single screen of a library of 116 novel compounds was carried out for modulation of production of the th1/th17 promoting inflammatory cytokines il-12p40 and il-6 by bone marrow - derived macrophages (bmms) in response to the pamps, lps (tlr4), bacterial lipoprotein (blp, tlr2/6), and cpg motifs (tlr9), to identify smas that mimicked the properties of es-62 (table 1). previously, we had successfully tested pc, pc - peptides (unpublished), and pc - lipids in the concentration range 110 g / ml for analysis of effects on cytokine production in vitro, and so a concentration of 5 g / ml was selected for the current study. activity at such a concentration would reflect a significant reduction in potency relative to es-62 (active at 12 g / ml), which has pc probably accounting for < 1% of its mass. this could possibly be due to the structure of es-62 and/or its mechanism of interaction with cells, somehow optimizing pc presentation and/or activity. bone marrow - derived macrophages preincubated for 18 h with 5 g / ml of compounds were stimulated with 100 ng / ml lps, 10 ng / ml blp, or 0.01 m cpg in the continued presence of the compounds. after 24 h, supernatants were collected and measured for their il-12p40 and il-6 content by elisa. arrows down () indicate statistically significant (at least p < 0.05) down - regulation and arrows up () statistically significant up - regulation of the levels of cytokine versus control ; blank squares = no significant change. abbreviations used in structural formulas : coumarin = (7-methoxy-2-oxo-2h - chromen-4-yl)methyl ; diam = n, n, n-1,1,2-tetramethylethylenediamino2-me - but = 2-methylbutylamino ; 4-me - pip = 4-methylpiperazinyl ; morph = morpholino ; 3-oh - n - propyl = 3-hydroxypropylamino ; pyrrol = pyrrolidino ; stilbenyl = 1-(4-[(e)-2-phenylethyl]benzyl ; thiomorph = thiomorpholino. many of the compounds were found to demonstrate immunomodulatory activity. however, perhaps unexpectedly, they were selective in terms of the pamp and/or cytokine responses they affected and, indeed, in some cases cytokine levels were elevated rather than reduced, a result not previously observed with es-62. nevertheless, a number of trends are detectable from the cytokine release data. in general, the tail group structure had no detectable influence on the cytokine release profile although a significant effect of the diamino tail group was observed for 21p and 21q as noted below. although severely limited by the range of aromatic substituents studied, there was a notable difference between the reduction of pro - inflammatory cytokine release (4-br and 4-me 11a11d) and the tendency to show a lack of an effect or even an increase in pro - inflammatory cytokine release (3-f 11e11h). in the sulfone series, compounds with a two - carbon methylene chain (11a11p and 12a12d) were generally more effective than comparable compounds with a three - carbon methylene chain (16a16f and 17a17e) at inhibiting pro - inflammatory cytokine release. shortening the distance between the aromatic ring and the amine essentially increased pro - inflammatory cytokine release (18a18l), but some reduction of cytokine release was observed, suggesting that there are different targets and mechanisms for these compounds in differently stimulated cells. the cytokine release signature of many of the sulfonamides was to stimulate the release of pro - inflammatory cytokines (csn type 19d19j, cns type 21c21i), and as with the sulfones, there was no evidence for an effect caused by the side chain structure. however there were two subsets of sulfonamides that caused predominant reduction in the release of pro - inflammatory cytokines ; in the cns type, compounds with electron withdrawing substituents (f, no221l21o) or both cns and csn types with large hydrophobic aromatic groups (naphthyl, 19u, coumarinyl, 21r21 t). a further special case of reduced pro - inflammatory cytokine release was found for compounds of the cns type with the dimethylaminoethylamino tail group (21p, 21q) for which some monoamino analogues had the opposite effect (21e, 21f, 21h). the shortened nsc type of sulfonamide (23a23h) responded with a decrease in release of il-12p40 in lps stimulated cells but an increase in il-6 release from cpg stimulated cells, again largely independent of tail group structure. the amides (24a24c, 25a25d) almost entirely caused a reduction in the release of pro - inflammatory cytokines. most strikingly, the phosphate and phosphonates, all of which are zwitterionic, in general caused an increase in pro - inflammatory cytokine release, suggesting that a distinctly different mechanism was stimulated by these compounds from that of the nonzwitterionic sulfones, sulfonamides, and amides. of direct relevance to the aims of the project, a number of the compounds reduced production of cytokines important for promoting th1 and/or th17 responses, il-12p40 and/or il-6, in response to one or more of the tlr ligands. however, some of these did not target either tlr4 (sulfonamide 21o) or tlr2 (sulfonamides 21n, 21o) responses. the most promising compounds were those that showed the broadest response to the pamps, the sulfones, 11a and 12b, the sulfonamide 21l, and the carboxamide 24b. 11a (see figure 3a) and 12b both mimicked es-62 in targeting il-12p40 production via each of tlr2, 4, and 9, and this cytokine is pathogenic in cia due to it being a component of both il-12p70 and il-23, which promote th1 and th17 responses, respectively, and hence a therapeutic target (ustekinumab) in inflammatory autoimmune diseases. by contrast, sulfonamide 21l and carboxamide 24b did not target lps- and blp - mediated il-12p40 responses, respectively. sulfone 11a additionally targeted il-6 production (see also figure 3b) in response to all three tlrs (although this did not reach statistical significance for tlr2 in all experiments), and this cytokine has also been shown to be pathogenic and thus a therapeutic target (tocilizumab) in ra. although sulfone 12b could suppress cpg - mediated il-6 responses, it was less effective at inhibiting such tlr2- or tlr4-coupled responses (decreases not reaching statistical significance), which have been shown to be important in the development of inflammatory th17 responses, including those in arthritis. moreover, as observed with es-62, (11a (figure 3c), but not 21l (results not shown), was able to inhibit tlr - mediated p65 nf-b activation in response to all three pamps. 11a - related changes in macrophage cytokine production and signaling of p65 nfb in response to lps, blp, and cpg. bmms were preincubated with 11a at 5 g / ml for 18 h prior to stimulation with 100 ng / ml lps, 10 ng / ml blp, or 0.01 m cpg for 24 h and analysis of levels of il-12p40 (a) and il-6 (b) performed by elisa. (c) stimulation with pamps as above for lps and blp, and 1 m cpg, but for 1 h and the level of p65 activation in duplicate samples measured by transam. for statistical analysis for (a) and (b), p < 0.05. thus, as both il-6 and il-12p40 (via il-23) promote the differentiation and maintenance of th17 responses, it was considered that 11a was most likely to mimic the protective effects of es-62 in cia by suppressing production of il-17, a cytokine that is pathogenic in ra and an emerging therapeutic target (for example via the humanized monoclonal antibody ly2439821 and the human monoclonal antibody ain457 ; reviews refs (42,5052)). limited analysis of potency revealed that 11a was still able to induce a statistically significant reduction of lps - stimulated production of il-6 by macrophages when the concentration was reduced to 1 g / ml, but significant effects were not consistently observed at 0.2 g / ml (data not shown). like es-62, 11a showed no evidence of toxicity under conditions mimicking the macrophage screen of cytokine production as determined using the cell viability indicator, 7-actinomycin d (7-aad) (figure 4). indeed, the sma showed some evidence of protecting against the loss of cell viability associated with exposure to lps. bmms in ultralow binding tissue culture plates were rested in rpmi complete medium for 24 h before culturing with fresh medium or medium containing 11a (5 g / ml) or es-62 (2 g / ml). after 18 h, the macrophages were stimulated with medium containing 100 ng / ml lps or 10 ng / ml blp or 0.01 m cpg for an additional 24 h before staining with 7-aad to assess their viability. the samples were analyzed by flow cytometry, and the data are presented as density plots with frequency of 7-aad positive (dead) cells indicated in the gates. the results shown are from a single experiment representative of two independent experiments. 11a, at a low dose of 50 g / kg per injection, was tested in the cia model and it was found that, as with es-62, it effectively reduced development of arthritis in treated mice. this was reflected in each of disease scores (figure 5a), hind paw width (figure 5b), disease incidence (figure 5c), and percentage of mice with high disease scores (figure 5d). moreover, whereas mice with cia exhibited a significant increase in dln cell number over the control group not exposed to collagen, this was significantly reduced by treatment with 11a (figure 5e) and to a level not significantly different from the control naive group. this result was mirrored by analysis of defined t cell populations, namely cd4 t cells (figure 5f), cd8 t cells (figure 5 g), and t cells (figure 5h), where 11a - treated mice showed reduced levels of cells relative to mice with cia and/or levels not significantly different to the control group of naive mice not exposed to collagen. previously we had found that cd4 and t cells from es-62-treated mice were similarly reduced, although statistical significance had not been reached. disease is shown by each of mean arthritis score ((a) pbs, n = 13 ; 11a, n = 13, data pooled from two independent experiments), hind paw width ((b) n = 7, data from a single experiment), and incidence (c, d) indicated by the % of mice developing a severity score 2 ((c) cumulative incidence) or 4 ((d) high score incidence). sem for pbs or 11a - treatment groups of mice exposed to collagen. the numbers of each of total (e), cd4 (f), cd8 (g), and (h) t cells in dln of individual mice from the nave (n = 7), pbs - treated (n = 13) and 11a - treated (n = 13) groups are shown. for statistical analysis, p < 0.01. our previous work with es-62 showed that it suppressed both ifn and il-17 responses and hence we investigated whether this was also the case for 11a. indeed, similarly to es-62, we found 11a to target ifn (figure 6a) and il-17 (figure 6b) responses in dln cells stimulated with pma / ionomycin ex vivo. first, whereas mice with cia displayed significantly higher numbers of dln, cd8 t, and cd4 t cells producing ifn in response to pma / ionomycin - stimulation than nave mice, this was not true of the 11a - treated mice exposed to collagen. moreover, the numbers of ifn-expressing cells in the dln and cd8 t cell populations were significantly reduced in the 11a group relative to the pbs - cia group (figure 6a). however, no significant difference in ifn-producing t cells was found between the mice exposed to collagen given 11a or not (results not shown). with respect to il-17, the effects observed were not as striking although it was still possible to see clear evidence that this pro - inflammatory cytokine was also being targeted. thus, for example, the numbers of dln, cd4, and t cells were significantly higher in pbs, but not 11a, treated mice undergoing cia than in mice not exposed to collagen (figure 6b). exemplar plots of gating strategy of intracellular ifn (a) and il-17 (b) expression by dln cells from representative individual pbs - treated mice with cia show forward scatter (fsc) or cd4, cd8, and expression on the y - axis versus cytokine expression on the x - axis as indicated. the number of (a) ifn-expressing total dln cells, cd8 t cells, and cd4 t cells and (b) il-17-expressing total dln cells, cd4 t cells, and t cells following stimulation with pma / ionomycin from individual mice are shown (nave, n = 7 ; pbs, n = 13 ; 11a, n = 13). for statistical analysis, p < 0.01 and p < 0.001. unlike in our earlier study with es-62,11a did not induce a statistically significant reduction in the serum levels of il-17 in these mice (figure 7a). however, in our previous study, it was shown that pre - exposure to es-62 inhibited the capacity of dendritic cells (bmdcs) to respond to lps by secretion of the pro - inflammatory cytokine tnf- and two cytokines, il-6 and il-23, associated with polarization and maintenance of th17 cells, respectively, and when these experiments were repeated with the bmdcs pre - exposed to 11a, production of all three cytokines was significantly reduced (figure 7b). as with es-62, this did not reflect downregulation of tlr4 expression or reduction in bmdc viability (data not shown). furthermore, consistent with the ability of 11a to inhibit production of the two th17-promoting cytokines, 11a - treated dcs demonstrate a reduced ability to drive nave antigen (ova)-specific th cells toward a th17 phenotype, as evidenced by suppression of ova - specific il-17 production in such bmdc - cd4 t cell cocultures (figure 7c, d). (a) serum il-17 levels are plotted as mean values of triplicate il-17 analyses from individual mice (pbs, n = 12 ; 11a, n = 13). (b) bmdcs from c57bl/6 mice were preincubated with or without (11a) (5 g / ml) for 2 h prior to stimulation with lps for 24 h, and tnf, il-6, and il-23 levels were then analyzed. data are the mean values (of triplicate samples) sem pooled from 4 independent experiments. (c) bmdcs from balb / c mice preincubated with or without 11a (5 g / ml) were pulsed with the indicated concentration of ova peptide and cocultured with naive ova - specific cd4 t cells (do.11.10/balb / c) for 3 days before measuring il-17 release. sd of duplicate samples pooled from two independent experiments (n = 4). (d) pooled normalized data from four independent experiments analyzing the effect of 11a on il-17 release from bmdc (c57bl/6)-ova - specific cd4 t cell (ot - ii / c57bl/6) cocultures. data are presented as the means of the mean percentage maximum (lps) response sem where data were normalized to the lps response at 10 nm (left), 100 nm (middle), and 300 nm (right) ova, respectively. p < 0.05 ; p < 0.01 ; p < 0.001. previous work published by our group has shown that es-62 s mechanism of action involves downregulation of the key tlr / il-1r signaling adaptor myd88 in th17 cells and mast cells. this is also true of macrophages (our unpublished results), the cell type used for our primary screen, and so we next investigated whether 11a was also able to cause downregulation of myd88 in these cells. as shown in figure 8a, b, the sma does indeed cause significant downregulation of the signaling adaptor in these cells. figure 8a c also shows that, as expected, lps causes an increase in myd88 expression within the cells, but as demonstrated in figure 8c, this is prevented by 11a. a simple schematic of the proposed mechanism of action of 11a is shown in figure 8d and described in detail in the figure legend. (a) bmms treated with medium (rpmi), 11a at 1 and 5 g / ml (11a-1 or 11a-5), or lps (100 ng / ml) for 20 h were analyzed by western blotting for expression of myd88 and loading control gapdh. levels of expression were determined by densitometry using image - j software and expressed as the ratio of myd88:gapdh and normalized to the rpmi value. (b) data from six analyses revealed that while lps significantly increased myd88 expression (p < 0.05), both 11a-1 and 11a-5 reduced it (p < 0.05) relative to the rpmi control. in addition, the level of myd88 in cells treated with either 11a-1 or 11a-5 was significantly different (p < 0.001) to that in those exposed to lps. (c) flow cytometric analysis of myd88 expression in permeabilised bmms relative to isotype control (20,000 bmms / treatment group). bmms were treated with rpmi or 11a (at 5 g / ml) for 2 h prior to exposure to lps (100 ng / ml) for a further 18 h, and consistent with the western blot data, lps upregulated levels of myd88 (127% relative to mfi of rpmi - treated bmms). moreover, bmms pretreated with 11a exhibited lower levels of myd88 expression (mfi for 11a + lps is 95.7% of the level of the rpmi value : the rpmi trace is not shown due to its overlap with that of 11a + lps) than cells treated with lps alone. 11a downregulates myd88 expression and hence induces a partial uncoupling of tlr / il-1r from nf-b activation and consequent pro - inflammatory cytokine production that both initiates pathogenic il-17-mediated inflammation and perpetuates chronic vascular permeability, inflammation, and pathology in the joints. thus 11a - mediated downregulation of myd88 impacting at one or more of these sites provides a molecular mechanism for the protection afforded in cia. the increasing awareness of the therapeutic potential of parasitic worms has resulted in a search to identify defined molecules that possess immunomodulatory properties and recently details of a number of these have appeared. although some of the molecules characterized to date are carbohydrate or lipid in nature, the majority are proteins, and hence there is the potential problem of their immunogenicity possibly interfering with activity. es-62, which is among the best characterized of helminth - derived immunomodulators, is active in mouse models of both allergic and autoimmune diseases and has been suggested as being the helminth - derived molecule for which there is most potential in testing in humans for treatment of such disorders. furthermore, pc - containing molecules circulate in the bloodstream of humans infected with filarial nematodes for decades without inducing any known adverse effects on general health or compromising the capacity to fight infection, therefore suggesting that es-62 might be safer than current immunosuppressive drugs, including glucocorticoids or biologics, used to treat human inflammatory diseases. however, as a large protein, es-62 is subject to the problem of immunogenicity referred to above and hence in reality is not suitable for use as a drug. nevertheless, our data obtained to date indicated that pc is the key anti - inflammatory moiety of es-62, and this was confirmed in the present study as when conjugated to bsa, it was found to mimic the suppressive effects of es-62 on il-17 and ifn responses. hence we decided to pursue a novel strategy of drug development : designing synthetic small drug - like compounds based around pc. the sma library was initially subjected to a simple in vitro screen that involved determining the effect of the compounds on pamp - induced macrophage pro - inflammatory cytokine responses, in particular focusing on those mediators that might contribute to th1/th17 responses, which are known targets of es-62 in cia. the results of this screen were surprising, in that although a number of the smas targeted macrophage responses, the effects were more selective than those recorded previously with es-62, in the sense that not all pamp - induced cytokine responses were inhibited. although unexpected, these may actually be potentially important observations, raising the possibility of generating drugs more specific than es-62 with respect to anti - inflammatory activity. another unexpected finding was that rather than inhibiting the cytokine responses, a number of the smas increased certain responses. although we have previously shown variation in inhibitory effects of small pc - containing molecules, we did not observe any stimulatory effects. the reasons underlying such sma - mediated promotion of tlr - driven cytokine responses, which potentially could reflect adjuvant - like applications for these compounds similar to those based on classical pro - inflammatory tlr ligands, have not been investigated at this stage. however, the data underline the point that structurally closely related compounds may vary with respect to immunomodulatory activity and hence require careful analysis at the individual level. on the basis of previous in vitro analysis of the effects of es-62 on macrophages, allied to its protective effects in cia, sulfone 11a was selected from the in vitro screen as a molecule showing properties that might suggest activity against cia similar to es-62. we employed the dosing schedule used successfully with es-62, which was based on the premise that exposing the mice to the parasite product both prior to and at the time of immunization will maximize the potential for modulating initiation of pathogenic immune responses while re - exposure at d21 in the antigen challenge phase will target ongoing and/or memory responses and therefore reflect a more therapeutic regime. this revealed that 11a not only demonstrated efficacy against disease development that mirrored that of the parent molecule but in inhibiting ifn and to a lesser extent, il-17 production, showed evidence of the same immunological mechanism of action. our aim in this study was simply proof of concept that an sma could afford protection using a regimen previously shown to be successful with es-62, and thus it is possible that the molecule can be shown to be even more effective with optimization of dosage regimen. further support for targeting of il-17 responses is shown by 11a mirroring es-62 in preventing secretion by dendritic cells, of cytokines involved in polarization and maintenance of th17 cells, and inhibiting the subsequent ability of such dcs to prime th17 responses. moreover, 11a is able to cause downregulation of the key signaling adaptor myd88 in macrophages. this is consistent with its effects on nf-b activation and explains its ability to mimic a primary mechanism of action of es-62 in suppressing tlr - mediated cytokine production. in addition, such targeting of myd88 provides a molecular rationale (figure 8d) for the protection against cia afforded by 11a as tlr / myd88-dependent signaling has been proposed to be pathogenic in both cia and ra. moreover, the recent use of myd88-deficient mice has shown this signaling element to be crucial to development of il-17-driven arthritis, both in terms of initiation of pathogenic il-17 responses and also the severity of joint inflammation and pathology. 11a is of course too small to be immunogenic, but it also possesses another feature that increases its candidature as a more effective version of es-62 for therapeutic purposes. it contains a tertiary dimethylamino group in contrast to the quaternary trimethylammonium choline component of pc ; previously we have shown that unlike choline, its dimethylamino analogue does not compete for binding to the myeloma protein tepc 15, an antibody which recognizes pc. this suggests that 11a will not bind to endogenous anti - pc antibodies that are found in humans and which could conceivably interfere with activity of pc - containing smas. furthermore, there is increasing evidence that natural anti - pc antibodies by an as yet unknown mechanism may in themselves be anti - inflammatory, protecting humans from diseases such as atherosclerosis, and so this represents another reason for designing drugs that avoid interacting with them. in this way, possessing the tertiary amine rather than quaternary ammonium salt choline may result in a safer as well as a more effective drug. finally, although based on a combination of epidemiological evidence and model studies there has been much enthusiasm for the idea that parasitic worms may represent a starting point for novel drug design for diseases associated with aberrant inflammation, relevant studies are at an early stage and to date no actual drugs have been produced. however, the work presented in this paper serves as a strong proof of concept that novel small molecules can be active against severe and complex diseases in vivo. moreover, the compounds described here benefit from ease of synthesis and active compounds such as 11a can be readily subjected to further chemical manipulation to optimize properties as required. in general, this study suggests that there is great medicinal chemical potential to be found in synthetic libraries based around active moieties of helminth - derived molecules. compounds were reconstituted at 100 mg / ml in cell culture - tested dmso (sigma - aldrich, uk), diluted in rpmi medium to 1 mg / ml, and stored in microcentrifuge tubes at 20 c. compounds were sterilized using a millex - gp (0.22 m) (millipore, uk) filter unit prior to use in culture. h and c nmr spectra were measured on a bruker dpx-400 mhz spectrometer with chemical shifts given in ppm (values) relative to proton and carbon traces in solvent. elemental analysis was carried out on a perkin - elmer 2400, analyzer series 2. anhydrous solvents were obtained from a puresolv purification system, from innovative technologies, or purchased as such from aldrich. melting points were recorded on a reichert hot - stage microscope and are uncorrected. chromatography was carried out using 200400 mesh silica gels or using reverse - phase hplc on a waters system using a c18 luna column. all final compounds were equal or more than 95% pure by hplc and h nmr. 1-bromo-4-(bromomethyl)benzene (3.10 g, 12.5 mmol) was suspended in triethylphosphite (2.4 ml, 13.7 mmol, 1.1 equiv), and the mixture was heated under reflux under n2 for 6 h. excess triethylphosphite was removed under reduced pressure to give the required product as a yellow oil (3.50 g, 91%). h nmr (dmso - d6) : 7.51 (2h, dd, j = 8.4 hz and j = 0.9 hz), 7.25 (2h, dd, j = 8.4 and 2.5 hz), 3.963.91 (4h, m), 3.25 (2h, d, j = 21.6 hz), 1.19 (6h, t, j = 7.1 hz). c nmr (dmso - d6) : 131.9, 131.8, 131.0, 119.7, 61.4, 32.2, 16.2. ir (nacl) : 3447, 2982, 1646, 1513, 1488, 1392, 1249, 1093, 1057, 963, 853, 766 cm. hresims : 1-(bromomethyl)-4-methylbenzene (2.60 g, 14.0 mmol) was suspended in triethylphosphite (2.9 ml, 16.7 mmol), and the mixture was heated under reflux under n2 for 20 h. excess triethylphosphite was removed under reduced pressure to give the required product as a colorless oil (3.20 g, 94%). h nmr (dmso - d6) : 7.17 (2h, d, j = 8.4 hz), 7.12 (2h, d, j = 8.4 hz), 3.963.88 (4h, m), 3.17 (2h, d, j = 20 hz), 2.27 (3h, s), 1.18 (6h, t, j = 7.1 hz). c nmr (dmso - d6) : 135.4, 129.5, 129.1, 128.9, 61.2, 32.4, 20.6, 16.1. ir (nacl) : 3439, 2982, 2901, 2902, 1651, 1516, 1443, 1391, 1251, 1163, 1058, 963, 841, 783 cm. 1-(bromomethyl)-4-nitrobenzene (2.44 g, 11.3 mmol) was suspended in triethylphosphite (5.0 ml, 28.79 mmol), and the mixture was heated under reflux under n2 for 20 h. excess triethylphosphite was removed under reduced pressure to give the required product as a brown oil (6.60 g, 89%). h nmr (dmso - d6) : 8.19 (2h, d, j = 8.7 hz), 7.58 (2h, dd, j = 8.7 hz and j = 2.3 hz), 4.07 (4h, q, j = 7.1 hz), 3.48 (2h, d, j = 22.4 hz), 1.19 (6h, t, j = 7.1 hz). c nmr (dmso - d6) : 146.2, 141.0, 130.9, 123.2, 62.6, 32.9, 16.2. ir (nacl) : 3466, 2983, 1600, 1520, 1347, 1248, 1027, 966, 860, 796, 694 cm. diethyl 4-bromobenzylphosphonate (3.74 g, 12.20 mmol) was suspended in 12 m hcl (30 ml), and the mixture was heated under reflux for 48 h. the reaction mixture was concentrated under reduced pressure to give the required product as an off - white solid (2.20 g, 72%), mp 167175 c [slow decomposition ]. h nmr (dmso - d6) : 9.71 (2h, br, s, oh), 7.47 (2h, d, j = 8.5 hz), 7.20 (2h, d, j = 8.5 hz), 2.97 (2h, d, j = 20.0 hz). c nmr (dmso - d6) : 35.4, 34.1, 119.1, 130.8, 131.9, 133.8. ir (kbr) : 2670, 2258, 1911, 1557, 1491, 1409, 1260, 1240, 1105, 999, 823, 703 cm. diethyl 4-methylbenzylphosphonate (3.48 g, 14.40 mmol) was suspended in 12 m hcl (30 ml), and the mixture was heated under reflux for 56 h. the reaction mixture was concentrated under reduced pressure to give the required product as a white solid (1.99 g, 74%), mp 186187 c. h nmr (dmso - d6) : 9.58 (2h, br, s, oh), 7.14 (2h, d, j = 8.5 hz), 7.08 (2h, d, j = 8.5 hz), 2.92 (2h, d, j = 21.2 hz), 2.26 (3h, s). c nmr (dmso - d6) : 135.2, 131.5, 129.95129.94, 35.9, 21.4. ir (kbr) : 2921, 2324, 1514, 1455, 1269, 1235, 1196, 1107, 996, 969, 946, 814, 740 cm. diethyl 4-nitrobenzylphosphonate (2.91 g, 10.60 mmol) was suspended in 12 m hcl (90 ml), and the mixture was heated under reflux for 18 h. the reaction mixture was concentrated under reduced pressure to give the required product as a brown solid (1.82 g, 93%), mp 208214 c [lit. h nmr (dmso - d6) : 9.45 (2h, br, s, 2h, oh), 7.28.2 (4h, m), 2.98 (2h, d, j = 20.0 hz). 4-bromobenzylphosphonic acid (0.700 g, 2.80 mmol) was suspended in thionyl chloride (5 ml), and the mixture was heated under reflux for 30 h under nitrogen. when the reaction mixture cooledd to room temperature, excess thionyl chloride was removed under reduced pressure and the residue was dissolved in chloroform (20 ml, dry). this solution was then transferred via syringe to a flame - dried, three - necked round - bottomed flask under nitrogen. this solution was then cooled to 0 c, followed by the addition of pyridine (2 ml, dry) and the portionwise addition of choline iodide (0.82 g, 3.55 mmol). the reaction mixture was concentrated under reduced pressure, and the residue was suspended in acetonitrile (2 ml). hplc purification gave the required product as a colorless oil (0.071 g, 56%). h nmr (dmso - d6) : 7.47 (2h, d, j = 7.5 hz), 7.24 (2h, d, j = 7.5 hz), 4.18 (2h, br, s), 3.54 (2h, m), 3.04 (9h, s), 2.99 (2h, d, j = 20 hz). c nmr (dmso - d6) : 139.7, 137.5, 136.4, 124.7, 70.9, 63.6, 58.7, 39.7, 38.4. ir (nacl) : 3383, 3030, 2957, 2913, 1896, 1767, 1686, 1485, 1337, 1241, 1202, 1163, 1093, 1012, 970, 830, 713 cm. 4-bromobenzylphosphonic acid (0.455 g, 1.81 mmol) was suspended in thionyl chloride (5 ml), and the mixture was heated under reflux for 20 h. when the reaction mixture cooled to room temperature, excess thionyl chloride was removed under reduced pressure and the residue was dissolved in dcm (10 ml, dry). to this solution was added, at 0 c, pyridine (400 l, 4.9 mmol, dry) and dimethylethanolamine (122 l, 2.2 mmol). the reaction mixture was stirred at room temperature for 5 h before the addition of water (1 ml). stirring was continued for a further 20 h, after which time the solvent was removed under reduced pressure until 1 ml of crude solution was left. the crude mixture was then purified by reverse phase hplc to give the required product as a colorless oil (295 mg, 37%) as a tfa salt. h nmr (dmso - d6) : 7.50 (2h, d, j = 7.8 hz), 7.26 (2h, dd, j = 2.3 hz and j = 8.4 hz), 4.154.10 (2h, m), 3.29 (2h, t), 3.15 (2h, d, j = 21.4 hz), 3.76 (6h, s). c nmr (dmso - d6) : 133.1, 131.8, 128.8, 119.4, 58.7, 56.7, 42.6, 32.1, 31.6. ir (nacl) : 3419, 3038, 2912, 2714, 2521, 1772, 1678, 1572, 1488, 1407, 1242, 1204, 1052, 1020, 836, 795, 720 cm. 4-methylbenzylphosphonic acid (0.39 g, 2.10 mmol) was suspended in thionyl chloride (5 ml), and the mixture was heated under reflux for 20 h. when the reaction mixture cold to room temperature, excess thionyl chloride was removed under reduced pressure and the residue was dissolved in chloroform (20 ml, dry). to this solution was added, at 0 c, pyridine (600 l, 7.3 mmol, dry) and dimethylethanolamine (300 l, 2.98 mmol). the reaction mixture was stirred at room temperature for 5 h before the addition of water (1 ml). stirring was continued for a further 20 h, after which time the solvent was removed under reduced pressure until around 2 ml of crude solution was left. the crude mixture was then purified by reverse phase hplc to give the required product as colorless oil (295 mg, 37%) as a tfa salt. h nmr (dmso - d6) : 7.17 (2h, dd, j = 2.1 hz and j = 8.0 hz), 7.10 (2h, d, j = 2.1 hz), 4.094.05 (2h, m), 3.24 (2h, t), 3.05 (2h, d, j = 21.2 hz), 2.72 (6h, s), 2.26 (3h, s). c nmr (dmso - d6) : 135.0, 130.6, 129.5, 128.6, 58.7, 56.9, 42.5, 33.7, 32.3, 20.6. ir (nacl) : 3418, 3030, 2964, 2921, 2714, 2528, 1766, 1682, 1515, 1472, 1416, 1321, 1203, 1088, 1020, 993, 832, 799, 720 cm. 4-nitrobenzylphosphonic acid (0.345 g, 1.59 mmol) was suspended in thionyl chloride (5 ml), and the mixture was heated under reflux for 20 h. when the reaction mixture cold to room temperature, excess thionyl chloride was removed under reduced pressure and the residue was dissolved in dcm (10 ml, dry). pyridine (350 l, 4.3 mmol, dry) and dimethylethanolamine (192 l, 1.91 mmol) were added to the reaction mixture at 0 c. the reaction mixture was stirred at room temperature for 5 h before the addition of water (1 ml). stirring was continued for a further 20 h, after which time the solvent was removed under reduced pressure until 1 ml crude solution was left. the crude mixture was then purified by reverse phase hplc to give the required product as colorless oil (200 mg, 31%) as a tfa salt. h nmr (dmso - d6) : 8.18 (2h, d, j = 8.2 hz), 7.56 (2h, dd, j = 2.3 hz and j = 8.8 hz), 4.144.09 (2h, m), 3.31 (2h, d, j = 21.2 hz), 3.253.23 (2h, m), 2.75 (6h, s). c nmr (dmso - d6) : 146.0, 142.7, 130.9, 123.1, 58.7, 56.9, 42.5, 34.3, 33.8. ir (kbr) : 1767, 1680, 1514, 1472, 1414, 1320, 1202, 1087, 1020, 992, 832, 800, 721 cm. 4-methylbenzylphosphonic acid (0.25 g, 1.34 mmol), was suspended in thionyl chloride (7 ml), and the mixture was heated to reflux for 20 h. when the reaction mixture cold to room temperature, excess thionyl chloride was removed under reduced pressure and the residue was dissolved in chloroform (20 ml, dry). to this solution was added, at 0 c, pyridine (295 l, 3.6 mmol, dry) and 3-(dimethylamino)-1-propanol (190 l, 1.60 mmol). the reaction mixture was stirred at room temperature for 5 h before the addition of water (1 ml). stirring was continued for a further 20 h, after which time the solvent was removed under reduced pressure until around 2 ml of crude solution were left. the crude mixture was then purified by reverse phase hplc to give the required product as a colorless oil (0.191 g, 37%) as a tfa salt. h nmr (dmso - d6) : 7.16 (2h, dd, j = 8.0 hz and 2.0 hz), 7.10 (2h, d, j = 8.0 hz), 3.90 (2h, m), 3.03 (2h, d, j = 21.2 hz), 3.012.98 (2h, m), 2.71 (6h, s), 2.27 (3h, s), 1.901.84 (2h, m). c nmr (dmso - d6) : 135.1, 130.5, 129.6, 128.6, 61.2, 53.8, 42.1, 33.8, 32.5, 25.1, 20.6. ir (nacl) : 3419, 3021, 2961, 2906, 2719, 2637, 1682, 1515, 1470, 1410, 1202, 1133, 1049, 971, 830, 719 cm. 4-aminophenol (0.67 g, 6.14 mmol) was dissolved in dmf (15 ml, dry) under nitrogen and the mixture cooled to 0 c before the addition of boc anhydride (1.40 g, 6.45 mmol). the mixture was gradually allowed to warm to room temperature, and stirring was continued for 20 h at room temperature. solvent was removed under reduced pressure and the residue was dried in a drying pistol 60 c for 2 h, to give the required product as an off - white solid (1.19 g, 93%), mp 146148 c [lit. h nmr (dmso - d6) : 8.99 (1h, s, nh), 8.94 (1h, br, s, oh), 7.21 (2h, d, j = 8.6 hz), 6.64 (2h, d, j = 8.6 hz), 1.40 (9h, s). c nmr (dmso - d6) : 152.9, 152.5, 130.9, 120.0, 114.9, 78.4, 28.2. ir (kbr) : 3361, 2975, 2936, 2876, 1696, 1610, 1531, 1436, 1370, 1231, 1165, 1058, 1029, 1014, 829, 803 cm. hresims : calcd for c11h15nnao3, 232.0950 ; found, (m + na) : 232.0951. tert - butyl 4-hydroxyphenylcarbamate (0.42 g, 2.00 mmol) was dissolved in chloroform (30 ml, dry) to which potassium carbonate (0.31 g, 2.20 mmol) and phosphorus oxychloride (0. 240 ml, 2.57 mmol) were added at 0 c, under nitrogen. after 2 h, pyridine (2 ml, dry) and dimethylaminopropanol 0.30 ml, 2.54 mmol) were added. the mixture was stirred at 0 c for 30 min then allowed to warm to room temperature. stirring was continued for 72 h. solvent was removed under reduced pressure, and the residue by hplc purification gave the required product as white solid (0.012 g, 16%), mp 144146 c. h nmr (dmso - d6) : 9.25 (1h, br, s, nh), 7.38 (2h, d, j = 8.6 hz), 7.06 (2h, d, j = 8.6 hz), 1.90 (2h, t), 1.46 (9h, s). c nmr (dmso - d6) : 153.7, 147.3, 136.4, 121.1, 120.0, 79.8, 62.7, 54.9, 42.7, 29.0, 26.7. ir (kbr) : 3414, 2981, 2929, 1700, 1605, 1513, 1410, 1393, 1311, 1214, 1161, 1054, 967, 841, 773, 701 cm. 1-bromo-4-(bromomethyl)benzene (10.00 g, 40.00 mmol), 2-mercaptoethanol (3.13 g, 40.00 mmol), and k2co3 (5.53 g, 40.00 mmol, anhydrous) were added to dcm (50 ml, dry) at room temperature with stirring. stirring was continued for 48 h, after which time water was added and then the reaction mixture was extracted with dcm. the organic layer was collected, dried (mgso4), filtered, and the solvent removed under reduced pressure to give the required product as a pale - yellow oil (11.00 g, 99%). h nmr (cdcl3) : 7.46 (2h, d, j = 8.0 hz), 7.22 (2h, d, j = 8.0 hz), 3.71 (2h, s), 3.70 (2h, t, j = hz), 2.65 (2h, t, j = 8.0 hz), 1.93 (1h, br, s, oh). c nmr (cdcl3) : 137.4, 131.9, 130.8, 121.3, 60.6, 35.4, 34.6. ir (thin film) : 3390 (br), 2919, 1901, 1663, 1589, 1486, 1402, 1289, 1198, 1096, 1069, 1011, 881, 821 cm. 1-(bromomethyl)-4-methylbenzene (10.00 g, 54.03 mmol), 2-mercaptoethanol (4.22 g, 54.03 mmol), and k2co3 (7.47 g, 54.03 mmol, anhydrous) were added to dcm (50 ml, dry) at room temperature with stirring. the stirring was continued for 48 h. water was added, and the reaction mixture was extracted with dcm. the organic layer was collected, dried (mgso4), filtered, and the solvent removed under reduced pressure to give the required product as a pale - yellow oil (9.60 g, 98%). h nmr (dmso - d6) : 7.23 (2h, d, j = 8.0 hz), 7.14 (2h, d, j = 8.0 hz), 4.79 (1h, t, j = 14.3 hz, oh), 3.72 (2h, s), 3.57 (2h, m), 2.52 (2h, t, j = 14.3 hz), 2.30 (3h, s). c nmr (dmso - d6) : 135.7, 135.6, 128.8, 128.7, 60.6, 35.0, 33.3, 20.6. ir (kbr) : 1664, 1513, 1422, 1386, 1098, 1068, 1020, 878, 817, 726 cm. 2-[(4-bromobenzyl)sulfonyl]ethanol (11.00 g, 44.7 mmol) was dissolved in dcm (50 ml, dry) to which m - cpba (21.10 g, 122.3 mmol) was added at room temperature with stirring. then 15 min after the addition of m - cpba, a white solid material precipitated. the organic layer was extracted with brine and collected, dried (mgso4), filtered, and the solvent removed under reduced pressure. the crude product was applied to a gel column chromatography using ethyl acetate / n - hexane 1/1, rf = 0.1 to give the pure material as white crystals (4.47 g, 36%) after recrystallization from ethyl acetate / n - hexane, mp 9799 c. h nmr (dmso - d6) : 7.61 (2h, d, j = 8.0 hz), 7.37 (2h, d, j = 8.0 hz), 5.21 (1h, br s, oh), 4.48 (2h, s), 3.83 (2h, t, j = 14.3 hz), 3.18(2h, t, j = 14.3 hz). c nmr (dmso - d6) : 133.2, 131.3, 128.1, 121.8, 58.8, 54.9, 53.9. ir (kbr) : 3480 (br), 2991, 2928, 1490, 1408, 1388, 1291, 1262, 1235, 1115 (s), 1064, 1014, 848, 808, 705, 523 cm. 2-[(4-methylbenzyl)sulfanyl]ethanol (9.60 g, 52.7 mmol) was dissolved in dcm (50 ml, dry) to which m - cpba (18.18 g, 105.0 mmol, 2.0 mol equiv) was added at room temperature with stirring. then 15 min after the addition of m - cpba, a white solid material precipitated. the organic layer was extracted with brine and collected, dried (mgso4), filtered, and the solvent removed under reduced pressure. the crude product was recrystallized from ethyl acetate / n - hexane to give the pure material as white crystals (3.11 g). the mother liquor was applied to a silica gel column chromatography using ethyl acetate / n - hexane 1/1 (rf = 0.2 and rf = 0.5) to give an additional amount (0.40 g). the total amount obtained was (3.51 g, 31%), mp 102104 c. h nmr (dmso - d6) : 7.307.28 (2h, d, j = 8.0 hz), 7.217.19 (2h, d, j = 8.0 hz), 5.29 (1h, br, s, oh), 4.41 (2h, s), 3.823.79 (2h, t, j = 14.3 hz), 3.143.11 (2h, t, j = 14.3 hz), 2.31 (3h, s). c nmr (dmso - d6) : 137.6, 130.9, 128.9, 125.6, 59.3, 54.9, 53.7, 20.7. ir (kbr) : 1697, 1517, 1419, 1296 (s), 1263, 1175, 1116 (s), 1063, 1012, 848, 549, 489 cm. 2-[(4-bromobenzyl)sulfonyl]ethanol (2.00 g, 7.17 mmol) was dissolved in dcm (25 ml, dry) to which triethylamine (3 ml, 2.18 g, 21.52 mmol, 3.0 mol equiv, anhydrous) was added followed by methylsulfonyl chloride (2 ml, 3.12 g, 19.14 mmol, 2.7 mol equiv) at 0 c with stirring, which was continued at room temperature overnight. the organic layer was collected after the extraction, dried over (mgso4), filtered, and the solvent removed under reduced pressure. the crude product was applied to a silica gel column chromatography using ethyl acetate / n - hexane (1/2, rf = 0.4). the required product (1.60 g, 86%) h nmr (dmso - d6) : 7.60 (2h, d, j = 8.4 hz), 7.32 (2h, d, j = 8.4 hz), 6.96 (1h, dd, j = 16.8 hz and j = 9.9 hz), 6.21 (1h, d, j = 8.0 hz), 6.09 (1h, d, j = 16.6 hz), 4.55 (2h, s). c nmr (dmso - d6) : 136.2, 133.1, 131.3, 130.5, 128.1, 121.9, 58.0. ir (kbr) : 3437, 3057, 2981, 2932, 1489, 1408, 1309, 1260, 1161, 1121, 1072, 1014, 976, 843, 795, 714,519 cm. 2-[(4-methylbenzyl)sulfonyl]ethanol (3.110 g, 14.51 mmol) was dissolved in dcm (25 ml, dry) to which triethylamine (2.202 g, 21.77 mmol, 1.5 mol equiv, anhydrous) was added, followed by methylsulfonyl chloride (2.493 g, 21.77 mmol, 1.5 mol equiv) at 0 c with stirring, which was continued at room temperature overnight. the organic layer was collected after the extraction, dried (mgso4), filtered, and the solvent removed under reduced pressure. tlc showed two spots : rf = 0.3 and rf = 0.6 (ethyl acetate / n - hexane 1/1). 1-bromo-4-[(vinylsulfonyl)methyl]benzene (1.600 g, 6.12 mmol) was dissolved in dcm (25 ml, dry) to which dimethylamine (4 ml, 2 m in thf) was added at room temperature with stirring. the organic layer was collected, dried over (mgso4), and filtered, and the solvents were removed under reduced pressure, after which the crude product was applied to a silica gel column chromatography using ethyl acetate / n - hexane (1/1) in the first instance, followed by ethyl acetate / methanol (9/2, rf = 0.5). the product was obtained as a white solid material (1.36 g, 73%) after trituration with n - hexane, mp 5860 c. h nmr (dmso - d6) : 7.64 (2h, d, j = 8.0 hz), 7.37 (2h, d, j = 8.0 hz), 4.52 (2h, s), 3.22 (2h, t, j = 14.3 hz), 2.67 (2h, t, j = 14.3 hz), 2.17 (6h, s). c nmr (dmso - d6) : 133.1, 131.4, 127.9, 121.9, 57.9, 51.6, 49.4, 44.7. ir (kbr) : 3424, 2979, 2942, 2822, 2772, 1591, 1488, 1408, 1295, 1275, 1118, 1051, 1013, 843, 816, 640,513 cm. the product from the previous experiment (4.880 g) was dissolved in dcm (50 ml, dry) to which dimethylamine (4 ml, 2 m in thf) was added at room temperature with stirring. the stirring was continued at room temperature overnight, after which time the reaction mixture was extracted with a saturated solution of sodium carbonate. the organic layer was collected, dried (mgso4), and filtered, and the solvents were removed under reduced pressure and the crude product was applied to a silica gel column chromatography eluted with ethyl acetate / n - hexane (1/1, rf = 0.1) in the first instance, followed by ethyl acetate / methanol (9/1). the product was obtained as white solid material (2.200 g, 63% based on 2-[(4-methylbenzyl)sulfonyl]ethanol) after trituration with n - hexane, mp 6870 c. h nmr (dmso - d6) : 7.28 (2h, d, j = 8.0 hz), 7.21 (2h, d, j = 8.0 hz), 4.44 (2h, s), 3.17 (2h, t, j = 14.3 hz), 2.65 (2h, t, j = 14.3 hz), 2.31 (3h, s), 2.16 (6h, s). c nmr (dmso - d6) : 137.7, 130.8, 129.0, 125.4, 58.4, 51.6, 49.0, 44.9, 20.7. ir (kbr) : 1511, 1463, 1399, 1380, 1314, 1258, 1156, 1119, 1050, 892, 853, 822, 749 cm. 2-[(4-bromobenzy)lsulfonyl]ethyl methanesulfonate (0.06 g, 0.17 mmol) was suspended in trimethylamine (5 ml in toluene), and the mixture was stirred at room temperature under nitrogen for a period of 5 d, then heated to 60 c for 20 h. the solvent was concentrated under reduced pressure to give an off - white solid (0.045 g, 83%), mp 187188 c. h nmr (dmso - d6) : 7.667.64 (2h, d, j = 8.0 hz), 7.407.38 (2h, d, j = 8.0 hz), 4.64(2h, s), 3.853.80 (2h, m), 3.783.74 (2h, m), 3.12 (9h, s). c nmr (dmso - d6) : 133.1, 131.6, 126.3, 121.9, 57.6, 57.4, 52.5, 44.2. ir (kbr) : 1591, 1489, 1424, 1413, 1360, 1318, 1277, 1193, 1140, 1068, 1040, 1011, 790, 770 cm. n, n - dimethyl-2-[(4-methylbenzyl)sulfonyl]ethanamine (0.950 g, 3.94 mmol) was dissolved in dcm (25 ml, dry) to which iodomethane (4 ml) was added with stirring at room temperature. the white solid material formed was filtered off and dried under reduced pressure (1.490 g, 99%), mp 212214 c. h nmr (dmso - d6) : 7.33 (2h, d, j = 8.0 hz), 7.25 (2h, d, j = 8.0 hz), 4.58 (2h, s), 3.833.79 (2h, m), 3.743.71 (2h, m), 3.12 (9h, s), 2.33 (3h, s). c nmr (dmso - d6) : 138.1, 131.0, 129.2, 124.3, 57.9, 57.3, 52.5, 45.0, 18.6. ir (kbr) : 1514, 1486, 1326, 1257, 1153, 1123, 1015, 882 cm. similarly, the following were prepared : 11c11p, 12c12d, 16d16f, 17d17f. (vinylsulfonyl)benzene (commercially available) (100 mg, 0.60 mmol) was dissolved in dcm (4 ml, dry) to which n, n, n - trimethyl-1,2-ethanediamine (61 mg, 0.062 ml, 0.60 mmol) was added at room temperature with stirring. the solvent was removed under reduced pressure to give the required product as a clear pale - yellow oil in quantitative yield. h nmr (dmso - d6) : 7.93 (2h, t, j = 7.3 hz), 7.77 (1h, t, j = 7.3 hz), 7.68 (2h, t, j = 7.3 hz), 3.49 (2h, t, j = 7.2 hz), 2.68 (2h, t, j = 7.4 hz), 2.31 (2h, t, j = 7.4 hz), 2.13 (2h, t, j = 7.4 hz), 2.05 (9h, s). ir : 1682, 1606, 1449, 1300, 1199, 1143, 1084, 1037, 744, 731, 689 cm. similarly, the following were prepared : 18b18l. phenylmethanesulfonyl chloride [commercially available ] (1.034 g, 5.416 mmol) was dissolved in dichloromethane (10 ml, dry) to which n, n - dimethyl-1,2-ethanediamine [commercially available ] (0.477 g, 5.416 mmol) dissolved in dichloromethane (20 ml, dry) was added dropwise at room temperature with stirring under nitrogen. the reaction mixture was washed with aqueous sodium hydroxide solution (318 mg, 7.95 mmol in 10 ml). the organic layer was separated, dried (mgso4), and the solvent removed under reduced pressure to give the required product as a white microcrystalline solid (1.100 g, 84%), mp 127129 c, rf = 0.1 [tlc : basic, 99% ethyl acetate and 1% tea ]. h nmr (dmso - d6) : 7.387.32 (5h, m), 6.93 (1h, br), 4.35 (2h, s), 2.93 (2h, t, j = 6.7 hz), 2.25 (2h, t, j = 6.9 hz), 2.11 (6h, s). ir [kbr ] : 1495, 1458, 1415, 1327, 1261, 1128, 1149, 1050, 893, 784 cm. calculated for c11h18n2o2s : c, 54.52 ; h, 7.49 ; n, 11.56 ; s, 13.23. found : c, 54.76 ; h, 7.48 ; n, 11.62 ; s, 13.49. hrfabms : calcd for c11h19o2n2s, 243.1167 ; found, 243.1169. similarly, the following were prepared : 19b19z. 2-naphthylmethanesulfonyl chloride (50 mg, 0.208 mmol) was dissolved in dichloromethane (1 ml, dry) to which 2-(4-morpholinyl)ethanamine [commercially available ] (27 mg, 34 l, 0.208 mmol) was added neat dropwise at room temperature with stirring under nitrogen. the reaction mixture was washed with aqueous sodium hydroxide solution (123 mg, 3.08 mmol in 6 ml). the organic layer was separated, dried (mgso4), and the solvent removed under reduced pressure. the product was obtained as a white solid after recrystallization from ethyl acetate / n - hexane (60 mg, 86%), mp 131134 c, rf = 0.2 [ethyl acetate only ]. h nmr (dmso - d6) : 7.927.90 (4h, m), 7.547.51 (3h, m), 6.96 (1h, br), 4.55 (2h, s), 3.55 (4h, t, j = 4.8 hz), 3.05 (2h, t, j = 6.8 hz), 2.362.32 (6h, m). ir (kbr) : 1676, 1642, 1594, 1561, 1506, 1462, 1423, 1308, 1174, 1118, 1070, 980, 911, 870, 823, 752, 722 cm. calculated for c17h22n2o3s : c, 61.05 ; h, 6.63 ; n, 8.38. found : c, 61.12 ; h, 6.73 ; n, 8.01. benzylamine (1.275 g, 11.89 mmol) was dissolved in dcm (5 ml, dry) at 0 c with stirring under n2 to which a solution of 2-chloroethanesulfonyl chloride (0.969 g, 5.94 mmol) dissolved in dcm (5 ml, dry) was added at 0 c with stirring under n2, after which time the reaction mixture was stirred at room temperature for 2 h. the reaction mixture was extracted with dilute hydrochloric acid, and the organic layers were collected, dried (mgso4), filtered, and the solvent removed under reduced pressure. the crude product was purified by column chromatography (ethyl acetate / n - hexane 1/3, rf = 0.2). h nmr (dmso - d6) : 7.82 (1h, t, j = 6.3 hz), 7.357.25 (5h, m), 6.69 (1h, dd, j = 10.0 and 16.5 hz), 6.04 (1h, d, j = 16.5 hz), 5.95 (1h, d, j = 10.0 hz), 4.04 (2h, d, j = 6.2 hz). ir (nacl) : 1497, 1456, 1385, 1327, 1148, 1061, 971, 843, 740 cm. calculated for c9h11no2s : c, 54.80 ; h, 5.62 ; n, 7.10. found : c, 54.89 ; h, 5.72 ; n, 7.04. benzylethylenesulfonamide (140 mg, 0.709 mmol) was dissolved in dcm (5 ml, dry) at room temperature to which pyrrolidine (500 mg ; 590 l, 7.09 mmol) was added at room temperature with stirring. the reaction mixture was left standing at room temperature for 48 h. solvent and excess pyrrolidine were removed under reduced pressure. the white solid material obtained was triturated with n - hexane and filtered to give the required product as a white solid (135 mg, 71%), rf = 0.2 (ethyl acetate / nmm 100/1 ; basic tlc), mp 100103 c. h nmr (dmso - d6) : 7.60 (1h, t, j = 6.2 hz), 7.367.24 (5h, m), 4.15 (2h, d, j = 6.2 hz), 3.11 (2h, t, j = 7.5 hz), 2.71 (2h, t, j = 7.9 hz), 2.372.32 (4h, m), 1.671.63 (4h, m). ir (kbr) : 1642, 1458, 1331, 1133, 1072, 1017, 870, 761, 738, 705 cm. calculated for c13h20n2o2s : c, 58.18 ; h, 7.51 ; n, 10.44. found : c, 58.03 ; h, 7.84 ; n, 10.32. aniline (1.59 g, 17.12 mmol) was dissolved in dcm (25 ml, dry) at 0 c with stirring under n2 to which nmm (1.73 g ; 1.88 ml ; 17.12 mmol) was added with stirring. 2-chloroethanesulfonyl chloride (2.79 g, 1.80 l, 17.12 mmol) was added at room temperature with stirring under n2. the reaction mixture was stirred at room temperature for 2 h. the reaction mixture was then extracted with dilute hydrochloric acid, and the organic layers were collected, dried (mgso4), filtered, and the solvent removed under reduced pressure. the crude product was purified by column chromatography (ethyl acetate / n - hexane 1/3, rf = 0.3). the product was obtained as a light - brown solid (1.918 g, 61%) after trituration with n - hexane, mp 5457 c [lit. h nmr (dmso - d6) : 9.96 (1h, s), 7.317.26 (1h, 2, m), 7.157.12 (2h, m), 7.08 (1h, t, j = 7.3 hz), 6.79 (1h, dd, j = 10.0 and 16.5 hz), 6.11 (1h, d, j = 16.5 hz), 6.03 (1h, d, j = 10.0 hz). ir (kbr) : 1597, 1488, 1410, 1321, 1218, 1151, 968, 924, 751 cm. calculated for c8h9no2s : c, 52.44 ; h, 4.95 ; n, 7.64. found : c, 52.33 ; h, 4.85 ; n, 7.54. n - phenylethylenesulfonamide (300 mg, 1.64 mmol) was dissolved in dichloromethane (5 ml, dry) to which dimethylamine (2 ml, 2.0 m solution in thf) was added at room temperature with stirring. volatile material was removed under reduced pressure, and the crude product obtained was applied to a silica gel column chromatography. the product was eluted with ethyl acetate / nmm (100/1), rf = 0.3. the required product was obtained as a white solid after recrystallization from ethyl acetate / n - hexane (340 mg, 91%), mp 6871 c [lit. h nmr (dmso - d6) : 9.77 (1h, br), 7.337.06 (5h, m), 3.19 (2h, t, j = 7.4 hz), 2.62 (2h, t, j = 7.4 hz), 2.05 (6h, s). calculated for c10h16n2o2s : c, 52.61 ; h, 7.06 ; n, 12.27. found : c, 52.75 ; h, 7.25 ; n, 12.14. n, n - dimethyl-1,2-ethanediamine (500 mg, 620 l, 5.67 mmol) was dissolved in dcm (15 ml, dry). phenylacetyl chloride (877 mg, 5.67 mmol) was dissolved in dcm (15 ml, dry) then added dropwise with stirring at 0 c to the amine solution. the reaction mixture was extracted with sodium hydroxide solution (500 mg in 25 ml of water). the organic layer was collected and dried (mgso4), and the solvent was removed under reduced pressure. the crude product was purified by column chromatography using ethyl acetate / nmm / methanol (98/1/1) to give the product as white crystals (0.940 g, 80%), mp 4042 c. h nmr (dmso - d6) : 7.92 (1h, br), 7.297.17 (5h, m), 3.38 (2h, s), 3.14 (2h, q, j = 6.8 hz), 2.27 (2h, t, j = 6.8 hz), 2.11 (6h, s). ir (kbr) : 1652, 1554, 1459, 1354, 1314, 1268, 1188, 1086, 859, 701 cm. calculated for c12h18n2o : c, 69.87 ; h, 8.80 ; n, 13.58. found : c, 69.94 ; h, 8.84 ; n, 14.11. mice were bred and/or maintained in accordance with the home office uk licenses ppl60/3580, ppl60/3119, and pil60/12183 and the ethics review board of the universities of glasgow and strathclyde. collagen - induced arthritis (cia) was induced in male dba/1 mice (810 weeks old ; harlan olac ; bicester, uk) by intradermal immunization with bovine type ii collagen (cii, md biosciences, switzerland) in complete freund s adjuvant (cfa) on day 0 and by intraperitoneal challenge (in pbs) on day 21. mice were treated with purified endotoxin - free pc - bsa, bsa (both 2 g / dose ; prepared as previously described), and the absence of endotoxin confirmed using an endosafe kit (charles river laboratories, uk), 11a (1 g / dose), or pbs subcutaneously on days 2, 0, and 21 and arthritis scored as previously described. isolated draining lymph node (dln) cells (10/ml) were stimulated in rpmi medium supplemented with 10% heat - inactivated fetal calf serum (hi fcs), 2 mm l - glutamine, 50 u / ml penicillin, and 50 g / ml streptomycin (all from gibco life technologies, uk) (complete rpmi) 50 ng / ml phorbol 12-myristate 13-acetate (pma ; sigma - aldrich, uk) plus 500 ng / ml ionomycin (sigma - aldrich) before addition of 10 g / ml brefeldin a (sigma - aldrich) for 5 h at 37 c with 5% co2. phenotypic markers were labeled using anti - cd4-percp, anti - cd8-fitc, or anti--pe (biolegend, uk) antibodies before the cells were fixed and permeabilized using biolegend protocols. cells were then labeled using anti - ifn-pacific blue and anti - il-17a - apc (biolegend) antibodies for 30 min prior to flow cytometry and gated according to appropriate isotype controls. bone marrow was isolated from mouse femurs from 68 week - old, male balb / c mice and cells cultured for 7 days at 37/5% co2 in complete dulbecco s modified eagle s medium (dmem ; gibco life technologies) supplemented with 20% l929 cell culture supernatant (contains csf-1), 20% hi fcs, 2 mm l - glutamine, 50 u / ml penicillin, and 50 g / ml streptomycin with fresh medium being added on day 4. the cells were analyzed by flow cytometry and were shown routinely to be 99% positive for cd11b (bd pharmingen, uk) and f4/80 markers (ebioscience, uk). bmms were cultured in rpmi complete medium in triplicate (2 10 cells / well) in 96-well plates and were rested overnight prior to stimulation with 5 g / ml of the relevant compounds diluted in complete rpmi for 18 h. in some experiments, bmms were then stimulated with either 100 ng / ml salmonella minnesota lps (sigma - aldrich, uk), 10 ng / ml blp (pam3csk4 ; axxora ltd., uk), or 0.01 m cpg (source bioscience autogen, uk) for 24 h. culture supernatants were then removed and stored at 20 c until analysis. elisas for il-12p40 and il-6 cytokines (limit of sensitivity : 16 pg / ml) were performed using paired antibodies from bd bioscience pharmingen. bmms were cultured in 6-well plates (4 10 cells / well) in complete rpmi medium. after 24 h, the medium was changed and the cells were pretreated with the compounds (5 g / ml) for 18 h before being stimulated with 100 ng / ml lps, 10 ng / ml blp, or 1 m cpg for 1 h. the ability of the compounds per se to activate nfb p65 was also tested. activated nfb p65 was measured in nuclear fractions (isolated using a nuclear extraction kit ; activemotif, uk) by the elisa - based transam kit (activemotif) according to the manufacturer s instructions. cell viability was determined by 7-amino actinomycin d (7-aad ; bd pharmingen) staining. bmms (2 10/well) were pretreated with 11a (5 g / ml) or es-62 (2 g / ml) for 18 h prior to being stimulated with either 100 ng / ml lps, 10 ng / ml blp, or 0.01 m cpg for 24 h. the ability of the compounds alone to induce cell death was also tested. the cells were washed in pbs containing 1% fcs, then subsequently incubated with 5 l of 7-aad for 10 min on ice in the dark. flow cytometry was conducted using a facscanto system (becton dickinson pharmingen) and the data processed by flowjo software (tree star inc. flow cytometric analysis of myd88 expression was performed using a rabbit anti - mouse myd88 antibody (ab2068 ; abcam) and fitc - conjugated goat anti - rabbit igg (vector laboratories). prior to staining, dead cells were discriminated by use of live / dead stain (aqua ; invitrogen) before being fixed and permeabilized using biolegend reagents and protocols. flow cytometry was conducted using the lsrii system (becton dickinson pharmingen) and the data processed by flowjo software (tree star inc. bone marrow - derived dendritic cells (bmdcs) from femurs of c57bl/6 or balb / c mice (68 weeks old) were derived by in vitro culture in complete rpmi 1640 medium (containing 2 mm glutamine, 50 u / ml penicillin, 50 g / ml streptomycin, and 10% fcs) supplemented with recombinant murine gm - csf (10 ng / ml, peprotech inc.). nave cd4cd62l t cells were isolated from lymph nodes using magnetic bead technology according to the manufacturer s instructions (miltenyi). for bmdc - t cell cocultures, bmdcs were incubated 11a (5 g / ml) prior to treatment lps from s. minnesota and then pulsed with ovalbumin (ova) peptide (0300 nm) before incubation with nave t cells derived from ova - specific do.11.10/balb / c or ot - ii / c57bl/6 mice for 3 d. conditioned medium from dc cultures and dc - t cell cocultures were analyzed for cytokine production by elisa for il-17a (biolegend), tnf-, il-6, and il-23 (r&d systems) according to manufacturer s instructions. bmms (10 cells / sample) were lysed by the addition of ice - cold, modified ripa buffer (50 mm tris, ph 7.4, 150 mm sodium chloride, 2% (v / v) np-40, 0.25% (w / v) sodium deoxycholate, 1 mm egta, 10 mm sodium orthovanadate plus 0.5 mm phenylmethylsulfonyl fluoride, 10 g / ml chymostatin, leupeptin, antipapain, and pepstatin) and solubilized on ice for 30 min. protein (30 g) samples were resolved on the xcell surelock mini - cell kit with nupage novex high - performance precast bis - tris gels and nupage buffers and reagents (invitrogen life technologies) at 200 v for 50 min. proteins were transferred to nitrocellulose (ge), and membranes were blocked by incubating for 1 h in 5% nonfat milk in tbs / tween (0.5 m nacl and 20 mm tris ph7.5 with 0.1% (v / v) tween-20) at rt. membranes were incubated with primary antibody diluted in 5% bsa in tbs / tween buffer overnight at 4 c, washed with tbs / tween, and incubated with the appropriate horseradish peroxidase (hrp)-conjugated secondary antibody in 5% nonfat milk for 1 h at rt. membranes were then washed with tbs / tween, and protein bands were visualized using the ecl detection system. quantification of the bands was performed using imagej software (national institutes of health, usa). parametric data were analyzed by one - tailed student s t test or by one - way anova followed by bonferroni s post - test. whitney test was used for analysis of clinical cia scores where p < 0.05, p < 0.01 and p < 0.001 mice were bred and/or maintained in accordance with the home office uk licenses ppl60/3580, ppl60/3119, and pil60/12183 and the ethics review board of the universities of glasgow and strathclyde. collagen - induced arthritis (cia) was induced in male dba/1 mice (810 weeks old ; harlan olac ; bicester, uk) by intradermal immunization with bovine type ii collagen (cii, md biosciences, switzerland) in complete freund s adjuvant (cfa) on day 0 and by intraperitoneal challenge (in pbs) on day 21. mice were treated with purified endotoxin - free pc - bsa, bsa (both 2 g / dose ; prepared as previously described), and the absence of endotoxin confirmed using an endosafe kit (charles river laboratories, uk), 11a (1 g / dose), or pbs subcutaneously on days 2, 0, and 21 and arthritis scored as previously described. isolated draining lymph node (dln) cells (10/ml) were stimulated in rpmi medium supplemented with 10% heat - inactivated fetal calf serum (hi fcs), 2 mm l - glutamine, 50 u / ml penicillin, and 50 g / ml streptomycin (all from gibco life technologies, uk) (complete rpmi) 50 ng / ml phorbol 12-myristate 13-acetate (pma ; sigma - aldrich, uk) plus 500 ng / ml ionomycin (sigma - aldrich) before addition of 10 g / ml brefeldin a (sigma - aldrich) for 5 h at 37 c with 5% co2. phenotypic markers were labeled using anti - cd4-percp, anti - cd8-fitc, or anti--pe (biolegend, uk) antibodies before the cells were fixed and permeabilized using biolegend protocols. cells were then labeled using anti - ifn-pacific blue and anti - il-17a - apc (biolegend) antibodies for 30 min prior to flow cytometry and gated according to appropriate isotype controls. bone marrow was isolated from mouse femurs from 68 week - old, male balb / c mice and cells cultured for 7 days at 37/5% co2 in complete dulbecco s modified eagle s medium (dmem ; gibco life technologies) supplemented with 20% l929 cell culture supernatant (contains csf-1), 20% hi fcs, 2 mm l - glutamine, 50 u / ml penicillin, and 50 g / ml streptomycin with fresh medium being added on day 4. the cells were analyzed by flow cytometry and were shown routinely to be 99% positive for cd11b (bd pharmingen, uk) and f4/80 markers (ebioscience, uk). bmms were cultured in rpmi complete medium in triplicate (2 10 cells / well) in 96-well plates and were rested overnight prior to stimulation with 5 g / ml of the relevant compounds diluted in complete rpmi for 18 h. in some experiments, bmms were then stimulated with either 100 ng / ml salmonella minnesota lps (sigma - aldrich, uk), 10 ng / ml blp (pam3csk4 ; axxora ltd., uk), or 0.01 m cpg (source bioscience autogen, uk) for 24 h. culture supernatants were then removed and stored at 20 c until analysis. elisas for il-12p40 and il-6 cytokines (limit of sensitivity : 16 pg / ml) were performed using paired antibodies from bd bioscience pharmingen. bmms were cultured in 6-well plates (4 10 cells / well) in complete rpmi medium. after 24 h, the medium was changed and the cells were pretreated with the compounds (5 g / ml) for 18 h before being stimulated with 100 ng / ml lps, 10 ng / ml blp, or 1 m cpg for 1 h. the ability of the compounds per se to activate nfb p65 was also tested. activated nfb p65 was measured in nuclear fractions (isolated using a nuclear extraction kit ; activemotif, uk) by the elisa - based transam kit (activemotif) according to the manufacturer s instructions. cell viability was determined by 7-amino actinomycin d (7-aad ; bd pharmingen) staining. bmms (2 10/well) were pretreated with 11a (5 g / ml) or es-62 (2 g / ml) for 18 h prior to being stimulated with either 100 ng / ml lps, 10 ng / ml blp, or 0.01 m cpg for 24 h. the ability of the compounds alone to induce cell death was also tested. the cells were washed in pbs containing 1% fcs, then subsequently incubated with 5 l of 7-aad for 10 min on ice in the dark. flow cytometry was conducted using a facscanto system (becton dickinson pharmingen) and the data processed by flowjo software (tree star inc. flow cytometric analysis of myd88 expression was performed using a rabbit anti - mouse myd88 antibody (ab2068 ; abcam) and fitc - conjugated goat anti - rabbit igg (vector laboratories). prior to staining, dead cells were discriminated by use of live / dead stain (aqua ; invitrogen) before being fixed and permeabilized using biolegend reagents and protocols. flow cytometry was conducted using the lsrii system (becton dickinson pharmingen) and the data processed by flowjo software (tree star inc. bone marrow - derived dendritic cells (bmdcs) from femurs of c57bl/6 or balb / c mice (68 weeks old) were derived by in vitro culture in complete rpmi 1640 medium (containing 2 mm glutamine, 50 u / ml penicillin, 50 g / ml streptomycin, and 10% fcs) supplemented with recombinant murine gm - csf (10 ng / ml, peprotech inc.). nave cd4cd62l t cells were isolated from lymph nodes using magnetic bead technology according to the manufacturer s instructions (miltenyi). for bmdc - t cell cocultures, bmdcs were incubated 11a (5 g / ml) prior to treatment lps from s. minnesota and then pulsed with ovalbumin (ova) peptide (0300 nm) before incubation with nave t cells derived from ova - specific do.11.10/balb / c or ot - ii / c57bl/6 mice for 3 d. conditioned medium from dc cultures and dc - t cell cocultures were analyzed for cytokine production by elisa for il-17a (biolegend), tnf-, il-6, and il-23 (r&d systems) according to manufacturer s instructions. bmms (10 cells / sample) were lysed by the addition of ice - cold, modified ripa buffer (50 mm tris, ph 7.4, 150 mm sodium chloride, 2% (v / v) np-40, 0.25% (w / v) sodium deoxycholate, 1 mm egta, 10 mm sodium orthovanadate plus 0.5 mm phenylmethylsulfonyl fluoride, 10 g / ml chymostatin, leupeptin, antipapain, and pepstatin) and solubilized on ice for 30 min. protein (30 g) samples were resolved on the xcell surelock mini - cell kit with nupage novex high - performance precast bis - tris gels and nupage buffers and reagents (invitrogen life technologies) at 200 v for 50 min. proteins were transferred to nitrocellulose (ge), and membranes were blocked by incubating for 1 h in 5% nonfat milk in tbs / tween (0.5 m nacl and 20 mm tris ph7.5 with 0.1% (v / v) tween-20) at rt. membranes were incubated with primary antibody diluted in 5% bsa in tbs / tween buffer overnight at 4 c, washed with tbs / tween, and incubated with the appropriate horseradish peroxidase (hrp)-conjugated secondary antibody in 5% nonfat milk for 1 h at rt. membranes were then washed with tbs / tween, and protein bands were visualized using the ecl detection system. quantification of the bands was performed using imagej software (national institutes of health, usa). parametric data were analyzed by one - tailed student s t test or by one - way anova followed by bonferroni s post - test. whitney test was used for analysis of clinical cia scores where p < 0.05, p < 0.01 and p < 0.001 | in spite of increasing evidence that parasitic worms may protect humans from developing allergic and autoimmune diseases and the continuing identification of defined helminth - derived immunomodulatory molecules, to date no new anti - inflammatory drugs have been developed from these organisms. we have approached this matter in a novel manner by synthesizing a library of drug - like small molecules based upon phosphorylcholine, the active moiety of the anti - inflammatory acanthocheilonema viteae product, es-62, which as an immunogenic protein is unsuitable for use as a drug. following preliminary in vitro screening for inhibitory effects on relevant macrophage cytokine responses, a sulfone - containing phosphorylcholine analogue (11a) was selected for testing in an in vivo model of inflammation, collagen - induced arthritis (cia). testing revealed that 11a was as effective as es-62 in protecting dba/1 mice from developing cia and mirrored its mechanism of action in downregulating the tlr / il-1r transducer, myd88. 11a is thus a novel prototype for anti - inflammatory drug development. |
oxidized low - density lipoprotein (oxldl) plays a critical role in atherosclerotic plaque formation, which is triggered by the persistence of lipid - laden macrophages as well as interfering endothelial cell motility in arterial wall. the differentiation of monocytes into macrophages, which accumulate oxldl to form foam cells, is induced by oxldl [1, 3 ]. the process starts with endothelial activation in the subendothelial areas by oxldl deposition [4, 5 ]. these macrophages then take up modified lipoproteins and, as they accumulate excess lipids, they form foam cells. the foam cells die and release intracellular contents, which induce an inflammatory reaction., ldl may be oxidized by enzymes, for example, myeloperoxidase [8, 9 ], or by reactive oxygen species (ros) in the inflammatory microenvironment of the plaque. although oxldl is important for atherosclerotic inflammation, there is no report about the oxidation degree of ldl and macrophage differentiation. monocytes that are recruited into the intima are exposed to a variety of stimuli, such as cytokines, lipids, iron, and calcium, which exist in complex microenvironments. these factors can influence the phenotypic polarization of macrophages and their subsequent activation. classically activated (m1) macrophages and alternatively activated (m2) macrophages are well - defined phenotypes. in addition, recent studies suggested that several subtypes of m2 macrophages (m2a, b, c, and d) and stimuli - induced subpopulations such as mox, mhem, m (hb), and m4 macrophages exist in atherosclerotic lesions. therefore, we hypothesized that ldl with different oxidation levels exists in the early atherosclerotic milieu and that the oxidation degree of ldl influences macrophage differentiation into classically activated (m1) or alternatively activated (m2) macrophages, which determines the inflammatory fate for atherosclerotic development. in this study, monocytes were treated with high - oxidized ldl (hi - oxldl) or low - oxidized ldl (low - oxldl) and their surface receptor expression and cytokine secretion profiles were analyzed to determine if they differentiated into m1 or m2 macrophages., low - oxldl migrates further than the native ldl and is also defined by a low tbar value (8.711.8 nmol / mg protein). hi - oxldl migrates 2.7 fold further than the native ldl and is defined by a high tbar value (55.775.6 nmol / mg protein). anti - human cd86-pe and anti - human cd206- (mannose receptor-) fitc antibodies were purchased from beckman coulter inc. (brea, ca, usa) and bd biosciences (san jose, ca, usa), respectively. anti - lox-1 and anti - tubulin antibodies were purchased from abcam (cambridge, ma, usa) and biolegend (san diego, ca, usa), respectively. recombinant human m - csf was purchased from biolegend (san diego, ca, usa). lps (e. coli o26:b6) was purchased from sigma - aldrich (st. louis, mo, usa). the human monocytic thp-1 cell line was purchased from atcc and cultured in rpmi1640 medium containing 10% fbs and penicillin - streptomycin (100 units / ml and 100 g / ml, resp.) at 37c in a humidified 5% co2 incubator. the cell proliferation rate was measured using the colorimetric cell counting kit-8 (cck-8) (dojindo laboratories, kyoto, japan). thp-1 cells were cultured at 1 10 cells per well in 24-well plates and treated with native ldl, low - oxldl, or hi - oxldl (50 g / ml each). after 24, 48, or 96 h, the cck-8 reagent was added to each well and cells were incubated for 30 min at 37c. the supernatant was harvested and transferred to 96-well plates. the o.d. at 450 thp-1 cells were cultured at 2 10 cells per well in 12-well plates and treated with native ldl, low - oxldl, or hi - oxldl (50 g / ml each). after 24 or 96 h, cells were harvested and analyzed using flow cytometry to measure granularity and autofluorescence. to detect surface markers, cells were treated with native ldl, low - oxldl, or hi - oxldl (50 g / ml each) for 96 h and stained with pe - conjugated anti - human cd86 antibodies. flow cytometric analysis was performed by a bd lsr ii flow cytometer (bd bioscience). thp-1 cells were cultured in 2-well chambered cover glasses at 2 10 cells per well and treated with native ldl, low - oxldl, or hi - oxldl (50 g / ml each) for 96 h. attached cells were stained with fitc - conjugated anti - human mannose receptor (cd206) antibodies and analyzed using an fv1000 microscope (olympus, tokyo, japan). blood was obtained from healthy donors after acquiring internal review board approval and informed consent (number 4 - 2012 - 0088). peripheral blood mononuclear cells (pbmcs) were separated by ficoll - hypaque density gradient centrifugation (density = 1.077) at 1,600 rpm for 30 min and human monocytes were purified from pbmcs using monocyte isolation kit ii (miltenyi biotec, bergsch gladbach, germany). staining for cd14 was performed to confirmed monocyte population and usually > 95% of cells were positive. (100 units / ml and 100 g / ml, resp.) at 37c in a humidified 5% co2 incubator. human monocytes were cultured in 12-well plates at 2 10 cells per well and incubated with both m - csf (50 ng / ml) and hi - oxldl or low - oxldl (50 g / ml each) for 7 days. morphological changes were observed using bright - field microscope (ix71, olympus, tokyo, japan). thp-1 cells were cultured at 2 10 cells per well in 12-well plates and pretreated with native ldl, low - oxldl, or hi - oxldl (50 g / ml each) for 24, 48, or 96 h. the medium was replaced with fresh medium and then cells were stimulated with lps (100 ng / ml). primary monocytes were cultured at 2 10 cells per well in 12-well plates and pretreated with both m - csf (50 ng / ml) and native ldl, low - oxldl, or hi - oxldl (50 g / ml each) for 24, 48, or 72 h. primary monocytes were stimulated with lps (20 ng / ml). eighteen hours after lps stimulation, culture supernatants were harvested and stored at 80c. elisa was performed with a human cytokine tnf-, il-12p40, il-6, and monocyte chemoattractant protein-1 (mcp-1) assay kit (bd bioscience). the o.d. at 450 thp-1 cells were seeded at 2 10 cells per well and treated with native ldl, low - oxldl, or hi - oxldl (50 g / ml each). after 24 and 48 h, cells were harvested and lysed with lysis buffer. each 50 g of protein samples was loaded in 12% sds - page and transferred onto nitrocellulose membrane (ge healthcare, nj, usa). protein bands were detected using a west save up western blot detection kit (ab frontier, seoul, korea). to investigate the effects of the degree of ldl oxidation, we assessed differentiation and proliferation of thp-1 cells after treatment with native ldl, low - oxldl, or hi - oxldl. cell granularity (figure 1(a)) and autofluorescence (figure 1(b)) were significantly increased in cells treated with hi - oxldl compared to cells treated with native ldl or low - oxldl. after treatment with hi - oxldl, the cell proliferation rate did not change compared to those of cells treated with native ldl. however, cell proliferation was significantly decreased in cells treated with low - oxldl compared to that in cells treated with hi - oxldl (figure 1(c)). these results indicate that hi - oxldl increases granularity and autofluorescence whereas low - oxldl decreases cell proliferation, suggesting that monocytes respond differently to oxidation degree of ldl. next, we tested whether the degree of ldl oxidation influences the differentiation of monocytes into macrophages. when cells were treated with low - oxldl, the expression level of the marker for m1 macrophages, cd86, was increased compared to that in cells treated with hi - oxldl (figure 2(a)). in contrast, the expression level of the marker for m2 macrophages, mannose receptor (cd206), was significantly increased in cells treated with hi - oxldl compared to that in cells treated with native ldl or low - oxldl (figure 2(b)). these results indicate that the degree of ldl oxidation affects the differentiation of monocytes into different subtypes of macrophages. to assess cytokine production, thp-1 cells were preexposed to differentially oxidized ldl for 24, 48, or 96 h and then stimulated with lps. the production of tnf- and il-12p40 was decreased in cells pretreated with hi - oxldl compared to those in cells pretreated with low - oxldl (figure 3(a)). however, the production of il-6 and mcp-1 was significantly increased in cells pretreated with hi - oxldl compared to cells pretreated with low - oxldl (figure 3(b)). these results suggest that macrophages secrete different sets of cytokines depending on the oxidation level of ldl. oxldl binds to scavenger receptors, such as lox-1, and also induces the expression of lox-1 in macrophages. lox-1 expression increased in cells treated with hi - oxldl compared to cells treated with native ldl or low - oxldl after 24 or 48 h (figure 4). the expression of lox-1 was slightly but not significantly decreased in cells treated with low - oxldl compared to that in cells treated with native ldl (figure 4). when human monocytes were treated with low - oxldl, cells have changed into spindle - like shape, which is a unique feature of m1 macrophages whereas the treatment of hi - oxldl led to round cells, which represents m2 macrophages (figure 5(a)). the production of tnf- and il-12p40 was decreased in monocytes pretreated with hi - oxldl compared to cells pretreated with low - oxldl (figure 5(b)). moreover, the production of il-6 and mcp-1 was significantly increased in monocytes pretreated with hi - oxldl (figure 5(c)). these results support that the oxidation degree of ldl affects primary monocytes in a similar way to the monocytic cell line, thp-1 cells. during atherosclerotic plaque development, ldl can be oxidized to various degrees by ros or enzymes released by macrophages in the subendothelial area. this modified ldl, along with growth factors such as m - csf, affects differentiation of monocytes into macrophages. depending on the extent and duration of oxidation, the physicochemical properties of ldl, such as the charge, particle size, and lipid content, in addition, depending on whether the lipid or protein component is oxidized, oxidized ldl can induce different signaling pathways. we used two types of oxldl, which were classified as low - oxldl or hi - oxldl based on the tbar value for lipid peroxidation and relative electrophoretic mobility of ldl particles on agarose gels [14, 15 ]. oxldl is known to stimulate cell proliferation and induces differentiation of monocytes and macrophages [16, 17 ]. treatment of thp-1 cells with hi - oxldl led to an increase in cell granularity and autofluorescence, which is a feature of differentiated macrophages. moreover, hi - oxldl did not change the cell proliferation rate compared to native ldl. in contrast, in cells treated with low - oxldl, the granularity and autofluorescence were not increased and cell proliferation was significantly decreased (figure 1). next, as shown in figures 2(a) and 2(b), cd86 (m1 macrophages) was induced by low - oxldl whereas mannose receptor (m2 macrophages) was induced by hi - oxldl. the results of the cytokine profiling showed an increased production of tnf- as well as il-12p40 (figure 3(a)), which reflects the typical phenotype of m1 macrophages, whereas increased production of il-6 and mcp-1 (figure 3(b)) reflects the m2 macrophage phenotype. m2 macrophages can be further classified into four subtypes, namely, m2a, m2b, m2c, and m2d. among them, m2b macrophages secrete high levels of il-6 and are referred to as regulatory macrophages. il-6 is a pleiotropic cytokine that induces either proinflammatory or antiinflammatory responses and is involved in a variety of conditions, such as obesity, arthritis, colitis, and sepsis. generally, il-6 is considered a proinflammatory cytokine, but its antiinflammatory roles have been suggested in specific conditions. recent studies have shown that il-6 promotes m2 activation while attenuating m1 activation. when myeloid cells were primed with il-6, the stat3-il-4-stat6 axis, which is a potent inducer of m2 polarization, was activated, whereas when monocytes were pretreated with il-6, lipopolysaccharide - induced production of proinflammatory cytokines was attenuated. moreover, il-6 induces the polarization of tumor - associated macrophages, which are similar to obesity - associated m2 macrophages. counter - inflammatory role in controlling metabolic homeostasis in low grade and chronic conditions, such as obesity. mcp-1-mediated recruitment of monocytes contributes to pathogenesis, but several studies have shown that mcp-1 has a beneficial role in various inflammatory conditions. in the gut, lamina propria macrophages, which revealed an m2-like phenotype, produce large amounts of mcp-1. additionally, a subset of lamina propria macrophages are recruited in response to mcp-1 and produce a large amount of il-10 to maintain gut homeostasis in the steady state as well as terminate excess inflammation in the intestine. furthermore, mcp-1 exerts beneficial effects in other inflammatory diseases such as ischemic heart diseases by inhibiting the generation of ros and by healing necrotic areas in the myocardium [24, 25 ]. the exact roles of il-6 and mcp-1 are still controversial. however, previous studies on the anti - inflammatory effects of il-6 and mcp-1 support our results that hi - oxldl - induced differentiation of macrophages skews towards the m2-like phenotype rather than the m1 phenotype (figures 2(b) and 3(b)). our results show that hi - oxldl induces il-6 and mcp-1 production in macrophages, indicating that the expression of these cytokines may be a feature of m2 macrophages. while the interpretation of cytokine profiles might be difficult, most of our results for cytokine production and surface marker expression suggest that low - oxldl induces m1 polarization, whereas hi - oxldl leads to m2 polarization. lox-1 is a receptor for oxldl and is expressed in a variety of cells, such as endothelial cells, muscle cells, monocytes, and macrophages [2, 26, 27 ]. after binding to oxldl, lox-1 is internalized ; this induces endoplasmic reticulum (er) stress that causes upregulation of lox-1, which occurs via a positive feedback loop between er stress and lox-1 expression and macrophage differentiation into m2 macrophages together with foam cell formation [2729 ]. figure 4 shows that treatment with hi - oxldl increased the lox-1 expression of macrophages. these high lox-1 levels amplify the signals from hi - oxldl and thus induce increased differentiation into m2 macrophages. a report using mesenchymal stem cells has demonstrated that treatments with oxldl markedly increase expressions of lox-1 and mcp-1, but in which the oxidation degree of ldl was not clearly defined. our study showed primary monocytes have changed into spindle shape after treatment with low - oxldl and into round cells after treatment with hi - oxldl. it is well known that the shape of m1 macrophages adopt more spindle - like shape, whereas m2 macrophages are more rounded [31, 32 ]. in addition to the morphological changes, the cytokines profiles and the decreased production of tnf- and il-12p40 as well as increased production of il-6 and mcp-1 after treatment with hi - oxldl (figure 5(b)) strongly support the role of differentially oxidized ldl in monocyte differentiation to m1 or m2 macrophages. taken together, our results for granularity, autofluorescence, cytokine profiles, and surface makers suggest that low - oxldl induces macrophage polarization towards m1 phenotype whereas hi - oxldl induces m2 phenotype. as summarized in figure 6, our results suggest that the degree of oxidation of ldl is the important factor for differentiation of monocytes and macrophages and may determine the inflammation status or even the development of atherosclerosis at early stages of the disease. | ldl plays an important role in atherosclerotic plaque formation and macrophage differentiation. however, there is no report regarding the oxidation degree of ldl and macrophage differentiation. our study has shown that the differentiation into m1 or m2 macrophages is related to the lipid oxidation level of ldl. based on the level of lipid peroxidation, ldl is classified into high - oxidized ldl (hi - oxldl) and low - oxidized ldl (low - oxldl). the differentiation profiles of macrophages were determined by surface receptor expression and cytokine secretion profiles. low - oxldl induced cd86 expression and production of tnf- and il-12p40 in thp-1 cells, indicating an m1 macrophage phenotype. hi - oxldl induced mannose receptor expression and production of il-6 and monocyte chemoattractant protein-1, which mostly match the phenotype of m2 macrophages. further supporting evidence for an m2 polarization by hi - oxldl was the induction of lox-1 in thp-1 cells treated with hi - oxldl but not with low - oxldl. similar results were obtained in primary human monocytes. therefore, our results strongly suggest that the oxidation degree of ldl influences the differentiation of monocytes into m1 or m2 macrophages and determines the inflammatory fate in early stages of atherosclerosis. |
this study included 21 cases (18 patients ; 15 females and 3 males ; age range, 26 - 70 years ; mean patient age, 55 years) that were surgically diagnosed as an mtc between 2002 and 2007. sixteen patients had a sporadic mtc, and two patients had type iia multiple endocrine neoplasia (men). one men type iia patient had bilateral adrenal pheochromocytomas and another patient had bilateral adrenal hyperplasia. the basal calcitonin level of the 15 mtc patients was measured prior to surgery and in 14 patients the serum calcitonin level was elevated (range, 24.7 - 355 pg / ml ; median level, 171.4 pg / ml) ; the normal range for an immunoradiometric assay is 10 pg / ml. as the mtc us findings of mtc were to be compared with the ptc us findings, a, control group of 114 ptc cases that were surgically diagnosed during 2007 (99 patients ; 85 females and 14 males ; age range, 27 - 76 years ; mean age, 50.4 years) was established. high resolution us examinations of the thyroid were performed with a hdi 3000 scanner (advanced technology laboratories, philips medical systems, bothell, wa) and a hdi 5000 scanner (philips medical systems) equipped with a 5 - 10 mhz linear array transducer. examinations were performed by radiology residents and by the radiologists in charge of thyroid imaging (with one to 16 years of experience). the us images were analyzed retrospectively in a blind review by two radiologists in charge of thyroid imaging with two and 16 years of experience, respectively. by summarization of the classifications of us findings (size, internal content, shape, margin, echogenicity and calcifications) as recommended by the thyroid study group of the korean society of neuroradiology and head and neck radiology (ksnhnr), each nodule was identified as suspicious malignant, indeterminate or probably benign (6). an analysis for distant or regional metastases, including lymph node metastases, was not performed in this study. with respect to the size of the nodules, the longest diameter measured by us was selected and was expressed as the mean standard deviation. the internal contents of the nodules were defined as solid (a solid component more than 90%), predominantly solid (a solid component more than 50% but less than 90%), predominantly cystic (a solid component more than 10% but less than 50%) and cystic (solid component less than 10%). the shape of nodules was defined as ovoid to round, taller than wide (the anteroposterior dimension was longer than the transverse diameter), or irregular (neither round to oval nor taller than wide). echogenicity was defined as markedly hypoechoic (less than the strap muscle echogenicity), hypoechoic (less than the thyroid echogenicity), isoechoic (equal to the thyroid echogenicity) or hyperechoic (greater than the thyroid echogenicity). calcifications were defined as microcalcifications (tiny punctate hyperechogenicities either with or without acoustic shadowings of less than 1 mm in diameter), macrocalcifications (larger than 1 mm in diameter) or rim calcifications. malignant sonographic characteristics were defined as a shape that was taller than wide, with a spiculated border, marked hypoechogenicity and micro calcifications or macrocalcifications. if a nodule had any one of these features that are suggestive of a malignancy, the lesion was classified as a suspicious malignant nodule. a probably benign nodule was classified as either completely cystic or predominantly cystic with a comet tail artifact. an indeterminate nodule was defined as a nodule that was not suspicious for malignancy or showed probably benign findings. the findings of medullary and papillary carcinomas were compared with use of the chisquared test. this study included 21 cases (18 patients ; 15 females and 3 males ; age range, 26 - 70 years ; mean patient age, 55 years) that were surgically diagnosed as an mtc between 2002 and 2007. sixteen patients had a sporadic mtc, and two patients had type iia multiple endocrine neoplasia (men). one men type iia patient had bilateral adrenal pheochromocytomas and another patient had bilateral adrenal hyperplasia. the basal calcitonin level of the 15 mtc patients was measured prior to surgery and in 14 patients the serum calcitonin level was elevated (range, 24.7 - 355 pg / ml ; median level, 171.4 pg / ml) ; the normal range for an immunoradiometric assay is 10 pg / ml. as the mtc us findings of mtc were to be compared with the ptc us findings, a, control group of 114 ptc cases that were surgically diagnosed during 2007 (99 patients ; 85 females and 14 males ; age range, 27 - 76 years ; mean age, 50.4 years) was established. high resolution us examinations of the thyroid were performed with a hdi 3000 scanner (advanced technology laboratories, philips medical systems, bothell, wa) and a hdi 5000 scanner (philips medical systems) equipped with a 5 - 10 mhz linear array transducer. examinations were performed by radiology residents and by the radiologists in charge of thyroid imaging (with one to 16 years of experience). the us images were analyzed retrospectively in a blind review by two radiologists in charge of thyroid imaging with two and 16 years of experience, respectively. by summarization of the classifications of us findings (size, internal content, shape, margin, echogenicity and calcifications) as recommended by the thyroid study group of the korean society of neuroradiology and head and neck radiology (ksnhnr), each nodule was identified as suspicious malignant, indeterminate or probably benign (6). an analysis for distant or regional metastases, including lymph node metastases, was not performed in this study. with respect to the size of the nodules, the longest diameter measured by us was selected and was expressed as the mean standard deviation. the internal contents of the nodules were defined as solid (a solid component more than 90%), predominantly solid (a solid component more than 50% but less than 90%), predominantly cystic (a solid component more than 10% but less than 50%) and cystic (solid component less than 10%). the shape of nodules was defined as ovoid to round, taller than wide (the anteroposterior dimension was longer than the transverse diameter), or irregular (neither round to oval nor taller than wide). echogenicity was defined as markedly hypoechoic (less than the strap muscle echogenicity), hypoechoic (less than the thyroid echogenicity), isoechoic (equal to the thyroid echogenicity) or hyperechoic (greater than the thyroid echogenicity). calcifications were defined as microcalcifications (tiny punctate hyperechogenicities either with or without acoustic shadowings of less than 1 mm in diameter), macrocalcifications (larger than 1 mm in diameter) or rim calcifications. malignant sonographic characteristics were defined as a shape that was taller than wide, with a spiculated border, marked hypoechogenicity and micro calcifications or macrocalcifications. if a nodule had any one of these features that are suggestive of a malignancy, the lesion was classified as a suspicious malignant nodule. a probably benign nodule was classified as either completely cystic or predominantly cystic with a comet tail artifact. an indeterminate nodule was defined as a nodule that was not suspicious for malignancy or showed probably benign findings. the findings of medullary and papillary carcinomas were compared with use of the chisquared test. a p - value of less than 0.05 was defined as statistically significant. the us findings of the mtcs and ptcs are summarized in the table 1. for the mtcs, a solid internal content (19 of 21, 90.5%), ovoid to round shape (12 of 21, 57.1%), marked hypoechogenicity (11 of 21, 52.4%), microcalcifications or macrocalcifications (11 of 21, 52.4%) and a spiculated or ill - defined margin (17 of 21, 80.9%) were the prevailing findings seen on us. for 21 mtcs, 17 (81%) were categorized as suspicious malignant nodules, and four (19%) were categorized as indeterminate nodules (figs. 1, 2). the prevailing findings as seen on us were a solid internal content (110 of 114, 96.5%), taller than wide shape (48 of 114, 48%), irregular border (48 of 114, 42.1%), marked hypoechogenicity (65 of 114, 57.0%) and microcalcifications or macrocalcifications (74 of 114, 64.9%). for 114 ptcs, 107 (94%) were categorized as suspicious malignant nodules and seven (6%) were categorized as indeterminate nodules (fig. 3). the mean size (19 13.9 mm vs. 11 7.5 mm) and ovoid to round shape (57.1% vs. 25.4%) were statistically different between the mtcs and ptcs. however, the internal content, margin, echogenicity and calcifications were not statistically significantly different between the two types of nodules. most of the mtc and ptc nodules were classified as suspicious malignant ; however, the frequency of an indeterminate nodule was higher for the mtcs than for the ptcs. eighty to ninety percent of mtcs have been considered as sporadic and the rest have been classified as hereditary (1). the familial form of the tumor usually occurs as a component of type iia men (sipple syndrome), which is characterized by the combination of an mtc, adrenal pheochromocytoma and hyperplasia of the parathyroid glands. men type iib is a highly unusual form of the men ii syndrome and consists of elements of type iia and additional features of marfanoid habitus, mucosal neuromas and intestinal ganglioneuromatosis. in the present study, 16 patients were categorized as having the sporadic form and two patients were categorized as having men type iia. fine needle aspiration (fna) of thyroid nodules has become one of the most useful, safe and accurate methods used to diagnose thyroid pathology. us findings have been reported to be of great help to distinguish malignant from benign nodules (6 - 11, 13). the us findings that have been reported for malignant thyroid nodules have included completely solid or predominantly solid nodules, hypoechogenicity as compared with the strap muscles, an irregular margin, intranodular microcalcifications, a taller than wide orientation and increased intranodular vascularity. (6) reported in a multicenter retrospective study the us findings of benign and malignant thyroid nodules (6). the thyroid study group of the ksnhnr organized this study, which was performed on patients from nine university hospitals and large general hospitals in the republic of korea. the findings suggested malignancy in nodules with a taller than wide shape, spiculated margin, marked hypoechogenicity, and intranodular calcifications (including microcalcifications or macrocalcifications). the findings of the study demonstrated a high sensitivity (93.8%) and a high negative predictive value (95.9%). in most cases, the malignant nodules (6) study and similar studies were ptcs, and most of the ptcs correlated with the us findings of malignant thyroid nodules (10 - 13). however, studies on the us findings of mtc are not common (14, 15). gorman. (14) reported on the high - resolution us findings of mtc in 1987. all of the tumors (six lesions in six patients) appeared hypoechoic and were relatively well defined. bright echogenic foci were reported in five patients (83.3%), and this finding was supported by the histological finding of focal deposits of calcium surrounded by amyloid. in 2002, saller. the present study evaluated 21 mtc cases, a higher number than in previous studies. most mtc nodules (95.3%) were markedly hypoechoic (52%) or hypoechoic (42.9%), and our findings are similar to the findings of the above two studies (14, 15). in the current study, intranodular calcifications were observed in 52% of the mtc patients. as compared to the results of the gorman. studies comparing the us findings of mtc with ptc have not been previously conducted and we compared and analyzed the us findings of 21 mtc cases and 114 ptc cases. the mean size of mtc nodules was larger than that of ptc nodules (19 13.9 mm vs. 11 7.5 mm, p < 0.05). in a comparison of the us findings, 57% of mtcs and 25% of ptcs exhibited an ovoid to round shape, which was significantly higher for the mtc nodules (p < 0.05). however, internal content, margin, echogenicity and frequency of calcifications were not significantly different between mtcs and ptcs. most mtc (80.9%) and ptc (93.9%) nodules were classified as suspicious malignant nodules. however, in a comparison of mtcs with ptcs, suspicious malignant nodules were found less frequently and indeterminate nodules were found more frequently in mtc than in ptc. this finding can be attributed to a significantly higher frequency of ovoid to round shape in mtc, which is not a suspicious malignant feature. the association of an mtc with amyloid deposition pathologically distinguishes this type of tumor from other thyroid malignancies (16). amyloid deposits may be associated with reactive fibrosis and calcified deposits that give rise to characteristically dense, irregular foci throughout the tumor mass. this observation is in contrast with the faint homogeneous calcifications seen in other thyroid tumors (1). dense and irregular calcifications are known as a characteristic finding of mtc ; these macrocalcifications were seen in five mtc nodules (23.8%) and in 21 ptc nodules (18.4%). 15) defined calcifications longer than 2 mm as macrocalcifications, and reported the presence of macrocalcifications in 53% of mtc patients in their study ; this frequency was higher than in our study. chan. (11) found that among 55 ptc cases, 11% were associated with macrocalcifications ; this frequency was similar to the frequency seen in the present study. the presence of microcalcifications is a known characteristic finding of ptc and microcalcifications have been reported in 29 - 59% of all thyroid carcinomas (17). in our study, microcalcifications were found in 29% of mtcs and 47% of ptcs. according to the findings in the study by chan. (11), 42% of ptcs were associated with microcalcifications, a finding that is similar to our results. 15) reported that microcalcifications less than 2 mm in length were seen in 42% of mtcs. serum calcitonin levels rise in mtc patients as mtcs are derived from parafollicular c - cells that produce calcitonin (2). however, not all mtcs secrete calcitonin and in particular, more advanced tumors may dedifferentiate with a subsequent decrease in calcitonin production (3). in the present study, the basal calcitonin level was elevated in 14 patients and was within the normal range in one patient. the patient with a normal calcitonin level had a huge, 7 cm thyroid mass with an extensive necrotic lymph node metastasis and perithyroidal soft tissue invasion. the pathological result was a poorly differentiated pattern of mtc. in conclusion, the common us features of mtc are a solid internal content, an ovoid to round shape, marked hypoechogenicity and calcifications. most mtcs showed us findings of suspicious malignant nodules, as reported by the thyroid study group of the ksnhnr. the us findings were not greatly different between mtcs and ptcs, except that the ovoid to round shape was more abundant in mtcs. | objectivethis study was designed to evaluate the ultrasonographic (us) findings of medullary thyroid carcinoma (mtc) as compared to findings for papillary thyroid carcinoma (ptc).materials and methodsthe study included 21 cases of mtc that were surgically diagnosed between 2002 and 2007 and 114 cases of ptc that were diagnosed in 2007. two radiologists reached a consensus in the evaluation of the us findings. the us findings were classified as recommended by the thyroid study group of the korean society of neuroradiology and head and neck radiology (ksnhnr) and each nodule was identified as suspicious malignant, indeterminate or probably benign. the findings of medullary and papillary carcinomas were compared with use of the chi - squared test.resultsthe common us findings for mtcs were solid internal content (91%), an ovoid to round shape (57%), marked hypoechogenicity (52%) and calcifications (52%). among the 21 cases of mtc nodules, 17 (81%) were classified as suspicious malignant nodules. the mean size (longest diameter) of mtc nodules was 19 13.9 mm and the mean size (longest diameter) of ptc nodules was 11 7.4 mm ; this difference was statistically significant (p < 0.05). an ovoid to round shape was more prevalent for mtc lesions than for ptc lesions (p < 0.05).conclusionthe us criteria for suspicious malignant nodules as recommended by the thyroid study group of the ksnhnr correspond to most mtc cases. the us findings for mtc are not greatly different from ptc except for the prevalence of an ovoid to round shape. |
ixodid or hard ticks cause most toxicoses, affecting humans and animals around the world [2, 3 ]. the most severe form of toxicosis results in the paralysis of the infested host. globally, just under 70 species of ticks have been described as being capable of inducing paralysis, the most important being ixodes holocyclus in australia, dermacentor andersoni, d. variabilis, and argas (persicargas) radiatus in north america, i. rubicundus in south africa, rhipicephalus evertsi evertsi and a. (p.) walkerae in ethiopia, and a. (p.) radiatus in the nearctic region of north america. in australia, i. holocyclus can cause paralysis in a range of domestic animals and livestock, affecting up to 10,000 companion animals and up to 100,000 livestock per year. it is considered highly toxic with one female able to kill a dog or sheep. i. holocyclus is found along the eastern seaboard of australia and is most abundant from early spring to late summer. paralysis is induced by a neurotoxin that is transmitted to the host in the saliva of a female i. holocyclus when the tick takes a blood meal. during feeding, toxicity in the salivary glands the treatment of paralysed hosts requires removal of the parasite and administration of a commercial tick anti - serum (tas) that is prepared from hyperimmune dogs. to standardise the toxin - neutralising activity of each batch of tas, a mouse bioassay has been traditionally used however, the bioassay is expensive, time consuming, and subjective, relying on observations of paralytic signs in neonatal mice over a 24-hour period. developing a suitable in vitro immunoassay to quantify toxin - specific antibodies in commercial tas could replace the in vivo test. knowledge of the immune status of dogs before and after treatment for tick paralysis will help to evaluate a minimal, effective dose of tas, avoiding possible adverse reactions and making treatment more affordable. in this paper, we describe the development of an enzyme - linked immunosorbent assay (elisa) to measure antibody levels against tick salivary gland antigens in the serum of dogs and rodents after natural or laboratory tick infestation. ten adult unfed female i. holocyclus were allowed to feed on 10- to 11-week - old female wistar rats following the methods of stone.. after 5 days, engorged ticks were carefully removed with tweezers, and salivary glands were excised and stored at 80c. one hundred frozen salivary gland pairs were homogenised (dounce, 7 ml) with 3 ml of sterile pbs and the homogenate clarified at 1500 g for 30 minutes at 4c. the pellet was washed three times with 0.5 ml pbs and centrifuged as above. the pooled supernatant was sonicated to further disrupt remaining particulate matter with a soniprep 150 mse ultrasonicator using 30-second bursts followed by 2 minutes cooling on ice for a total of 10 minutes. the sonicated homogenate was pelleted at 109,000 g for 1 hour at 4c, and the resulting supernatant was stored in aliquots at 80c. protein concentrations in antigen preparations were estimated with the bca protein assay kit as per protocol. all batches of i. holocyclus toxin were tested in the neonatal mouse bioassay to determine the level of paralysing activity. (b.) microplus, which had fed for 5 days on cattle, were prepared in the same manner and used as a nontoxin control in all assays. these extracts were also tested in the bioassay, and no paralysis signs were observed in neonatal mice. a standardised reference serum was prepared by pooling several batches of commercially available tas prepared from ten hyperimmune (hi) dogs and confirmed for toxin - neutralising activity in the mouse bioassay. ten nonreactive dog sera were obtained from perth in western australia, an area where i. holocyclus does not naturally occur. sera were also obtained from dogs that presented at manly road veterinary hospital, brisbane, for treatment against tick paralysis. samples were collected at the time of admission, prior to treatment, and again approximately 16 days later. the basic protocol was as follows : tick salivary gland antigen was diluted in coating buffer (0.05 m carbonate / bi - carbonate ph 9.6) and 50 l added to wells of elisa plates (falcon flexible, 96 wells, u bottom, becton dickinson) at 4c overnight. after washing (phosphate - buffered saline containing 0.05% tween 20) the plates twice to remove unbound antigen, 100 l of blocking buffer (0.05 m tris, 0.001 m edta, 0.15 m nacl, 0.2% w / v casein, 0.05% v / v tween 20, ph 8.0) was applied to all wells for one hour at 37c. after the removal of blocking buffer, 50 l of dog sera diluted in blocking buffer was added per well for 1 h at 37c. after 4 washes, rabbit antidog igg conjugated to horse - radish peroxidase (whole molecule, hrpo, sigma) was diluted in blocking buffer, added (50 l per well), and allowed to bind to captured dog igg for a further hour at 37c. after an additional 6 washes, bound conjugate was detected with 100 l of abts substrate (2% of 2,2-azino - bis (3-ethylbenzo - thiazoline-6-sulfonic acid) diammonium salt, sigma ; 2% of 1.26% h2o2 in substrate buffer : 2.8% na2hpo4 and 2.1% citric acid, ph 4.2). after a 1-h reaction at 37c, the spectroscopic absorbance of each well was measured in an automated plate reader (multiskan) at a wavelength of 405 nm. a modified elisa was used to test sera from rats experimentally infested with i. holocyclus ticks. serum was collected from rats prior to and at various times after ticks had fed on the animals for five days. pre- and post- tick infestation sera were then tested in the elisa. in the absence of rat serum known to be reactive to tick toxin, the optimised protocol for dog sera was employed, with the exception that horseradish peroxidase - conjugated antirat igg diluted 1 : 1000 was used for the second antibody step. initial analysis of hi dog sera revealed that high levels of nonspecific binding to the plastic in the wells of the elisa plate occurred at dilutions below 1/50 (results not shown). therefore, all dog sera were subsequently tested at dilutions of 1/50 or greater. to determine the optimal dilution of i. holocyclus salivary gland antigen, pooled hi dog serum and antigen were titrated in a checkerboard format. serum dilutions of 1/501/200 demonstrated maximum binding to ixodes antigen at 6.25 g / ml with optical densities (od) between 1.5 and 2.3 (figure 1(a)). negligible binding was observed for each dilution of the negative control dog serum at all antigen concentrations examined (od 1.0) (table 1). only the day 0 sera from dog c showed significantly less reactivity with rhipicephalus antigen, while the corresponding sample from dogs d and h reacted similarly with both antigens. antibody levels to ixodes antigen from the other five dogs fell below the cutoff value determined earlier. testing of serum samples from day 16 (after administration of tas) revealed a sharp increase of antibody levels to ixodes antigen in dogs c and h, while a similar increase of antibody levels to rhipicephalus antigen was also observed in dog c. antibodies in sera from dog g did not react with ixodes antigen on day of admission but rose just above the cutoff threshold on day 16, after treatment with tas. antibody levels from all other dogs did not rise above the levels from day 0. serum from seven rats taken prior to and 21 days after infestation with ticks was tested in an elisa modified to detect rat immunoglobulin to tick toxin. all samples tested on ixodes antigen in elisa showed slightly increased antibody levels in postinfestation samples as compared to preinfestation serum, although od levels remained well below values seen with hi dog sera. ods for rhipicephalus antigen did not increase in three rats while increased only marginally in the remaining four rats (see table 2). these preliminary data indicated that we could detect, albeit weakly, antibodies to the toxin - associated antigen in rat sera after a single infestation with multiple ticks. further analysis of the seroconversion of a rat after sequential infestations with multiple ticks was also undertaken. serum collected from this animal after two rounds of infestations over 248 days exhibited strong, specific reaction to ixodes antigen when tested in elisa compared to serum taken prior to infestation (see figure 3). collectively, these results reveal that the elisa can be used to detect serum antibodies specific for ixodes antigen, induced by single or multiple exposures to i. holocyclus ticks. a partially purified antigen preparation from the salivary glands of female i. holocyclus ticks was assessed for use as a diagnostic reagent in elisa to measure tick toxin - specific antibodies in dog sera. the results from these assays indicated that sera from hi dogs reacted strongly to the ixodes antigen, compared to negligible reactions of sera from dogs not exposed to i. holocyclus ticks. furthermore, a control antigen prepared from the salivary glands of the cattle tick rh. (b.) microplus, which does not produce paralysis toxin, showed relatively weak reactions for both hi (positive) and control (negative) sera. (b.) microplus seen in elisa was to be expected due to common antigens in the salivary glands in both tick species. indeed, amino acid sequences between ixodes and rhipicephalus (boophilus) ticks show a high degree of homology, > 85% indicating a close antigenic relationship. these data suggest that the elisa detected antibodies specific to toxin - associated antigens and that in vitro analysis of commercially prepared tas may be a viable alternative to the expensive and subjective mouse bioassay. previously, the australian pesticides and veterinary medicines authority (apvma) has declared that an in vitro immunoassay to evaluate commercial tas would only be approved if a highly specific antigen was used (b. stone, pers comm.). indeed, morrison (unpublished data) used a partially purified antigen extracted from i. holocyclus salivary glands in an elisa and claimed good correlation with the bioassay. however, in the absence of a control antigen (a similar extract from toxin - free ticks), the author was unable to demonstrate the reaction to the antigen was toxinspecific. in contrast to the strong reactivity of hi dog sera in the elisa, an apparent lack of antibody responsiveness was observed in sera from dogs naturally infested with ticks and presenting with paralysis signs at a brisbane veterinary clinic. these sera were tested in the elisa to assess the level of toxin - specific antibodies at the onset of signs and approximately 16 days later. according to elisa od readings, three out of eight dogs showed significant antibodies to tick toxin antigen at the time of presentation, but only two of seven exhibited a significant rise in antitick antibody 16 days later. a fourth serum was negative to ixodes antigen on presentation but seroconverted after treatment to just above threshold level. in the absence of additional information on the general health status of the dogs before and after treatment, including weight, breed, sex, age, history of previous tick exposure, and the dosage and batch of tas used, interpretation of the data was limited. however, based on the observation that at least half (4/7) of the animals showed no increase in serum antibody levels to the ixodes antigen after treatment with tas, we may conclude that the increased elisa response to this antigen detected in some animals was more likely due to an immune response to tick infestation rather than the passive transfer of antibodies through the tas treatment. based on the low rate of seroconversion to toxin - specific antigens in dogs naturally exposed to i. holocyclus, even after treatment with tas, it is unlikely that the assay will be a useful tool for clinicians to monitor the immune status of animals or the efficacy of treatment. this is consistent with findings of stone and wright who observed slow and infrequent neutralising antibody responses (measured in a mouse bioassay) in dogs over 14 weeks of repeated tick infestation during the priming phase towards hyperimmunity. the lack of immune response is likely due to the equilibrium achieved between host immunity to the tick and the tick 's suppression of the host 's immune system. the feeding tick modulates the host 's haemostasis, inflammation, and immunity to enable uninterrupted engorgement. this is supported by the fact that i. holocyclus ticks are not rejected (no weight loss) and dogs show very little cutaneous hypersensitivity despite repeated exposure. in summary, an elisa was established which differentiated strongly between hi dog sera and sera from dogs that had not been infested with i. holocyclus ticks. since the ixodes salivary gland antigen was only partially purified, the specificity of this elisa was supported by parallel testing of the sera on an antigen prepared in the same way from nontoxic rhipicephalus ticks. hi and nonreactive sera bound substantially weaker to the rhipicephalus antigen, suggesting that reactions of hi sera to the ixodes antigen were specific. sera from dogs experimentally or naturally infested with a limited number of ticks, however, produce only weak reactions to the ixodes antigen in elisa, suggesting that antibody levels to low - frequency tick infestations are not detected in this assay. in the absence of highly purified toxin antigen, the specificity of the assay may be enhanced by the use of tick toxin - reactive monoclonal antibodies to capture toxin - specific antigen to the solid phase in a modified elisa format. to this end, the elisa described above was successfully adapted for the detection of antibodies in rats exposed to tick infestation. indeed, the detection of strong antibody responses in rats after two exposures to ticks indicates that this animal model will be useful for producing hybridomas to i. holocyclus toxin antigens and the elisa an effective means for screening the resultant monoclonal antibodies. | the ixodes holocyclus tick causes paralysis in up to 10,000 companion and domestic animals each year in australia. treatment requires the removal of the parasite and the administration of a commercial tick antiserum that is prepared from hyperimmune dogs. each batch of this serum is initially tested for toxin - neutralising potency in a mouse bioassay that is expensive, time consuming, and subjective. with the aim of developing a rapid in vitro assay to replace the bioassay, we used a partially purified antigen prepared from i. holocyclus salivary glands to develop an elisa to detect toxin - reactive antibodies in hyperimmune dog sera. the optimised elisa reliably detected antibodies reactive to i. holocyclus salivary gland antigens. parallel testing of sera with a negative control antigen prepared from the salivary glands of the nontoxic tick rhipicephalus (boophilus) microplus provided further evidence that we were detecting toxin - specific antibodies in the assay. using the elisa, we could also detect antibodies induced in rats after experimental infestation with i. holocyclus. this assay shows promise as an alternative means of assessing the potency of batches of hyperimmune dog serum and to screen for toxin - reactive monoclonal antibodies produced from immunised rodents. |
rational drug discovery requires early methods of all factors influencing on the likely success of a drug candidate in the subsequent stages of the drug development. the study of absorption, distribution, metabolism, excretion, and pharmacokinetics (adme / pk) has become a major discipline in drug discovery through the application of well - established in vitro and in vivo methodologies. pk plays a crucial role in the pharmaceutical research and development and, because of the major role of drug metabolism, drug discovery research in this area is covered by groups coalesced around the name drug metabolism and pharmacokinetics (dmpks) [26 ]. pharmacokinetic properties of drugs may be affected by factors such as the administration site and the administered drug dose ; these may influence the rate of absorption. one more process, liberation, plays an important role in pharmacokinetics : liberation means the release of drug from the formulation. the primary goal of the drug discovery and development process is to find a molecule possessing both good pharmacodynamics and good pharmacokinetic properties. ideally, a new drug should be efficacious and selective, target tissue(s) specific, and orally absorbed, cause minimal or no adverse effects due to metabolite activity or toxicity, and be distributed / excreted in such a fashion as to permit dosage once a day. several computational models have also been reported for such biopharmaceutical properties as % hia, blood - brain barrier, skin and ocular permeation, pharmacokinetics and metabolism. however, all these studies involved sets of closely related structural analogues, and models based on limited chemical space generally lack predictive value outside their structural classes. broadly applicable qspr models of biopharmaceutical properties must be built using compounds which cover both a wide range of the property being modeled as well as of the chemical structure space. bioavailability is a key pharmacokinetic parameter which expresses the proportion of a drug administered by any nonvascular route that gains access to the systemic circulation. therefore, there is a need of a robust and accurate computational model (qsar / qspr) which can predict the oral bioavailability of compounds without carrying out any experiments. development of model using linear and nonlinear multivariate statistics to predict the oral bioavailability of a compound. the qspr methodology used in this project consists of three main parts : representation of molecular structure, feature selection, and mapping. the general assumption in qspr modeling is that molecular structure causes the observed behavior of a compound, linking a series of chemical structures to properties of interest ; in this case, oral bioavailability should provide a method for modeling the property. general regression neural network method present in dtreg was used to generate the oral bioavailability model. this neural network has 3 layers and 1 hidden layer, and it uses kernel function. the set of 217 drug and drug - like compounds and their experimentally derived percentage of oral bioavailability values used in this project were gathered from the literature sources. the 217 compounds in the working data set were classified by using sphere exclusion algorithm into a training set of 159 compounds, test set of 50 compounds, and an external prediction set of 8 compounds. the external prediction set was chosen in such a way as to cover the range of percentage of oral bioavailability values in the data set, and it spans the range of 0%100%. the compounds in the external prediction set were never used during the model development process but were reserved to validate potential models. the structures of the 217 compounds were extracted from the small molecule chembiobase database with isis which consists of kinase, protease inhibitors, and gpcr antagonists. accurate geometries are necessary for the calculation of certain descriptors thought to be necessary for modeling physical and chemical properties. as a result, ligprep tool was used to generate accurate three - dimensional geometries, and conformational analysis was performed in order to get the local minima structure which is necessary for calculating geometric descriptors. a total of 11 descriptors were generated for each of the 217 compounds using codessa 2.1 tool. topological descriptors like number of single bonds, number of aromatic bonds, number of oxygen atoms, nitrogen atoms, relative number of oxygen atoms, and relative number of nitrogen atoms are derived from the information of the two - dimensional structure of the molecule. electronic descriptors were calculated by mopac using the am1 hamiltonian electronic descriptors include maximum partial charge for hydrogen atoms. geometric descriptors include cube root of gravitational index, saaa (surface area of hydrogen bond acceptor atoms / number of hydrogen bond acceptor atoms), and normalized 2d projection on yz plane. accurate three - dimensional geometries of the molecules are necessary to calculate descriptors of this nature. a fourth class of descriptors can be derived by combining electronic and geometric information to form hybrid descriptors. by combining the molecular surface area with partial atomic charges, charged - partial surface area (cpsa) descriptors can be calculated. eleven descriptors span the following ranges : number of single bonds (2690), number of aromatic bonds (012), yz shadow (29.9110.46), grav-3 (25.9742.16), saaa (28.3832.14), number of oxygen atoms (07), number of nitrogen atoms (18), relative number of oxygen atoms (00.12), relative number of nitrogen atoms (0.020.17), maximum partial charge for a hydrogen atom (0.030.1), and hasa-2 (2.8932.2). first two descriptors indicate amount of structural flexibility ; next two descriptors encode molecular size, shape, and bulk properties. topological descriptors include number of single bonds, number of oxygen atoms, relative number of oxygen atoms, number of nitrogen atoms, relative number of nitrogen atoms, and number of aromatic bonds.geometric descriptors include the following : grav-3 : cube root of gravitational index, saaa-2 : surface area of hydrogen bond acceptor atoms / number of hydrogen bond acceptor atoms ; these two are calculated using java program, shdw-6 : normalized 2d projection of molecule on yz plane, maximum partial charge for hydrogen atoms, hasa-2 : area - weighted surface charge of hydrogen bonding acceptor atoms. topological descriptors include number of single bonds, number of oxygen atoms, relative number of oxygen atoms, number of nitrogen atoms, relative number of nitrogen atoms, and number of aromatic bonds. geometric descriptors include the following : grav-3 : cube root of gravitational index, saaa-2 : surface area of hydrogen bond acceptor atoms / number of hydrogen bond acceptor atoms ; these two are calculated using java program, shdw-6 : normalized 2d projection of molecule on yz plane, grav-3 : cube root of gravitational index, saaa-2 : surface area of hydrogen bond acceptor atoms / number of hydrogen bond acceptor atoms ; these two are calculated using java program, shdw-6 : normalized 2d projection of molecule on yz plane, maximum partial charge for hydrogen atoms, hasa-2 : area - weighted surface charge of hydrogen bonding acceptor atoms. the nsb descriptor is encoding single bonds, and this may be an indication of the amount of structural flexibility. the shdw-6 and grav-3 descriptors are encoding molecular size, shape, and bulk properties. these size descriptors may be important with respect to the ability of the drug to penetrate cell membranes. in this model, alogp and psa descriptors were used as predictor variables. well - absorbed and poor - absorbed compounds were plotted against alogp versus psa. a 95% confidence ellipse for the well - absorbed dataset was also computed and plotted. the 95% confidence ellipse represents the region of chemical space where we can expect to find well - absorbed compounds (90%) 95 out of 100 times (figure 1). it gives more than 60% of misclassification ; from this, we conclude that the alogp and psa (polar surface area) descriptors are not enough for this dataset to classify well - absorbed and poor - absorbed molecules. 95% confidence means that we get 95 times out of 100 molecules having good absorption inside the ellipse. dtreg software was used to generate single decision tree (figure 2) ; this model also gives a lot of misclassification (more than 40%). alogp and psa descriptors are not enough to classify well - absorbed and poor compounds. partial least square method and multiple linear regression present in cimpl tool were employed to get good linear models ; none of the models found was satisfactory. it became evident that the diversity of this data set and the number of data points possessing greater than 50% absorption values are largely responsible for producing poor quality linear models. therefore, this data set became a good candidate for developing nonlinear neural network model. 11-member reduced pool of descriptors was fed to the general regression neural network method for the purpose of developing a nonlinear model. the original regression data set was split randomly into a neural network training set of 159 compounds and a test set of 50 compounds using sphere exclusion method. the test set was used to monitor overtraining of the network, and the training set was used to actually train the network (figures 3 and 4). to decrease the possibility of chance effects influencing neural network training, the ratio of observations to total adjustable parameters should be at or above 2.0. a neural network consisting of 11 input neurons (descriptors), 3 hidden neurons, and 1 output neuron (target and percentage of bioavailability), thus producing an 11 - 3 - 1 architecture, was used since it produced the maximum number of adjustable parameters recommended for a dataset of this size. for this 11 - 3 - 1 architecture, the ratio of training set observations to adjustable parameters f was 67/33 or 2.03 (table 1). table 1 refers to statistics results of r, rmse, mae, and standard deviation for training set, test set, external prediction set. 11-descriptor general regression neural network model has been developed for the estimation of the percentage of oral bioavailability values for a data set of 217 drug and drug - like compounds. the training set rms error was 4.55% oral bioavailability units, and the test set rms error was 14.32% oral bioavailability units. based on the rms errors of the training and test sets, it is clear that a link between the structure and the percentage of oral bioavailability does exist. however, the strength of that link is best measured by the quality of the external prediction set. with rms error of 19.0% hia units and a good visual plot, given the structural diversity and bias of the data set, this is a good first attempt at modeling oral bioavailability using qspr methods. the optimization of individual properties along the absorption process must be integrated in a multiobjective scenario for studying oral bioavailability behavior in the early drug discovery and development. the model can be used as a potential virtual screen or property estimator. with a larger data supply less biased toward the high end values of the percentage of oral bioavailability, a more successful and reviling model can be developed. this study illustrates the potential of using qspr methods to aid in the drug development process. | oral bioavailability of a drug compound is the significant property for potential drug candidates. measuring this property can be costly and time - consuming. quantitative structure - property relationships (qsprs) are used to estimate the percentage of oral bioavailability, and they are an attractive alternative to experimental measurements. a data set of 217 drug and drug - like compounds with measured values of the percentage of oral bioavailability taken from the small molecule chembiobase database was used to develop and test a qspr model. descriptors were calculated for the compounds using codessa 2.1 tool. nonlinear general regression neural network model was generated using the dtreg predictive modeling program software. the calculated percentage of oral bioavailability model performs well, with root - mean - square (rms) errors of 4.55% oral bioavailability units for the training set, 14.32% oral bioavailability units for the test set, and 19.12% oral bioavailability units for the external prediction set. given the structural diversity and bias of the data set, this is a good first attempt at modeling oral bioavailability using qspr methods. the model can be used as a potential virtual screen or property estimator. with a larger data supply less biased toward the high end values of the percentage of oral bioavailability, a more successful model could likely be developed. |
reverse micelles are monodisperse ensembles of surfactant - encapsulated nanoscale water pools that result from spontaneous organization of a mixture of appropriate surfactants, water, and nonpolar solvent such as the alkanes (fig. the size of the water pool can be tightly controlled by varying the molar ratio of water to surfactant (water loading or wo). proteins can be encapsulated within the water pool of reverse micelles reproducibly and with high structural fidelity and at concentrations amenable to high - resolution solution nmr methods14,15. when prepared in low viscosity solvents, the full arsenal of solution multi - dimensional heteronuclear nmr techniques becomes accessible16,17. reverse micelles have served as a powerful tool for nmr - based studies of protein and nucleic acid structure and biophysics under a range of conditions14,16,1823. for water loadings such as those used here (w0 ~ 10), the reverse micelle contains approximately one thousand water molecules on average24 and the re - orientational dynamics of encapsulated water is slowed relative to bulk water by approximately one order of magnitude25,26. this suggested that the combined effects of the protein surface and nanoscale confinement could slow the water dynamics to allow efficient detection of hydration water - protein cross - relaxation. at the same time, if reorganization of water contributes to the rate limiting step, hydrogen exchange chemistry in the reverse micelle should also be somewhat slowed, potentially minimizing complications arising from hydrogen - exchange. finally, reverse micelles also provide an appropriate experimental condition to directly assess the contributions from long - range intermolecular dipolar interactions. ubiquitin is a small 76 residue protein comprised of a five stranded mixed beta sheet, a long alpha helix and a short 310 helix27. we first examined the hydration of ubiquitin in free aqueous solution at ph 5 and 25 c using three - dimensional n - resolved noesy29 and roesy30 experiments (fig. only a few amide - water weak cross peaks were observed, which is generally indicative of very short residence times for protein hydration water in bulk aqueous solution3. at this ph and temperature, the exchange of even non - hydrogen bonded amide hydrogens with solvent is generally too slow to impact these experiments. however, every amide hydrogen - water cross peak observed in the free solution spectra involves an amide hydrogen that is within the distance detectable by the noe (see below) of a labile side chain hydrogen. chemical exchange of side chain hydrogens with solvent water followed by intramolecular noe produces cross peaks at the water resonance that are indistinguishable from direct protein - water noes2,3. water noe correlations seen in bulk solution are the result of such exchange - relayed interactions5. we have previously demonstrated that the structure of ubiquitin is unperturbed by appropriate encapsulation in reverse micelles composed of bis-2-ethylhexyl sulfosuccinate (aot)14. water interactions revealed by the noesy spectrum of ubiquitin in free solution, the protein encapsulated in aot reverse micelles displays literally dozens of protein - water interactions under similar temperature and ph conditions (fig. dipolar exchange is identified by the opposite phase of the cross peaks in the orthogonal frames (shown here as positive noe, negative roe), while direct hydrogen exchange with solvent gives rise to peaks of identical phase (shown as positive in both spectra)2,3. calibration of the noe obtained at the 40 ms mixing time used here placed it in the linear regime and corresponds to a maximum noe - detected distance of approximately 4.3. more than half of detected amide hydrogen - water cross peaks involve amides that are over 4.3 from any labile side chain hydrogen, as indicated by of the ensemble of structures determined for encapsulated ubiquitin (pdb 1g6j)14. these can therefore be unequivocally assigned to direct noe interactions between hydration water and the protein. in addition, the hydrogen exchange chemistry catalyzed by hydroxide ion within the reverse micelle water pool is effectively slowed by two orders of magnitude as indicated by the ph dependence of the onset of line broadening due to amide hydrogen exchange (supplementary fig. it should be noted that the pka values of side chain hydroxyls and primary amines are low enough that general base catalysis by buffer or even the head groups of aot is also a potential concern. however, the fact that lysine side chain amine groups are observed (fig. 2) and that most side chain hydroxyls could be identified by direct noe (supplementary fig. given the relative concentrations of protein and water, this restricts the hydrogen exchange rates to the 1 s or slower rate regime. the single labile side chain hydrogen whose chemical exchange may be faster than this rate is the hydroxyl of tyr-59, which is anticipated to have a considerably lower pka. however, contributions from hydrogen exchange - mediated pathways are sufficiently minimized to allow quantitative interpretation of the vast majority of the noe and roe cross peak intensities. interpretation of the noe / roe ratio (see below) as directly representative of local hydration dynamics also requires that the contributions from long - range dipolar couplings be negligible. noesy spectra of highly deuterated ubiquitin lack any measurable cross peaks to organic solvent hydrogens, demonstrating that long - range interactions beyond the exterior of the reverse micelle are minimal. it is this long - range contribution in bulk aqueous solution that has been argued to provide the vast majority of the noe intensity to the water resonance10. it is also of note that only a handful of amide hydrogens show noes with hydrogens of the surfactant headgroup. with the exception of gln 47 and gln 49, the grouping of such interactions suggests that they arise from local close approach of the protein to the surfactant shell rather than general long - range dipolar exchange contributions. it is important to note that these regions of the surface are not dehydrated (fig. the spatial geometry of the reverse micelle and encapsulated protein also cause the number of water molecules to increase roughly as the square of the distance from a solvent - exposed protein hydrogen in contrast to the cubic dependence seen in bulk solution (supplementary fig. these fundamental differences in the physical parameters of the reverse micelle system versus bulk solution combine to largely eliminate contamination of short - range dipolar interactions between protein and hydration water by contribution from long - range couplings to solvent. in summary, the residence times of water on the surface of an encapsulated ubiquitin molecule are sufficiently increased, the effective rates of specific and general base catalysis of hydrogen exchange sufficiently decreased, and the potential contribution by long - range coupling to bulk solvent sufficiently reduced to allow confident and comprehensive site - resolved measurements of dipolar cross - relaxation between hydration waters and amide hydrogens of the protein. this enables the first site - resolved analysis of relative hydration water mobilities across an entire protein surface. a quantitative measure of hydration dynamics can be obtained from the ratio of the noe to roe cross - relaxation rates, noe and roe2,3. here ratios of the intensities of the water cross peaks are used (supplementary tables 1 and 2). in the slow correlation time limit (1), a rigidly bound water molecule would result in a noe / roe ratio of 0.5 (ref. we find that a minority of the resolved protein - water correlations have ratios at this limit. previous relaxation studies indicate that the effective macromolecular tumbling time for these reverse micelle particles dissolved in liquid pentane is about 10 ns16. the majority of detected hydration sites showed a range of noe / roe values between 0 and 0.5 indicating the presence of substantial motion of the hydration water on the surface of the protein and/or a somewhat shorter residence time3,31. in previous studies of protein - water cross relaxation, it was unclear to what degree indirect magnetization exchange contributed to noe / roe ratios more positive than 0.5. as measurements made in the reverse micelle are generally uncomplicated by indirect exchange effects, we conclude that the range of observed noe / roe ratios arise from a distribution of hydration water dynamics that reflect both local geometrical factors and the residence times of the individual water molecules on the surface of ubiquitin31. approximately three fourths of the amide hydrogens within the noe - detection distance limit (4.3) of solvent showed measurable cross peaks to water. no cross peaks to water from buried amide hydrogens were detected, confirming the absence of spin - diffusion or appreciable penetration of water into the interior of the protein. a detailed view of the relative hydration dynamics of ubiquitin is shown in figure 3 where the amide hydrogens within noe distance of the molecular surface are represented as spheres and are color - coded according to dynamic classes. sites that have noe / roe ratios at the bound limit, representing the slowest regions of hydration dynamics, are colored dark blue. sites with ratios between 0.47 and 0.3 (relatively slow or spatially restricted hydration water) or between 0.3 and 0 (relatively fast or spatially less restricted hydration water) are colored cyan and yellow, respectively. sites that show no cross peaks to water and are therefore representative of the fastest hydration dynamics are colored green. a clear grouping of very slow and spatially restricted hydration is evident along the c - terminal tail of the protein (top panel), and a cluster of very fast hydration sites is evident along the surface of the alpha helix (center panel). clusters of intermediate dynamics are also visible along the mixed beta sheet (bottom panel). these groupings illustrate the relative hydration dynamics essentially across the surface of the entire protein. importantly, a minority of solvent - exposed sites lacked detectable cross - relaxation to water. these sites, again clustered along the solvent - exposed ridge of the alpha helix, also did not show measurable cross peaks to surfactant hydrogens suggesting that long - lived interactions with the inner surface of the reverse micelle shell are absent and that the fastest and least spatially restricted hydration dynamics occur at these amide sites. reverse micelles are monodisperse ensembles of surfactant - encapsulated nanoscale water pools that result from spontaneous organization of a mixture of appropriate surfactants, water, and nonpolar solvent such as the alkanes (fig. the size of the water pool can be tightly controlled by varying the molar ratio of water to surfactant (water loading or wo). proteins can be encapsulated within the water pool of reverse micelles reproducibly and with high structural fidelity and at concentrations amenable to high - resolution solution nmr methods14,15. when prepared in low viscosity solvents, the full arsenal of solution multi - dimensional heteronuclear nmr techniques becomes accessible16,17. reverse micelles have served as a powerful tool for nmr - based studies of protein and nucleic acid structure and biophysics under a range of conditions14,16,1823. for water loadings such as those used here (w0 ~ 10), the reverse micelle contains approximately one thousand water molecules on average24 and the re - orientational dynamics of encapsulated water is slowed relative to bulk water by approximately one order of magnitude25,26. this suggested that the combined effects of the protein surface and nanoscale confinement could slow the water dynamics to allow efficient detection of hydration water - protein cross - relaxation. at the same time, if reorganization of water contributes to the rate limiting step, hydrogen exchange chemistry in the reverse micelle should also be somewhat slowed, potentially minimizing complications arising from hydrogen - exchange. finally, reverse micelles also provide an appropriate experimental condition to directly assess the contributions from long - range intermolecular dipolar interactions. ubiquitin is a small 76 residue protein comprised of a five stranded mixed beta sheet, a long alpha helix and a short 310 helix27. we first examined the hydration of ubiquitin in free aqueous solution at ph 5 and 25 c using three - dimensional n - resolved noesy29 and roesy30 experiments (fig. only a few amide - water weak cross peaks were observed, which is generally indicative of very short residence times for protein hydration water in bulk aqueous solution3. at this ph and temperature, the exchange of even non - hydrogen bonded amide hydrogens with solvent is generally too slow to impact these experiments. however, every amide hydrogen - water cross peak observed in the free solution spectra involves an amide hydrogen that is within the distance detectable by the noe (see below) of a labile side chain hydrogen. chemical exchange of side chain hydrogens with solvent water followed by intramolecular noe produces cross peaks at the water resonance that are indistinguishable from direct protein - water noes2,3. water noe correlations seen in bulk solution are the result of such exchange - relayed interactions5. we have previously demonstrated that the structure of ubiquitin is unperturbed by appropriate encapsulation in reverse micelles composed of bis-2-ethylhexyl sulfosuccinate (aot)14. water interactions revealed by the noesy spectrum of ubiquitin in free solution, the protein encapsulated in aot reverse micelles displays literally dozens of protein - water interactions under similar temperature and ph conditions (fig. dipolar exchange is identified by the opposite phase of the cross peaks in the orthogonal frames (shown here as positive noe, negative roe), while direct hydrogen exchange with solvent gives rise to peaks of identical phase (shown as positive in both spectra)2,3. calibration of the noe obtained at the 40 ms mixing time used here placed it in the linear regime and corresponds to a maximum noe - detected distance of approximately 4.3. more than half of detected amide hydrogen - water cross peaks involve amides that are over 4.3 from any labile side chain hydrogen, as indicated by of the ensemble of structures determined for encapsulated ubiquitin (pdb 1g6j)14. these can therefore be unequivocally assigned to direct noe interactions between hydration water and the protein. in addition, the hydrogen exchange chemistry catalyzed by hydroxide ion within the reverse micelle water pool is effectively slowed by two orders of magnitude as indicated by the ph dependence of the onset of line broadening due to amide hydrogen exchange (supplementary fig. it should be noted that the pka values of side chain hydroxyls and primary amines are low enough that general base catalysis by buffer or even the head groups of aot is also a potential concern. however, the fact that lysine side chain amine groups are observed (fig. 2) and that most side chain hydroxyls could be identified by direct noe (supplementary fig. given the relative concentrations of protein and water, this restricts the hydrogen exchange rates to the 1 s or slower rate regime. the single labile side chain hydrogen whose chemical exchange may be faster than this rate is the hydroxyl of tyr-59, which is anticipated to have a considerably lower pka. however, contributions from hydrogen exchange - mediated pathways are sufficiently minimized to allow quantitative interpretation of the vast majority of the noe and roe cross peak intensities. interpretation of the noe / roe ratio (see below) as directly representative of local hydration dynamics also requires that the contributions from long - range dipolar couplings be negligible. noesy spectra of highly deuterated ubiquitin lack any measurable cross peaks to organic solvent hydrogens, demonstrating that long - range interactions beyond the exterior of the reverse micelle are minimal. it is this long - range contribution in bulk aqueous solution that has been argued to provide the vast majority of the noe intensity to the water resonance10. it is also of note that only a handful of amide hydrogens show noes with hydrogens of the surfactant headgroup. with the exception of gln 47 and gln 49, the grouping of such interactions suggests that they arise from local close approach of the protein to the surfactant shell rather than general long - range dipolar exchange contributions. it is important to note that these regions of the surface are not dehydrated (fig. the spatial geometry of the reverse micelle and encapsulated protein also cause the number of water molecules to increase roughly as the square of the distance from a solvent - exposed protein hydrogen in contrast to the cubic dependence seen in bulk solution (supplementary fig. these fundamental differences in the physical parameters of the reverse micelle system versus bulk solution combine to largely eliminate contamination of short - range dipolar interactions between protein and hydration water by contribution from long - range couplings to solvent. in summary, the residence times of water on the surface of an encapsulated ubiquitin molecule are sufficiently increased, the effective rates of specific and general base catalysis of hydrogen exchange sufficiently decreased, and the potential contribution by long - range coupling to bulk solvent sufficiently reduced to allow confident and comprehensive site - resolved measurements of dipolar cross - relaxation between hydration waters and amide hydrogens of the protein. this enables the first site - resolved analysis of relative hydration water mobilities across an entire protein surface. a quantitative measure of hydration dynamics can be obtained from the ratio of the noe to roe cross - relaxation rates, noe and roe2,3. here ratios of the intensities of the water cross peaks are used (supplementary tables 1 and 2). in the slow correlation time limit (1), a rigidly bound water molecule would result in a noe / roe ratio of 0.5 (ref. we find that a minority of the resolved protein - water correlations have ratios at this limit. previous relaxation studies indicate that the effective macromolecular tumbling time for these reverse micelle particles dissolved in liquid pentane is about 10 ns16. the majority of detected hydration sites showed a range of noe / roe values between 0 and 0.5 indicating the presence of substantial motion of the hydration water on the surface of the protein and/or a somewhat shorter residence time3,31. in previous studies of protein - water cross relaxation, it was unclear to what degree indirect magnetization exchange contributed to noe / roe ratios more positive than 0.5. as measurements made in the reverse micelle are generally uncomplicated by indirect exchange effects, we conclude that the range of observed noe / roe ratios arise from a distribution of hydration water dynamics that reflect both local geometrical factors and the residence times of the individual water molecules on the surface of ubiquitin31. approximately three fourths of the amide hydrogens within the noe - detection distance limit (4.3) of solvent showed measurable cross peaks to water. no cross peaks to water from buried amide hydrogens were detected, confirming the absence of spin - diffusion or appreciable penetration of water into the interior of the protein. a detailed view of the relative hydration dynamics of ubiquitin is shown in figure 3 where the amide hydrogens within noe distance of the molecular surface are represented as spheres and are color - coded according to dynamic classes. sites that have noe / roe ratios at the bound limit, representing the slowest regions of hydration dynamics, are colored dark blue. sites with ratios between 0.47 and 0.3 (relatively slow or spatially restricted hydration water) or between 0.3 and 0 (relatively fast or spatially less restricted hydration water) are colored cyan and yellow, respectively. sites that show no cross peaks to water and are therefore representative of the fastest hydration dynamics are colored green. a clear grouping of very slow and spatially restricted hydration is evident along the c - terminal tail of the protein (top panel), and a cluster of very fast hydration sites is evident along the surface of the alpha helix (center panel). clusters of intermediate dynamics are also visible along the mixed beta sheet (bottom panel). these groupings illustrate the relative hydration dynamics essentially across the surface of the entire protein. importantly, a minority of solvent - exposed sites lacked detectable cross - relaxation to water. these sites, again clustered along the solvent - exposed ridge of the alpha helix, also did not show measurable cross peaks to surfactant hydrogens suggesting that long - lived interactions with the inner surface of the reverse micelle shell are absent and that the fastest and least spatially restricted hydration dynamics occur at these amide sites. until now, experimental efforts to develop a site resolved understanding of the hydration layer around proteins have relied largely on crystallographic data. because most protein crystal structures are heavily hydrated and contain complex ensembles of solvent, the general view seems to be that the environment in protein crystals should be representative of the crowded conditions within the cell and that therefore crystallographic water locations should be representative of the native hydration behavior of the protein. extensive analysis of crystallographic waters has been combined with simulation data in several systems to propose a picture of the molecular nature of the hydration layer32. two crystal structures for wild type human ubiquitin are available (pdb codes 1ubq and 1ubi)27,33. only about half of the defined waters in the two crystal structures were conserved to within 1 between the structures even though the proteins were crystallized in the same space group under almost identical conditions. furthermore, there is little correlation between the waters detected by solution nmr and the locations and occupancies of the crystallographic waters in these crystal structures (supplementary fig. 4). to illustrate the degree of correspondence between the long - lived hydration waters detected by nmr to the locations of the defined waters common to both crystal structures of ubiquitin, a color - coded ribbon diagram of 1ubq is shown in figure 4. orange backbone corresponds to amides that are buried deeply enough to be outside noe detection distance from solvent. regions of the backbone that have solvent - accessible amide hydrogens but did not show cross peaks to water are colored green. as mentioned above, these sites must have only short - lived interactions with hydration water. portions of the backbone that have nmr - detected hydration water and also have water common to both crystal structures that are within noe distance are colored blue, as are the corresponding waters. crystallographic waters that do not have long - lived counterparts in the nmr hydration data are colored green. finally, yellow regions correspond to those amide hydrogens that have nmr - detected hydration water but have no counterpart in the waters common to both crystal structures. interestingly, only ~60% of the crystallographic waters that are common to both crystal structures appear within the 4.3 noe detection distance of sites where nmr - detected hydration waters are long - lived. in addition, more than half of the nmr - detected long - lived protein - water interactions involve amide hydrogens that are outside noe distance from the nearest conserved crystallographic water molecule. additionally, we have found no correlation between the noe / roe values for the nmr - detected sites of long - lived hydration and the location of the nearest conserved crystallographic water. in the case of ubiquitin, there seems to be limited correlation between the locations of the defined water molecules in the two crystal structures and the nmr - detected hydration waters of encapsulated ubiquitin. indeed, magnetic relaxation dispersion experiments have suggested one very long - lived water molecule is present in ubiquitin, which on the basis of the crystal structure (1ubq) was identified with a water molecule (water 28) localized near the amide nh of leu 43 (ref. though this water is illuminated by noes in encapsulated ubiquitin, its residence time is short enough to cause complete chemical shift averaging with the bulk water resonance. in contrast, encapsulated ubiquitin shows a very long lived water molecule that is localized to a relatively deep surface pocket and is highlighted by short distance noes between the amide hydrogens of val 69 and lys 6 and a resonance with a chemical shift (5.4 p.p.m.) resolved from the water resonance (4.32 p.p.m.) 2). these amide hydrogens are not within the noe detection distance of any hydroxyl group and we therefore identify this water, which is not resolved in the crystal structure, as the long - lived water molecule suggested by the magnetic resonance dispersion experiments. interestingly, this water resides in a pocket that is lined with hydrophobic side chains, which may account for the lack of ordered crystallographic water at this site35. in recent years, there has been an increasing appreciation of the inherent complexity of the cellular context in which proteins must function36. it is now well known that cells are packed with macromolecular surfaces that confine proteins to nanometer - scale volumes. confinement on this length scale can potentially alter a number of fundamental properties of proteins36 and creates an intracellular hydration environment that is potentially highly heterogeneous and distinct from bulk water1. reverse micelle encapsulation provides a convenient way to undertake detailed studies of the effects of confinement on protein hydration, folding and stability and dynamics at the atomic - scale using solution nmr methods20,37,38. here we have found a previously unseen range of hydration behavior across the surface of the protein. this represents an exciting alternate view for the ongoing effort to unravel the interplay between proteins and solvent that is so vital to living systems. in recent years, there has been an increasing appreciation of the inherent complexity of the cellular context in which proteins must function36. it is now well known that cells are packed with macromolecular surfaces that confine proteins to nanometer - scale volumes. confinement on this length scale can potentially alter a number of fundamental properties of proteins36 and creates an intracellular hydration environment that is potentially highly heterogeneous and distinct from bulk water1. reverse micelle encapsulation provides a convenient way to undertake detailed studies of the effects of confinement on protein hydration, folding and stability and dynamics at the atomic - scale using solution nmr methods20,37,38. here we have found a previously unseen range of hydration behavior across the surface of the protein. this represents an exciting alternate view for the ongoing effort to unravel the interplay between proteins and solvent that is so vital to living systems. methods and any associated references are available in the online version of the paper at http://www.nature.com / nsmb/. | the interactions of biological macromolecules with water are fundamental to their structure, dynamics and function. historically, characterization of the location and residence times of hydration waters of proteins in solution has been quite difficult. confinement within the nanoscale interior of a reverse micelle slows water dynamics, allowing detection of global protein - water interactions using nuclear magnetic resonance techniques. complications that normally arise from hydrogen exchange and long - range dipolar coupling are overcome by the nature of the reverse micelle medium. characterization of the hydration of ubiquitin demonstrates that encapsulation within a reverse micelle allows detection of dozens of hydration waters. comparison of nuclear overhauser effects obtained in the laboratory and rotating frames indicate a considerable range of hydration water dynamics is present on the protein surface. in addition, an unprecedented clustering of different hydration dynamic classes of sites is evident. |
entamoeba moshkovskii, entamoeba dispar and entamoeba histolytica are morphologically identical but biochemically and genetically are different and microscopic examination is unable to detect and differentiate these three enatamoeba spp. although e. histolytica is known to be pathogen, the other two species are non - pathogen or the ability of them to cause disease is unclear (1, 2). before redescription of e. histolytica and e. dispar in 1997. infection rate of about 2.2 to 30 percent (5 - 7). in the past decade, these three entamoeba have been differentiated and reported by molecular methods in some areas of iran (2, 8 - 17). multiplex pcr is a molecular biology technique for amplification of multiple targets in a single pcr experiment. the multiplex pcr was used extensively for pathogen identification, single nucleotide polymorphism (snp) genotyping, mutation analysis, gene deletion analysis, template quantitation, linkage analysis, rna detection, forensic studies and diet analysis (18, 19). in a multiplexing assay, more than one target sequence can be amplified by using multiple primer pairs in a reaction mixture (18). while the single template pcr reaction uses a single template along with several pairs of forward and reverse primers the multiple template pcr reaction uses multiple templates and several primer sets in the same reaction tube (18, 19). extraction of dna is often an early and important step in many diagnostic processes used to detect bacteria, viruses and parasites in the environment as well as diagnosing disease and genetic disorders (20). in the presence study, we investigated the presence of e. histolytica, e. dispar and e. moshkovskii by single - round and multiplex pcr using six different dna extraction methods. twenty - seven dna templates from 20 entamoeba histolytica and 7 entamoeba dispar samples were analyzed. all the 20 e. histolytica dnas were extracted previously in japan from japanese patients (21). the dna of 7 e. dispar was also extracted previously from iranian isolates (22). dna of e. histolytica hm-1 : imss, e. dispar saw760, and e. moshkovskii laredo cells as positive controls that were maintained alive in liquid nitrogen tank in department of medical parasitology and mycology, shahid beheshti university of medical sciences, were recovered in tyi - s-33 medium and their dnas were extracted to study the single and multiplex pcr assays. about 200 l of cultured trophozoites was washed with phosphate - buffered saline (ph=7.2). the genomic dna of cultured trophozoites was extracted and compared using five dna extraction kits : dng plus and dnp kit(cinnagen inc., tehran, iran), chelex (bio - rad), qiaamp dna mini kit and qiaamp dna stool mini kit (qiagen, germany) according to the manufacturer s directions and also the traditional phenol - chloroform method (23). the procedure of dng -plus and dnp requires about 60 minutes and does not require phenol extraction or proteinase digestion for dng-plus. in qiaamp dna stool mini kit and qiaamp dna mini kit for tissue extraction, purification requires no phenol - chloroform extraction or alcohol precipitation, and dna is eluted in low - salt buffer and is free of protein, nucleases, and other impurities or inhibitors. the chelex protects the sample from dnaases that might remain active after the boiling and could subsequently destroy the dna. the concentration and purity of the extracted dna was assessed by spectrophotometer wpa (biowave ii, eng) reading of the absorbance at the 260/280 nm. the dna concentration and the corresponding a260 values, for single - round pcr amplification as well as multiplex pcr were used in the study. the sequence of a forward primer used (entaf, 5-atgcacgagagcgaaagcat-3) was conserved in all three entamoeba spp., whereas the specific reverse primers, ehr (5-gatctagaaacaatgcttctct-3 x64142), edr (5-caccacttactatccct - acc-3 z49256), and emr (5-tgaccggagccagagacat-3 af149906), were specific for e. histolytca, e. dispar, e. moshkovskii, respectively (24). pcr was performed using amplicon (taq dna polymerase master mix red, denmark) as a ready - made solution. the reaction mixture contained 5 l of distilled water, 7.5 l of amplicon, 20 pmol of forward and reverse primers, and about 5 - 10 ng of extracted dna template to achieve a final volume of 15 l. amplification of each species - specific dna fragment started with an initial denaturation at 94c for 5 min, followed by 30 cycles of 94c for 1 min, 55c for 1 min, and 72c for 1 min, with a final extension at 72c for 7 min. twenty - seven dna templates from 20 entamoeba histolytica and 7 entamoeba dispar samples were analyzed. all the 20 e. histolytica dnas were extracted previously in japan from japanese patients (21). the dna of 7 e. dispar was also extracted previously from iranian isolates (22). dna of e. histolytica hm-1 : imss, e. dispar saw760, and e. moshkovskii laredo cells as positive controls that were maintained alive in liquid nitrogen tank in department of medical parasitology and mycology, shahid beheshti university of medical sciences, were recovered in tyi - s-33 medium and their dnas were extracted to study the single and multiplex pcr assays. about 200 l of cultured trophozoites was washed with phosphate - buffered saline (ph=7.2). the genomic dna of cultured trophozoites was extracted and compared using five dna extraction kits : dng plus and dnp kit(cinnagen inc., tehran, iran), chelex (bio - rad), qiaamp dna mini kit and qiaamp dna stool mini kit (qiagen, germany) according to the manufacturer s directions and also the traditional phenol - chloroform method (23). the procedure of dng -plus and dnp requires about 60 minutes and does not require phenol extraction or proteinase digestion for dng-plus. in qiaamp dna stool mini kit and qiaamp dna mini kit for tissue extraction, purification requires no phenol - chloroform extraction or alcohol precipitation, and dna is eluted in low - salt buffer and is free of protein, nucleases, and other impurities or inhibitors. the chelex protects the sample from dnaases that might remain active after the boiling and could subsequently destroy the dna. the concentration and purity of the extracted dna was assessed by spectrophotometer wpa (biowave ii, eng) reading of the absorbance at the 260/280 nm. the dna concentration and the corresponding a260 values, for the six dna isolation methods of three positive controls are shown in table 1. single - round pcr amplification as well as multiplex pcr were used in the study. the sequence of a forward primer used (entaf, 5-atgcacgagagcgaaagcat-3) was conserved in all three entamoeba spp., whereas the specific reverse primers, ehr (5-gatctagaaacaatgcttctct-3 x64142), edr (5-caccacttactatccct - acc-3 z49256), and emr (5-tgaccggagccagagacat-3 af149906), were specific for e. histolytca, e. dispar, e. moshkovskii, respectively (24). pcr was performed using amplicon (taq dna polymerase master mix red, denmark) as a ready - made solution. the reaction mixture contained 5 l of distilled water, 7.5 l of amplicon, 20 pmol of forward and reverse primers, and about 5 - 10 ng of extracted dna template to achieve a final volume of 15 l. amplification of each species - specific dna fragment started with an initial denaturation at 94c for 5 min, followed by 30 cycles of 94c for 1 min, 55c for 1 min, and 72c for 1 min, with a final extension at 72c for 7 min. the forward primer in combination with the appropriate reverse primer amplified a 166-bp pcr product with e. histolytica, a 752-bp pcr with e. dispar, and a 580-bp with e. moshkovskii dnas. by using the separate entamoeba spp. dna template, with species - specific e. histolytica primers (entaf / ehr) amplified dna from the hm-1:imss strain with all of the six dna extracted methods observed, but no band were seen when e. dispar saw760 or e.moshkovskii laredo dna were used. the e. dispar species - specific primers (entaf / edr) and the e. moshkovskii primers (entaf / emr) also showed the expected specificities in single - round pcr. similar results were also observed when the forward and reverses primers for e. histolytica, e. dispar, and e. moshkovskii were mixed in a single dna template reaction. amplified pcr bands of the six dna extracted methods using the three entamoeba isolates visualized on a 1.5% agarose gel are presented in fig. 1. when dnas of e. histolytica (hm1:imss), e. dispar (saw760), and e. moshkovskii laredo strain were mixed together in a multiplex pcr assay using the entire forward and three reverses primers, the same fragments of pcr results were observed. however, the pcr bands intensity of some of the dna extracted methods for e. histolytica and e. dispar were weak (fig. 2). overall, seventeen (62.96%) samples from 27 isolates including ten e. histolytica and seven e. dispar as well as extracted dnas from e. histolytica hm-1:imss, e. dispar saw 760, and e. moshkovskii laredo cells as positive control using single - round pcr reaction were identified (fig. a mixture dna of some of those positive e. histolytica, e. dispar isolates, and e. moshkovskii laredo strain, which were tested using multiplex pcr assay, are also shown in figs. 4. multiplex pcr fragments of the e. histolytica (hm-1:imss), e. dispar (saw760 and e. moshkovskii laredo strain amplified in a single reaction using forward primer combined with the three reverse primers are shown in fig. the forward primer in combination with the appropriate reverse primer amplified a 166-bp pcr product with e. histolytica, a 752-bp pcr with e. dispar, and a 580-bp with e. moshkovskii dnas. by using the separate entamoeba spp. dna template, with species - specific e. histolytica primers (entaf / ehr) amplified dna from the hm-1:imss strain with all of the six dna extracted methods observed, but no band were seen when e. dispar saw760 or e.moshkovskii laredo dna were used. the e. dispar species - specific primers (entaf / edr) and the e. moshkovskii primers (entaf / emr) also showed the expected specificities in single - round pcr. similar results were also observed when the forward and reverses primers for e. histolytica, e. dispar, and e. moshkovskii were mixed in a single dna template reaction. amplified pcr bands of the six dna extracted methods using the three entamoeba isolates visualized on a 1.5% agarose gel are presented in fig. 1. when dnas of e. histolytica (hm1:imss), e. dispar (saw760), and e. moshkovskii laredo strain were mixed together in a multiplex pcr assay using the entire forward and three reverses primers, the same fragments of pcr results were observed. however, the pcr bands intensity of some of the dna extracted methods for e. histolytica and e. dispar were weak (fig. overall, seventeen (62.96%) samples from 27 isolates including ten e. histolytica and seven e. dispar as well as extracted dnas from e. histolytica hm-1:imss, e. dispar saw 760, and e. moshkovskii laredo cells as positive control using single - round pcr reaction were identified (fig. 3). a mixture dna of some of those positive e. histolytica, e. dispar isolates, and e. moshkovskii laredo strain, which were tested using multiplex pcr assay, are also shown in figs. 4. multiplex pcr fragments of the e. histolytica (hm-1:imss), e. dispar (saw760 and e. moshkovskii laredo strain amplified in a single reaction using forward primer combined with the three reverse primers are shown in fig. a single - round pcr - based approach for differential diagnosis of three species of entamoebas, e. moshkovskii, e. histolytica, and e. dispar, which share identical morphology as both cysts and trophozoites were described in this study. this simple diagnostic pcr technique does not require extra steps, as is the case with nested pcr (25), pcr restricted fragment length polymorphism (26), and pcr with reverse line blot hybridization (27). six simple methods including phenol chloroform, dng -plus, dnp, qiaamp dna mini kit for tissue, qiaamp dna stool mini kit and chelex kit for the extraction of dna from entamoeba spp. were also compared and evaluated. in all 6 methods, dna was extracted from cultured trophozoites of the three cryopreserved entamoebas. in manual phenol chloroform dna extraction method which is time consuming, two bands were seen above the expected band of e. histolytica cultured trophozoites (fig 1, a). the study indicated that the multiplex - pcr consisted of multiple primer sets within single template had a better result compared with multiple primer plus multiple template pcr reaction. molecular tools are extensively used for epidemiological studies, particularly in differentiation of the pathogenic from the non - pathogenic species of the entamoeba species. this study and a few reports by the other researchers showed single and multiplex pcr assay in a single sample is able to detect and differentiate e. histolytica, e. dispar and e. moshkovskii (28 - 30). recently usefulness of nested multiplex pcr method for differentiation of e. histolytica from e. dispar on 31 stool samples was reported by fallah. although a remarkable results were obtained for differentiation of the three microscopy identical entamoeba species by a single - round and multiplex pcr in this study, but further studies on more positive stool entamoeba samples as well as normal subjects and non - entamoeba isolates in iran are needed to evaluate sensitivity and specificity of those primers in a multiplex pcr. we recommend the application of multiplex pcr assay as an alternative tool in routine diagnosis and epidemiological studies of amoebiasis. it is expected that this will provide better epidemiological data and a greater understanding of infections with these three amoebae in humans. | background entamoeba moshkovskii and e. dispar are impossible to differentiate microscopically from the pathogenic species e. histolytica. multiplex polymerase chain reaction (multiplex pcr) is a widespread molecular biology technique for amplification of multiple targets in a single pcr experiment.methodsfor detection and differentiation of the three - microscopy indistinguishable entamoeba species in human, multiplex pcr assay using different dna extraction methods was studied. a conserved forward primer was derived from the middle of the small - subunit rrna gene, and reverse primers were designed from signature sequences specific to each of these three entamoeba species.resultsa 166-bp pcr product with e. histolytica dna, a 580-bp product with e. moshkovskii dna and a 752-bp product with e. dispar dna were generated in a single - round and multiplex pcr reaction.conclusionwe recommend this pcr assay as an accurate, rapid, and effective diagnostic method for the detection and discrimination of these three entamoeba species in both routine diagnosis of amoebiasis and epidemiological surveys. |
solid lipid nanoparticles (slns) were developed in the early 1990s as an alternative carrier system to traditional colloidal systems, including emulsions, liposomes, and polymeric microparticles and nanoparticles.1 tremendous progress has been made in the treatment of disorders using new drug delivery systems, including slns, which are made from solid lipids (ie, lipids solid at room temperature as well as body temperature) and stabilized by surfactant(s).2 they are generally composed of a solid hydrophobic core with a monolayer of phospholipids or phospholipid - derived coating. the solid core contains the active drug dissolved or dispersed in a solid fat matrix with the hydrophobic ends of the phospholipid chains embedded in the fat matrix. thus, they have the potential to carry lipophilic or hydrophilic drug(s) for diagnostic purposes.35 a clear advantage of slns over polymeric nanoparticles is the fact that the lipid matrix is made from physiologically tolerated lipid components, which decreases the potential for acute and chronic toxicity.5,6 slns have fewer storage and drug leakage problems compared with systems such as liposomes and also have good stability for 23 years.69 it is noteworthy that tumors and inflamed tissues are often associated with leaky vascular architecture as a result of the poorly regulated nature of tumor angiogenesis. therefore, the use of acid - sensitive drug carriers has been one of the most extensively studied active triggering strategies.10,11 dong and hoffman prepared and reported ph - sensitive hydrogels for drug delivery.12 oh and lee created a novel vector for delivery of anticancer therapy to solid tumors using diafiltration to synthesize ph - sensitive polymeric micelles, and observed that release of doxorubicin as micelles was triggered at ph 6.8.13 reddy and low prepared a ph - sensitive lipid formulation from dioleylphosphatidylethanolamine and citraconic anhydride, and used this to prepare liposomes. they observed that the resulting liposomes were stable at neutral ph but fusogenic at ph 5.11 the nature of tumor angiogenesis enables submicronsized particulate matter, such as slns, to penetrate preferentially into tumor tissue and be retained there. consequently, tumor - specific drug delivery is attracting considerable attention in cancer therapy.7,1416 moreover, with advances in surface engineering technology, the biodistribution of slns can be further manipulated by modifying the surface physicochemical properties of slns to target them to the tissue of interest, particularly to tumors and inflamed tissue.7,17 so far, researchers have prepared various derivatives of phospholipids and used them on the outer shells of slns.18 the aim of the present study was to investigate the feasibility of the inclusion of triamcinolone acetonide into surface - modified solid lipid nanoparticles. for this purpose triamcinolone acetonide is usually administered via the parenteral route to treat inflammatory disorders and cancer of the lymph nodes. it is a drug with poor water solubility and high lipophilicity, which makes it suitable for sln encapsulation and in vitro characterization. modified high shear homogenization and ultrasound techniques have been used to produce low particle sizes and high encapsulation efficiency,19 while photon correlation spectroscopy, differential scanning calorimetry, atomic force microscopy, and scanning electron microscopy have been used for their characterization. the following materials were obtained from the indicated sources and used in our study without further purification. tripalmitin glyceride was purchased from alfa aesar (germany), and phosphatidylethanolamine and triamcinolone acetonide from sigma - aldrich (st louis, mo). glutaric anhydride, polysorbate 80, and sucrose were obtained from merck (darmstadt, germany). high - pressure liquid chromatography (hplc) grade methanol and analytical grade chloroform and ethanol were also purchased from merck. double - distilled water was prepared in our laboratory. for preparation of modified phosphatidylethanolamine, 5 mg of lipid was dissolved in 10 ml of chloroform, and an approximately five - fold molar excess (3.85 mg) of glutaric anhydride was then added in the presence of 7.5 l pyridine, and the mixture was incubated at 20c for 5 hours.20 modified phosphatidylethanolamine was identified by iodine staining.21 n - glutaryl phosphatidylethanolamine was isolated using plate chromatography and chloroform - methanol (65:35 v / v) as the solvent, with a yield of 90% : h nmr (200 mhz, cdcl3) 0.88 (6h), 1.30 (32 h), 1.59 (4h), 1.99 (2h), 2.16 (8h), 2.27 (6h), 2.61 (2h), 3.48 (2h), 3.95 (2h), 4.13 and 4.38 (2h), 4.20 and 4.51 (2h), 4.71 (1h), 5.33 (4h), and 7.38 (1h) that peaked in 7.38 ppm indicated amidic hydrogen. triamcinolone acetonide (0.75% w / v), tripalmitin glyceride (2% w / v), and n - glutaryl phosphatidylethanolamine (0.5% w / v) were dissolved in 10 ml of chloroform and methanol mixture (1:1). organic solvents were completely removed using a rotoevaporator (laborota 4003, heidolph, schwabach, germany). the drug - embedded lipid layer was melted by heating at 70c above the melting point of the lipid. an aqueous phase was prepared by dissolving polysorbate 80 (1% w / v) in double - distilled water and heated to the same temperature as the oil phase. the hot aqueous phase was added to the oil phase, and homogenization was achieved (at 10,000 rpm and 70c) using a diax 900 homogenizer (heidolph) for 2 minutes. the resulting coarse hot oil in water emulsion was ultrasonicated using a sonoplus ultrahomogenizer (bandelin, germany) for 15 minutes. triamcinolone acetonide - slns were obtained by allowing the hot nanoemulsion to cool off at room temperature. sucrose was used in the freeze - drying process as a cryoprotector at a concentration of 3 wt%.22 the sln suspension was frozen in an aqueous sucrose solution at 70c overnight, and the sample was subsequently transferred to the freeze - drier (zirbus technology vaco 5, d-37539) at 50c for 72 hours, and finally the sln powder was collected for further experiments. the percentage of incorporated triamcinolone acetonide (entrapment efficiency) was determined by spectrophotometric determination at 234 nm using an agilent 8453-spectrophotometer, after ultracentrifugation of the aqueous dispersion (40,000 rpm for 45 minutes). the amount of free drug was detected in the supernatant and the amount of incorporated drug was calculated as the initial drug minus the free drug. the drug entrapment efficiency and drug - loading in the slns were calculated using equations (1) and (2) : where ee is entrapment efficiency, dl is drug - loading, and wa, ws, and wl are the weight of drug added into the system, analyzed weight of drug in the supernatant, and weight of lipid added into the system, respectively.23 measurement of hydrodynamic diameter, polydispersity index, and zeta potential of the nanoparticles was accomplished using photon correlation spectroscopy (nano zs4700, malvern instruments, worcestershire, uk). for size measurement, all formulations were diluted by deionized water to eliminate the effect of viscosity caused by the ingredients. hydrodynamic diameter and zeta potential were reported as the mean of three measurements at 25c. the refractive index of the slns and water was set at 1.35 and 1.33, respectively. in order to verify the result, the morphology of the triamcinolone acetonide - slns was studied by scanning electron microscopy using a philips xl30 microscope at an accelerating voltage of 10 kv. one droplet of sln nanosuspension was placed on an aluminum specimen stub. after oven - drying for 12 hours, the sample was coated with a platinum layer using an scdoos sputter coater (bal - tec, sweden) in an argon atmosphere. the surface properties of the drug - loaded slns were visualized using an atomic force microscope (mobile s, nanosurf, switzerland). explorer atomic force microscopy was in noncontact mode, using high resonant frequency (f0 170 khz) pyramidal cantilevers with silicon probes having dynamic force. the sample was diluted with distilled water and then dropped onto a freshly cleaved mica plate, followed by vacuum drying for 24 hours at 25c. the thermal characteristics of the slns and pure drug were determined using a differential scanning calorimeter (shimadzu dsc-60, single heating ramp 0c300c, heated at 5c / minute) under a dry nitrogen atmosphere. samples (78 mg) were placed in an aluminum pan and heated at a rate of 5c / minute. empty aluminum pans were used as references, and the entire thermal behavior was studied under a nitrogen purge. a simple and sensitive hplc method with ultraviolet detection was used for analysis of triamcinolone acetonide.20 the triamcinolone acetonide concentration was determined using an hplc system (lc-10as liquid chromatograph, scl- 10a system controller, sil-10av uv - vis detector, c - r6a chromatopac, shimadzu, japan). the analytical conditions were as follows : column c18, hplc pack column, inertsil ods, 5 m, 4.6 250 mm (gl sciences inc, japan) ; ultraviolet detection, 240 nm ; mobile phase, methanol ; water 50:50 (v / v) ; flow rate 1 ml / minute ; and injection volume 25 l. this experiment was conducted using a static horizontal franz diffusion cell to evaluate the amount of triamcinolone acetonide released from each formulation.24 a cellulose acetate membrane with a molecular weight cutoff of 12,000 da and a surface area of 2.0 cm was used and mounted on the franz diffusion cell. the receptor medium was precisely 50 ml in volume and composed of an aqueous solution of physiological saline, phosphate - buffered saline, and 20% ethanol at four ph values of 4.0, 5.0, 6.0, and 7.4, stirred by a magnetic bar at 500 rpm to homogenize the medium. each formulation, 10 mg in 1 ml volume, was loaded onto the membrane in the donor compartment. the temperature of the assay was controlled at precisely 37c. at predetermined time intervals, 1 ml aliquots of the release medium were withdrawn using a syringe needle, and the same volumes of freshly prepared receptor medium were added. the samples were analyzed using an hplc method described previously.20 the experiments were repeated three times and the results were expressed as means standard deviation. for preparation of modified phosphatidylethanolamine, 5 mg of lipid was dissolved in 10 ml of chloroform, and an approximately five - fold molar excess (3.85 mg) of glutaric anhydride was then added in the presence of 7.5 l pyridine, and the mixture was incubated at 20c for 5 hours.20 modified phosphatidylethanolamine was identified by iodine staining.21 n - glutaryl phosphatidylethanolamine was isolated using plate chromatography and chloroform - methanol (65:35 v / v) as the solvent, with a yield of 90% : h nmr (200 mhz, cdcl3) 0.88 (6h), 1.30 (32 h), 1.59 (4h), 1.99 (2h), 2.16 (8h), 2.27 (6h), 2.61 (2h), 3.48 (2h), 3.95 (2h), 4.13 and 4.38 (2h), 4.20 and 4.51 (2h), 4.71 (1h), 5.33 (4h), and 7.38 (1h) that peaked in 7.38 ppm indicated amidic hydrogen. triamcinolone acetonide (0.75% w / v), tripalmitin glyceride (2% w / v), and n - glutaryl phosphatidylethanolamine (0.5% w / v) were dissolved in 10 ml of chloroform and methanol mixture (1:1). organic solvents were completely removed using a rotoevaporator (laborota 4003, heidolph, schwabach, germany). the drug - embedded lipid layer was melted by heating at 70c above the melting point of the lipid. an aqueous phase was prepared by dissolving polysorbate 80 (1% w / v) in double - distilled water and heated to the same temperature as the oil phase. the hot aqueous phase was added to the oil phase, and homogenization was achieved (at 10,000 rpm and 70c) using a diax 900 homogenizer (heidolph) for 2 minutes. the resulting coarse hot oil in water emulsion was ultrasonicated using a sonoplus ultrahomogenizer (bandelin, germany) for 15 minutes. triamcinolone acetonide - slns were obtained by allowing the hot nanoemulsion to cool off at room temperature. sucrose was used in the freeze - drying process as a cryoprotector at a concentration of 3 wt%.22 the sln suspension was frozen in an aqueous sucrose solution at 70c overnight, and the sample was subsequently transferred to the freeze - drier (zirbus technology vaco 5, d-37539) at 50c for 72 hours, and finally the sln powder was collected for further experiments. the percentage of incorporated triamcinolone acetonide (entrapment efficiency) was determined by spectrophotometric determination at 234 nm using an agilent 8453-spectrophotometer, after ultracentrifugation of the aqueous dispersion (40,000 rpm for 45 minutes). the amount of free drug was detected in the supernatant and the amount of incorporated drug was calculated as the initial drug minus the free drug. the drug entrapment efficiency and drug - loading in the slns were calculated using equations (1) and (2) : where ee is entrapment efficiency, dl is drug - loading, and wa, ws, and wl are the weight of drug added into the system, analyzed weight of drug in the supernatant, and weight of lipid added into the system, respectively.23 measurement of hydrodynamic diameter, polydispersity index, and zeta potential of the nanoparticles was accomplished using photon correlation spectroscopy (nano zs4700, malvern instruments, worcestershire, uk). for size measurement, all formulations were diluted by deionized water to eliminate the effect of viscosity caused by the ingredients. hydrodynamic diameter and zeta potential were reported as the mean of three measurements at 25c. the refractive index of the slns and water was set at 1.35 and 1.33, respectively. in order to verify the result, the morphology of the triamcinolone acetonide - slns was studied by scanning electron microscopy using a philips xl30 microscope at an accelerating voltage of 10 kv. one droplet of sln nanosuspension was placed on an aluminum specimen stub. after oven - drying for 12 hours, the sample was coated with a platinum layer using an scdoos sputter coater (bal - tec, sweden) in an argon atmosphere. the surface properties of the drug - loaded slns were visualized using an atomic force microscope (mobile s, nanosurf, switzerland). explorer atomic force microscopy was in noncontact mode, using high resonant frequency (f0 170 khz) pyramidal cantilevers with silicon probes having dynamic force. the sample was diluted with distilled water and then dropped onto a freshly cleaved mica plate, followed by vacuum drying for 24 hours at 25c. the thermal characteristics of the slns and pure drug were determined using a differential scanning calorimeter (shimadzu dsc-60, single heating ramp 0c300c, heated at 5c / minute) under a dry nitrogen atmosphere. samples (78 mg) were placed in an aluminum pan and heated at a rate of 5c / minute. empty aluminum pans were used as references, and the entire thermal behavior was studied under a nitrogen purge. a simple and sensitive hplc method with ultraviolet detection was used for analysis of triamcinolone acetonide.20 the triamcinolone acetonide concentration was determined using an hplc system (lc-10as liquid chromatograph, scl- 10a system controller, sil-10av uv - vis detector, c - r6a chromatopac, shimadzu, japan). the analytical conditions were as follows : column c18, hplc pack column, inertsil ods, 5 m, 4.6 250 mm (gl sciences inc, japan) ; ultraviolet detection, 240 nm ; mobile phase, methanol ; water 50:50 (v / v) ; flow rate 1 ml / minute ; and injection volume 25 l. this experiment was conducted using a static horizontal franz diffusion cell to evaluate the amount of triamcinolone acetonide released from each formulation.24 a cellulose acetate membrane with a molecular weight cutoff of 12,000 da and a surface area of 2.0 cm was used and mounted on the franz diffusion cell. the receptor medium was precisely 50 ml in volume and composed of an aqueous solution of physiological saline, phosphate - buffered saline, and 20% ethanol at four ph values of 4.0, 5.0, 6.0, and 7.4, stirred by a magnetic bar at 500 rpm to homogenize the medium. each formulation, 10 mg in 1 ml volume, was loaded onto the membrane in the donor compartment. the temperature of the assay was controlled at precisely 37c. at predetermined time intervals, 1 ml aliquots of the release medium were withdrawn using a syringe needle, and the same volumes of freshly prepared receptor medium were added. the samples were analyzed using an hplc method described previously.20 the experiments were repeated three times and the results were expressed as means standard deviation. in this study, homogenization was performed at above the melting point of the lipid followed by ultrasonication for preparation of modified sln.25,26 to disperse the triamcinolone acetonide homogeneously in the lipid, a chloroform / methanol solvent system was used and homogenized for 2 minutes.27 subsequently, the prepared slns were subjected to further lyophilization. our preliminary studies showed that reconstitution of freeze - dried slns was impossible when they were freeze - dried without addition of cryoprotectants. therefore, a 1:1 dilution with 3% w / w of sucrose as a cryoprotectant was performed.28 many different drugs have been incorporated in the slns.17 the prerequisite for obtaining a sufficient loading capacity is a sufficiently high solubility of the drug in the lipid melt. relatively higher encapsulation efficiency constitutes one of the major advantages of slns. for calculations of entrapment efficiency and loading capacity, calibration curves for the ultraviolet assays of triamcinolone acetonide were conducted on eight solutions in the concentration ranges of 1.5 10 to 1.25 10 mol / l ; encapsulation efficiency and drug - loading were 94.32% and 25.32%, respectively. the high entrapment efficiency of the drug is thought to be the result of the lipophilic characteristics and high compatibility between the drug and the lipid. furthermore, the high encapsulation efficiency and drug - loading in comparison with previous work24 is a result of the method of preparation and mixing of the drug and lipid matrix in the organic solvents. the mean particle size, polydispersity index and zeta potential of the slns with and without drug were measured by photon correlation spectroscopy, as summarized in table 1. the mean particle sizes of 165.8 nm and 97.44 nm were obtained for drug - loaded and free slns, respectively (table 1). the increase in size after drug loading must be due to incorporation of the drug in the nanoparticle matrix.23 the polydispersity index is a ratio providing information about the homogeneity of particle size distribution in a given system, and ideally, this should be < 0.3.29 triamcinolone acetonide - loaded and free sln had polydispersity index values of 0.254 and 0.337, respectively, indicating that the nanoparticles had a narrow size distribution, and suggesting nanoparticle monodispersity.30,31 the zeta potential is a key factor for evaluation of the stability of a colloidal dispersion.23 when the absolute value of the zeta potential is higher than 30 mv for a colloidal formulation, the particles are likely to be electrochemically stable under the investigated condition.25 zeta potential values of 41.6 mv and 43.0 mv were obtained for the drug - loaded and free slns, respectively (figure 1). as table 1 demonstrates, as time passes, increases are observed in size, polydispersity index, and zeta potential as a result of light and other factors that cause gelation.32 however, it is obvious from these values that the prepared nanosuspension has an acceptable electrochemical and physical stability potential for 3 and 6 months of storage. similarly, in order to evaluate the stability of slns lyophilized powder after 6 months, 10 mg of sample was dispersed in distilled water and characterized. the values for particle diameter, polydispersity index, and zeta potential were 223.3 nm, 0.321, and 30.1, respectively. these results indicate that, using sucrose as a cryoprotectant, the particle size remains in the nanometric range. in order to obtain a scanning electron microscopic image, we used freshly prepared sln loaded with triamcinolone acetonide, and micrographs of the samples containing drug - loaded slns are shown in figure 2. the micrographs demonstrated a spherical shape and a smooth surface, with a particle size in the nanometric range.25 the atomic force microscopy technique has been widely used to obtain size, shape, and surface morphological information about nanoparticles. it is capable of resolving surface details down to 0.01 nm and producing a contrasted and three - dimensional image of the sample.23 a drop of diluted aqueous suspension (without preliminary ultrafiltration) was placed on a mica slide and dried out at room temperature for 24 hours. figure 3 gives the tapping mode atomic force microscopic images of freshly prepared sln containing triamcinolone acetonide after 24 hours (a and b), 48 hours (c and d), and 1 week (e and f). the nanoparticle images demonstrated spherical particles of 50600 nm in diameter, but only 66.5 nm in height. after 48 hours, the nanoparticles were uniformly grown on mica slides, as illustrated in figure 3 (c and d). one week after spreading slns onto the mica plates, no particles could be distinguished in the atomic force microscopic images, which showed homogeneous and fiat shapes, as illustrated in figure 3 (e and f).33 this result is not surprising, considering the well known ability of particles to fuse during evaporation of water consequent to the molecular diffusion on the surface of the particles. moreover, the presence of free surfactant in the suspension may increase the characteristic size.33 differential scanning calorimetry thermograms for triamcinolone acetonide, tripalmitin glyceride, a physical mixture of triamcinolone acetonide, tripalmitin glyceride, modified phosphatidylethanolamine, and lyophilized slns are shown in figure 4. a sucrose thermogram is also shown in this figure to demonstrate peaks of lyophilized slns. the triamcinolone acetonide powder showed a sharp melting process with an onset temperature of 284.72c and a peak of 286.04c, with a melting enthalpy of 64.73 j / g. this was confirmed by the presence of a melting peak for triamcinolone acetonide in the physical mixture. this suggested that there was no crystalline triamcinolone acetonide in the sln suspension, and triamcinolone acetonide might be present in an amorphous state. similar results have been reported by previous researchers, stating that rapid quenching of the nanoemulsion does not allow the drug to crystallize.26,27 an endothermic peak of the sucrose used as a cryoprotectant was observed at 170.21c in the triamcinolone acetonide- sln curve. meanwhile, the melting peak temperature of tripalmitin glyceride in lyophilized sln (57.73c) was diminished when compared with the temperature of the bulk lipid (64.38c). the decrease in melting point can be attributed to the small size (nanometer range), high specific surface area, and presence of a surfactant.15,30 this can also be attributed to the kelvin effect and is described by the thomson equation.3 moreover, the lower melting enthalpy value of 35.72 j / g indicates a less ordered lattice arrangement of lipids within the nanoparticles compared with the bulk materials.9 in this study, the in vitro release of triamcinolone acetonide from slns was investigated for 60 hours at 37c using a franz diffusion cell (figure 5). due to the low water solubility of triamcinolone acetonide, 20% ethanol was added to the phosphate - buffered solution at four ph values of 4.0, 5.0, 6.0, and 7.4.24 our results showed that up to 50% was released over the test period. the prolonged and lower release of slns after 12 hours can be explained by slower diffusion of triamcinolone acetonide from the inner solid core matrix of the formulations. however, the initial fast release was detected at a lower ph. in this experiment, we studied the release profile of pure drug compared with nanoparticles in acidic ph, and the release half - life of drug release was significantly diminished compared with nanoparticles (about 25 minutes). the release data were analyzed using the following higuchi kinetic equation:25 qt = kt0.5, where qt is the percentage of drug released at time t, and k is the release rate constant. regarding the release model of all formulations, it was found that the prolonged release characteristic of triamcinolone acetonide was well fitted to higuchi s square root model, as has been reported for drug - loaded sln systems.25,34,35 linear fits were obtained, indicating that the release profile of triamcinolone acetonide from homogenous and granular matrix systems is diffusion - controlled. the r values from in vitro release kinetics and the k values or release rate constant obtained from the higuchi model plot are presented in table 2. furthermore, as shown in figure 6 and the in vitro release data, we found that the burst release was increased with a decline in ph that demonstrates a change in structure of the phospholipids and release of the terminal anhydride. several previous studies have been conducted to identify a ph - sensitive carrier preparation, and often the amount of drug release from slns was small compared with our results, and the values for common slns without modification were below 25%.8,25,27 subedi investigated ph - sensitive slns and used them for doxorubicin delivery ; in their work, 30% of the drug was released after 24 hours.8 venkateswarlo and manjunath investigated slns of tripalmitin and used them as clozapine carriers, and in their work, up to 20% of drug was released after 48 hours.25 liu investigated slns containing triamcinolone acetonide in mixtures of monoglycerides, diglycerides, and triglycerides of palmitic acid and stearic acid, and found that the drug was released more slowly compared with our prepared carrier.24 the simple mechanism for terminal release of anhydride from modified polyethylene is shown in figure 6. nevertheless, it is certain that this carrier requires further in vitro and in vivo studies. in this study, homogenization was performed at above the melting point of the lipid followed by ultrasonication for preparation of modified sln.25,26 to disperse the triamcinolone acetonide homogeneously in the lipid, a chloroform / methanol solvent system was used and homogenized for 2 minutes.27 subsequently, the prepared slns were subjected to further lyophilization. our preliminary studies showed that reconstitution of freeze - dried slns was impossible when they were freeze - dried without addition of cryoprotectants. therefore, a 1:1 dilution with 3% w / w of sucrose as a cryoprotectant was performed.28 many different drugs have been incorporated in the slns.17 the prerequisite for obtaining a sufficient loading capacity is a sufficiently high solubility of the drug in the lipid melt. relatively higher encapsulation efficiency constitutes one of the major advantages of slns. for calculations of entrapment efficiency and loading capacity, calibration curves for the ultraviolet assays of triamcinolone acetonide were conducted on eight solutions in the concentration ranges of 1.5 10 to 1.25 10 mol / l ; encapsulation efficiency and drug - loading were 94.32% and 25.32%, respectively. the high entrapment efficiency of the drug is thought to be the result of the lipophilic characteristics and high compatibility between the drug and the lipid. furthermore, the high encapsulation efficiency and drug - loading in comparison with previous work24 is a result of the method of preparation and mixing of the drug and lipid matrix in the organic solvents. the mean particle size, polydispersity index and zeta potential of the slns with and without drug were measured by photon correlation spectroscopy, as summarized in table 1. the mean particle sizes of 165.8 nm and 97.44 nm were obtained for drug - loaded and free slns, respectively (table 1). the increase in size after drug loading must be due to incorporation of the drug in the nanoparticle matrix.23 the polydispersity index is a ratio providing information about the homogeneity of particle size distribution in a given system, and ideally, this should be < 0.3.29 triamcinolone acetonide - loaded and free sln had polydispersity index values of 0.254 and 0.337, respectively, indicating that the nanoparticles had a narrow size distribution, and suggesting nanoparticle monodispersity.30,31 the zeta potential is a key factor for evaluation of the stability of a colloidal dispersion.23 when the absolute value of the zeta potential is higher than 30 mv for a colloidal formulation, the particles are likely to be electrochemically stable under the investigated condition.25 zeta potential values of 41.6 mv and 43.0 mv were obtained for the drug - loaded and free slns, respectively (figure 1). as table 1 demonstrates, as time passes, increases are observed in size, polydispersity index, and zeta potential as a result of light and other factors that cause gelation.32 however, it is obvious from these values that the prepared nanosuspension has an acceptable electrochemical and physical stability potential for 3 and 6 months of storage. similarly, in order to evaluate the stability of slns lyophilized powder after 6 months, 10 mg of sample was dispersed in distilled water and characterized. the values for particle diameter, polydispersity index, and zeta potential were 223.3 nm, 0.321, and 30.1, respectively. these results indicate that, using sucrose as a cryoprotectant, the particle size remains in the nanometric range. in order to obtain a scanning electron microscopic image, we used freshly prepared sln loaded with triamcinolone acetonide, and micrographs of the samples containing drug - loaded slns are shown in figure 2. the micrographs demonstrated a spherical shape and a smooth surface, with a particle size in the nanometric range.25 the atomic force microscopy technique has been widely used to obtain size, shape, and surface morphological information about nanoparticles. it is capable of resolving surface details down to 0.01 nm and producing a contrasted and three - dimensional image of the sample.23 a drop of diluted aqueous suspension (without preliminary ultrafiltration) was placed on a mica slide and dried out at room temperature for 24 hours. figure 3 gives the tapping mode atomic force microscopic images of freshly prepared sln containing triamcinolone acetonide after 24 hours (a and b), 48 hours (c and d), and 1 week (e and f). the nanoparticle images demonstrated spherical particles of 50600 nm in diameter, but only 66.5 nm in height. after 48 hours, the nanoparticles were uniformly grown on mica slides, as illustrated in figure 3 (c and d). one week after spreading slns onto the mica plates, no particles could be distinguished in the atomic force microscopic images, which showed homogeneous and fiat shapes, as illustrated in figure 3 (e and f).33 this result is not surprising, considering the well known ability of particles to fuse during evaporation of water consequent to the molecular diffusion on the surface of the particles. moreover, the presence of free surfactant in the suspension may increase the characteristic size.33 differential scanning calorimetry thermograms for triamcinolone acetonide, tripalmitin glyceride, a physical mixture of triamcinolone acetonide, tripalmitin glyceride, modified phosphatidylethanolamine, and lyophilized slns are shown in figure 4. a sucrose thermogram is also shown in this figure to demonstrate peaks of lyophilized slns. the triamcinolone acetonide powder showed a sharp melting process with an onset temperature of 284.72c and a peak of 286.04c, with a melting enthalpy of 64.73 j / g. this was confirmed by the presence of a melting peak for triamcinolone acetonide in the physical mixture. this suggested that there was no crystalline triamcinolone acetonide in the sln suspension, and triamcinolone acetonide might be present in an amorphous state. similar results have been reported by previous researchers, stating that rapid quenching of the nanoemulsion does not allow the drug to crystallize.26,27 an endothermic peak of the sucrose used as a cryoprotectant was observed at 170.21c in the triamcinolone acetonide- sln curve. meanwhile, the melting peak temperature of tripalmitin glyceride in lyophilized sln (57.73c) was diminished when compared with the temperature of the bulk lipid (64.38c). the decrease in melting point can be attributed to the small size (nanometer range), high specific surface area, and presence of a surfactant.15,30 this can also be attributed to the kelvin effect and is described by the thomson equation.3 moreover, the lower melting enthalpy value of 35.72 j / g indicates a less ordered lattice arrangement of lipids within the nanoparticles compared with the bulk materials.9 in this study, the in vitro release of triamcinolone acetonide from slns was investigated for 60 hours at 37c using a franz diffusion cell (figure 5). due to the low water solubility of triamcinolone acetonide, 20% ethanol was added to the phosphate - buffered solution at four ph values of 4.0, 5.0, 6.0, and 7.4.24 our results showed that up to 50% was released over the test period. the prolonged and lower release of slns after 12 hours can be explained by slower diffusion of triamcinolone acetonide from the inner solid core matrix of the formulations. however, the initial fast release was detected at a lower ph. in this experiment, we studied the release profile of pure drug compared with nanoparticles in acidic ph, and the release half - life of drug release was significantly diminished compared with nanoparticles (about 25 minutes). the release data were analyzed using the following higuchi kinetic equation:25 qt = kt0.5, where qt is the percentage of drug released at time t, and k is the release rate constant. regarding the release model of all formulations, it was found that the prolonged release characteristic of triamcinolone acetonide was well fitted to higuchi s square root model, as has been reported for drug - loaded sln systems.25,34,35 linear fits were obtained, indicating that the release profile of triamcinolone acetonide from homogenous and granular matrix systems is diffusion - controlled. the r values from in vitro release kinetics and the k values or release rate constant obtained from the higuchi model plot are presented in table 2. furthermore, as shown in figure 6 and the in vitro release data, we found that the burst release was increased with a decline in ph that demonstrates a change in structure of the phospholipids and release of the terminal anhydride. several previous studies have been conducted to identify a ph - sensitive carrier preparation, and often the amount of drug release from slns was small compared with our results, and the values for common slns without modification were below 25%.8,25,27 subedi investigated ph - sensitive slns and used them for doxorubicin delivery ; in their work, 30% of the drug was released after 24 hours.8 venkateswarlo and manjunath investigated slns of tripalmitin and used them as clozapine carriers, and in their work, up to 20% of drug was released after 48 hours.25 liu investigated slns containing triamcinolone acetonide in mixtures of monoglycerides, diglycerides, and triglycerides of palmitic acid and stearic acid, and found that the drug was released more slowly compared with our prepared carrier.24 the simple mechanism for terminal release of anhydride from modified polyethylene is shown in figure 6. nevertheless, it is certain that this carrier requires further in vitro and in vivo studies. as mentioned above, slns offer an attractive means of drug delivery, particularly for poorly water - soluble drugs. they combine the advantages of polymeric nanoparticles, fat emulsions, and liposomes. in this study, modified n - glutaryl- phosphatidylethanolamine was prepared, used for sln preparation, and characterized. the results indicate that the carrier has suitable stability in colloidal form and may increase drug release under acidic conditions. homogenization followed by ultrasonication is suitable for producing small slns with an average diameter of 165.8 nm. using a zetasizer, atomic force microscopy, and sem, after 6 months, zetasizer data did not indicate any obvious changes in diameter, polydispersity index, and zeta potential. an in vitro study indicated an increase in burst release and a ph decrement as well. in vitro release kinetics these observations suggest that the present system offers an exciting mode of target delivery for potent lipophilic anticancer drugs and anti - inflammatory agents. | backgroundsolid lipid nanoparticles (slns) are colloidal carrier systems which provide controlled - release profiles for many substances. in this study, we prepared aqueous dispersions of lipid nanoparticles using a modified, ph - sensitive derivative of phosphatidylethanolamine.methodsslns were prepared using polysorbate 80 as the surfactant and tripalmitin glyceride and n - glutaryl phosphatidylethanolamine as the lipid components. particle size, polydispersity index, and zeta potential were examined by photon correlation spectroscopy. morphological evaluation was performed using scanning electron microscopy, atomic force microscopy, and differential scanning calorimetry.resultsphoton correlation spectroscopy revealed a particle hydrodynamic diameter of 165.8 nm and zeta potential of 41.6.0 mv for the drug - loaded nanoparticles. atomic force microscopy investigation showed the nanoparticles to be 50600 nm in length and 66.5 nm in height. differential scanning calorimetry indicated that the majority of slns possessed less ordered arrangements of crystals compared with corresponding bulk lipids, which is favorable for improving drug - loading capacity. drug - loading capacity and drug entrapment efficiency values for the slns were 25.32% and 94.32%, respectively.conclusionthe slns prepared in this study were able to control the release of triamcinolone acetonide under acidic conditions. |
coats ' disease is characterized by telangiectatic and aneurysmal retinal vessels with retinal exudation and detachment. the conventional treatments of coats ' disease include laser photocoagulation and/or cryotherapy to the aneurysms and avascular retina in the early to middle stages. in the late stages with neovascular glaucoma suspected to have coats ' disease, a differential diagnosis from a retinoblastoma is critically important prior to any invasive therapy. considering the risk of a misdiagnosis and the low probability of preserving normal vision, eyes with advanced coats ' disease are sometimes enucleated even though the possibility of preserving the eye by surgical treatment exists. the patient was a 16-month - old boy whose parents noticed esotropia and a white pupil in his right eye at 1 year of age. he had visited 3 hospitals before he was finally referred to our hospital. at the first visit, the anterior segment of the right eye was normal by slit - lamp examinations, but a totally detached retina was observed. because the detached retina was close to the lens, b - mode ultrasonography showed a diffuse, high echo area in the vitreous cavity with no apparent sign of calcification (fig. the ct image that had been taken at a previous hospital 1 month earlier showed no evidence of calcification in the right eye (fig. the mri evaluations taken at 1 of the 2 previous hospitals showed high - intensity signals in the t1-weighted images and low - intensity signals in the t2-weighted images in the vitreous body of the right eye. however, high - intensity signals were observed in the t2-weighted images in the same area on the second mri, which was taken 3 months after the first mri examination (fig. there was no positive family history of any eye diseases, and no systemic abnormality was found. surgery was performed for exudative retinal detachment 2 months after the first visit to our hospital. first, external subretinal fluid (srf) drainage was performed, and srf was submitted for a rapid pathological examination, so this was a diagnostic procedure. the examination showed many histiocytes and a small number of epithelial cells but no signs of malignancy. second, lens - sparing vitrectomy was performed, and the retina was reattached under perfluoro - n - octane. thus, the fundus, especially the peripheral area, could be seen better, and the clinical characteristics of coats ' disease, including dilated retinal vessels, numerous microaneurysms, and intra- and subretinal exudates became visible (fig. endophotocoagulation was performed on the microaneurysms and the presumably avascular retina near the abnormal vessels. at the end of vitrectomy, 0.5 mg of bevacizumab postoperatively, the posterior retina was reattached ; however, the dilated retinal vessels and peripheral subretinal exudates had not improved even after 3 months. a reoperation with similar techniques to the first surgery thereafter, the condition of the eye remained unchanged during the 11 months of follow - up. this is especially true when media opacity or a total bullous retinal detachment makes the visibility of the retina difficult or impossible. typical mri findings of retinoblastomas are high - intensity signals in t1-weighted images and low - intensity signals in t2-weighted images. on the other hand, the typical mri findings in coats ' disease are high - intensity signals in both t1- and t2-weighted images. in our case, the characteristics of coats ' disease were difficult to detect ophthalmoscopically because of the bullous retinal detachment. the ct images did not show any signs of calcification ; however, the first mris showed features consistent with a retinoblastoma. the second mri evaluation at the second hospital showed features consistent with coats ' disease. lai. reported a case of coats ' disease in which mri showed low signal intensity in the t2-weighted images. they postulated that t2-weighted mris may vary depending on the nature of the subretinal exudates, which can change during the natural course of the disease process. the possibility of a retinoblastoma in our case was considered to be low due to the following reasons : first, there was no clear mass lesion in the mri findings, and the t2-weighted images of the vitreous cavity showed a change in signal intensities ; second, no sign of calcification was noted on the ct scans ; and third, no tumor mass was detected by ophthalmoscopy and ultrasonography. because a retinoblastoma could not be completely excluded, we used a rapid pathological examination of srf to ascertain the diagnosis. as a result, in addition, the removal of srf made the observation of the peripheral retina better, and typical features of coats ' disease became visible. in our case, reoperation was necessary because there was significant srf even at 3 months after surgery. the reason for this is unclear, however, and we consider that the presence of extensive subretinal exudates hindered the complete drainage of srf regardless of multiple srf drainage procedures. in addition, it is possible that laser ablation during primary vitrectomy was not enough to decrease the disease activity. several authors have reported that vitrectomy with posterior retinotomy, the use of intraocular silicone oil or gas, and scleral buckling were useful in treating eyes with coats ' disease [8, 9 ]. recommend less invasive treatments consisting of pars plana vitreous infusion, sclerotomy, and external srf drainage, followed by laser ablation and/or cryopexy for the treatment of coats ' disease. with regard to the surgical technique, srf is usually drained internally through an intentional or preexisted retinal hole in eyes with rhegmatogenous retinal detachment. however, in eyes with total retinal detachment associated with coats ' disease, the presence of inflammation may induce severe proliferative vitreoretinopathy after surgery if internal srf drainage and gas or silicone oil tamponade are performed. in the treatment of severe coats ' disease with total retinal detachment, although careful consideration should be given to the possibility of tumor cell dissemination, a combination of external srf drainage and a rapid pathological examination of the fluid may be beneficial both for diagnosis and preserving the eye. the subject of this case report has given informed consent for the publication of this article and the associated photographs. | severe forms of coats ' disease are often associated with total retinal detachment, and a differential diagnosis from retinoblastoma is critically important. in such eyes, laser- and/or cryoablation is often ineffective or sometimes impossible to perform. we report a case of advanced coats ' disease in which a rapid pathological examination of subretinal fluid was effective for the diagnosis, and external subretinal drainage combined with vitrectomy was effective in preserving the eye. |
heat intolerance has long been known to be a significant issue for people with ms, where an increase in core body temperature is associated with increased neurological symptoms [16 ]. people with ms rate high temperatures as one of the top three factors adversely affecting their symptoms, with stress and insufficient sleep being the other two. indeed, heat intolerance has a significant impact on the economic situation and quality of life of people with ms and their families, and managing heat - related problems is important to maintaining employment of people with ms. while neurological symptoms are frequently exacerbated by heat, these generally return to baseline when core temperature returns to normal, and colder temperatures usually reduce ms symptoms [911 ]. while it is clear that heat frequently exacerbates ms symptoms, patterns of relapse and exacerbation in relation to seasonal climate variations are variable and confounded by other factors such as viral infections and the modifying behaviours of people with ms, who routinely try to minimise their exposure to heat on hot days and nights [1214 ]. in australia, where the main climate variations range from temperate to tropical, residential air conditioning is widely used by people with ms to minimise the impact of hot days and nights on their symptoms. australian electricity prices are rising rapidly with annual increases of more than 10% annually between 2007 and 2010. thus, in addition to the other economic difficulties faced by people with ms [16, 17 ], affordability of residential cooling is a major issue for many, frequently compounded by an early exit from the labour force and loss of income. in light of the above, ms australia perceived a potential need for government - funded financial assistance for residential electricity costs australians with ms on low incomes and sought relevant evidence on this issue to determine whether assistance was required and to underpin public policy advocacy and government lobbying efforts if government assistance was warranted. an extensive search of peer - reviewed literature and other sources indicated no studies of air - conditioning use by people with ms. the keeping cool survey consisted of nine questions covering the following topics (see the supplementary material available online at doi:10.1155/2012/794310) : home air - conditioner use / nonuse and reasons why ; ambient temperature range when air conditioning is switched on ; age and type of air conditioner (to estimate efficiency) ; rooms of house that are air conditioned ; number of hours the air conditioner is used in particular months ; other home modifications to assist cooling ; the following question. as a person with ms, what happens when you get too hot ? i lack energy and require more rest. apart from fatigue, my other symptoms of ms become worse. i am unable to participate in normal social activities (time with family or friends). i am unable to do my normal household duties (e.g., cleaning, cooking, etc.). demographic data for survey respondents was available from the australian ms longitudinal study (amsls) database, including sex, age, and postcode. of the respondents to the keeping cool survey, 66% (1,578) had also participated in one or both amsls economic surveys conducted in 2003 and 2007. this, along with links to basic demographic information collected in other parts of the longitudinal survey, enabled additional analyses to be undertaken (see section 2.3 below). the amsls is a nationwide, longitudinal cohort study described elsewhere [18, 19 ]. briefly, the amsls maintains a large sample of volunteer participants from all australian states and territories ; 96% of whom have definite ms by the mcdonald diagnostic criteria, according to their neurologist or treating physician. the amsls project is approved by the act health human research ethics committee, an independent national health and medical research council - constituted human research ethics committee conforming to the ethical standards for human studies as per the 1964 declaration of helsinki. the survey was sent to 3,150 consenting participants of the amsls in september 2008, of whom 2,385 responded (76%). analyses were undertaken comparing responses on all survey questions between those who would likely be eligible for government - funded assistance and those that would not. nonparametric tests were used to test categorical variables (chi - square) and ordinal data (kruskal - wallis and mann - whitney). no p values approached significance, so all subsequent analysis considered the survey population as a whole. also, there were only three survey participants from the northern territory ; consequently their data were excluded from the analysis. economic modelling for estimating costs of air conditioner use by people with ms was undertaken utilising the survey results. this was necessary as it was found in piloting early versions of the questionnaire that it was not possible to get meaningful responses to direct questions about actual running costs. additionally, the bom created a new data set combining annual average maximum air temperatures and humidity into a single measure of apparent temperature (a commonly used biometeorological measure), for this study. air - conditioner use at home by people with ms is a direct response to day - to - day weather conditions. one of the difficulties of examining climatic impacts on the use of air conditioners by people with ms is the climatic variation across time and place. additionally, air temperature data has significant limitations because moderate to high levels of humidity, coupled with hot days and nights, make it more difficult for people to keep cool. the bom routinely reports data on the number of hot and very hot days and nights, and national annual means for these are reported below in table 1. australia has experienced an increased number of hot days and nights over time (table 1), which impacts on the air - conditioning needs of australians with ms and their associated costs. temperatures in 2008 when the survey was conducted were typical of the decade at 48.8 days 35c and 80.5 nights 20c. importantly, 35c is very hot for people with ms, as the survey results indicated that 29.9c is the mean external temperature at which australians with ms turn on their air conditioners. moreover, there are many more days over 30c than over 35c in most areas ; for example, across five weather stations in the sydney area there were 4.4 times more days 30c than there were 35c. table 2 presents the average maximum apparent temperature (at) data for australian states / territories and capital cities. at is an adjustment made to the ambient air temperature based on the level of humidity, which also impacts on personal comfort levels. the bom adjustments use absolute humidity with a dewpoint of 14c as the reference point (with slight adjustments depending on the temperature). if the humidity is higher than the reference point, then the at is higher than the air temperature, and if the humidity is lower than the reference point, then the at is lower than the air temperature. in table 2, at is ordered from highest to lowest, and ats for capital cities are included along with data for the states and territories within which at can vary by as much as 16c. the at for the capital cities may better represent the actual impacts on a state and territory populations. the demographic profile of survey participants was 79.0% female, and the mean age of the participants was 52 years, with a range of 2583 years. also for the 1,578 participants for whom economic data was available, 32.2% were likely to be eligible for a government - funded rebate on their electricity bills if a medical cooling rebate was in place. air conditioners were used by 81.9% of survey participants, with an additional 9.6% stating that they could not afford an air conditioner. in comparison, only 10.3% of respondents stated that they coped well in the heat and had no problems as a consequence of getting too hot. the most common issue was fatigue at 83.7%, followed by increases in symptoms other than fatigue at 61.9%. just under half of the respondents also reported reduced capacity for social, household and work activities, and self - care. small but notable proportions of people with ms also reported impacts on their use of medication (9.1%), doctor or other health professional visits (7.4%), and hospitalisation (3.4%). the mean national average external air temperature at which people turned on their air conditioners was 29.9c. tasmania was the state with the lowest mean at 26.4c, with the highest in south australia and western australia at 30.7c and 30.2c, respectively. figure 1 describes the imputed mean total hours of air - conditioner use per household across states and territories from september to april. imputed means were derived by using the midpoints from the categories selected by survey respondents : 0 = 0 ; 16 hrs = 3.5 hrs ; 712 hrs = 9.5 hrs ; 1318 hrs = 15.5 ; 1924 hrs = 21.5. the types of air conditioners (evaporative or heat exchanger) used were almost identical to that of the general population, with heat exchangers dominating (ranging from 60 to 98%) and higher levels of evaporative more common in the driest areas (western australia, south australia, victoria, and australian capital territory 3040%). newer air conditioners (03 years old) were used in 36.2% of households, with 49-year - old units at 42.8% and those 10 years old or more at 20.9%. when the survey was conducted, inverter technology, which increases heat exchanger air conditioner efficiency by 2535%, was only present in those units 03 years old. nationally, 34.1% were cooling one room only, 32.7% were cooling two rooms, and 33.1% were cooling 4 rooms or more (none stated that they were cooling 3 rooms). finally, respondents were also asked about their use of various measures to increase the thermal performance of their homes. nationally, 40.0% used external window coverings such as blinds and awnings, 80.5% used internal window coverings, 69.8% had ceiling insulation, 18.9% had roof vents, and 26.6% had wall insulation. while the figures for insulation are generally low by north american and european standards, ms households exceed national means for both wall (19%) and ceiling (59%) insulation. economic modelling was undertaken to convert the survey results into cost data (see section 2.3 above). the first two sets, based on $ 0.15/kwh and $ 0.20/kwh, represent the range of prices across australia when the survey was conducted in 2008. the other two sets of prices, $ 0.25 and $ 0.30, represent the range of prices in 2011. estimated nonevaporative air - conditioner cost data for households across australia in 2007 are included under the heading all. the present study has provided strong evidence that use of residential air conditioning by australians with ms is a major and common strategy for minimising the impact of a warm to hot climate. it also appears that people with ms in australia report a much higher incidence of heat sensitivity than those living in cooler climates. a recent survey in sweden found that only 58% reported heat sensitivity, compared to almost 90% in australia. this difference would appear to derive from climatic differences where australians are exposed to much higher temperatures (see tables 1 and 2) consistently compared to their swedish counterparts where the temperature rarely exceeds 25c at the peak of summer. other research across a variety of locales has typically found a range of 6080% of people with ms self - reporting as heat sensitive in relation to symptoms increasing with exposure to heat. this further emphasises the importance of air conditioning for people with ms to keep cool in warmer climates such as australia. australian households that include people with ms are more likely than households nationally to (a) have an air conditioner, (b) use the air conditioner more often, and (c) incur the concomitantly higher electricity bills. these data are fundamental to the argument that government - funded financial assistance should be available to those on low incomes requiring medical - related cooling in the form of residential air conditioning. moreover, our results show that ms households had taken additional steps relative to other households to improve the thermal efficiency of their homes, above and beyond any government - funded contribution. the survey has therefore provided information for policy makers about the considerable problems people with ms incur with warm to hot weather, including regional variation in usage and need. nationally, people with ms spend between 4 and 12 times more on running their air conditioners than other households, with a national average of about 4 times more (excluding evaporative air coolers). little research has been done to track household use of air conditioners generally, but one comparable survey (based on recall and the completion of a questionnaire similar to the keeping cool survey) performed in victoria in 2008 found that the average total hours of air conditioner use was 107 hours, compared to the present survey results in victoria of 1,508 hours. this indicates that ms households in victoria run their air conditioners by 14 times more hours annually than other households. for many households these additional costs must be met from relatively low incomes, with relatively high unemployment associated with ms. the high number of hours that ms households use their air conditioners relative to other households probably arises from several factors. people with ms are more likely to be at home as many are not working in paid employment, and they are more likely to be indoors during hot weather to escape the heat. the outside air temperature at which they turn on their air conditioners may also be at a lower threshold than other households given their heat sensitivity. while some of this higher use is probably slightly reduced through the higher levels of wall and ceiling insulation in ms households, these differences in thermal efficiency are relatively minor. strengths of the present study include the large national sample size (almost 2,400 households with people with ms) and the ability to subanalyse use of air conditioning by climatic and political regions. the latter enabled collation of data relevant to government policy makers in different australian states, leading to significant local outcomes in electricity subsidies for people with ms. limitations of the study include the use of retrospective questions on air - conditioning use, which may have introduced some recall bias, though in which direction remains unclear. the inability of households to identify the actual costs of running their air conditioners also necessitated economic modelling to obtain cost estimates. the sampling for the air - conditioning survey had some biases that are likely to have impacted on the results. previous examinations of the representativeness of participants in the amsls have found that the demographic characteristics of participants are similar to those of other recent studies, supporting the claim that the survey is broadly representative of australians with ms. nevertheless, there is likely an under - representation of those at the youngest and oldest ends of the age distribution and perhaps some bias towards over - representation of females. in australia people with ms use air conditioners extensively to keep cool at home when ambient external temperatures reach levels that begin to exacerbate their symptoms. the economic costs of this are increasingly problematic for low - income households, and the research results have been essential in demonstrating the need for government assistance to these households. subsequent campaigns by ms australia to obtain medical cooling rebates from several state governments have been successful, in part because of the presentation of such hard data to government policy makers. therefore, the use of patient self - reported data on salient life issues, facilitated by the existence of well - characterised national cohorts like the australian ms longitudinal study, can effectively be used for government lobbying to assist people with ms. finally, high levels of air - conditioner use by people with ms, and the need for assistance with electricity bills, are not the only policy implications of the survey. policy advocacy work also needs to be undertaken in relation to the replacement of old and inefficient air conditioners with more efficient units that have the capacity to cut operating costs substantially (up to 5065%). also, given that peak demand for electricity in australia is during the afternoon and early evening of hot days, this is also when electricity supply overloads and blackouts are most likely to occur, making it essential for people with ms to have plans in place to minimise their exposure to heat when electricity is not available to their homes. | despite the known difficulties many people with ms have with high ambient temperatures, there are no reported studies of air conditioning use and ms. this study systematically examined air conditioner use by australians with ms. a short survey was sent to all participants in the australian ms longitudinal study cohort with a response rate of 76% (n = 2,385). questions included hours of air - conditioner use, areas cooled, type and age of equipment, and the personal effects of overheating. air conditioners were used by 81.9% of respondents, with an additional 9.6% who could not afford an air conditioner. regional and seasonal variation in air conditioning use was reported, with a national annual mean of 1,557 hours running time. 90.7% reported negative effects from overheating including increased fatigue, an increase in other ms symptoms, reduced household and social activities, and reduced work capacity. households that include people with ms spend between 4 and 12 times more on keeping cool than average australian households. |
the traumatic rupture of the achilles tendon is more and more frequently encountered in current orthopedic practice. it occurs round the age of 40, the majority of the patients ranging between 30 and 50 years, mainly in the male gender (approximately 84% of the cases). the rupture of the achilles tendon is evidenced mostly in patients who occasionally perform physical activity, the weekend sportsman, due to a precarious preparation for the physical effort. however, in the recent years the tendinous lesions have been increasingly found among the elderly, without any connection to the sports activity. the ruptures of the achilles tendon appear in 85% of the cases at the level of a hypovascular area, 27 cm proximal to its calcaneal insertion. a number of degenerative lesions of the tendon are also involved, among which we can mention : hyaline, mucoid degeneration with chondroid metaplasia of the tenocytes. the changes in the structures of the collagen fibers are related to the age and in the majority of subjects they do not determine any clinical symptoms. the fatal role of the corticoids (either orally, or locally injected) and also of the antibiotics (fluoro - quinolones) must however be mentioned. in medical literature, increased frequencies of tendon lesions have been described, determined by conditions such as hyperthyroidism, gout, renal insufficiency, atherosclerosis. the ruptures of achilles tendon may be classified in total or partial, acute / traumatic or chronic. the main aim of this article is an analysis of both advantages and disadvantages of the modern solutions of treatment percutaneous surgery, in comparison with the classic methods of treatments described in the surgery of achilles tendon. we also propose a presentation of the role of the imaging methods of diagnosis, mri and ultrasonography including tridimensional, in establishing the diagnosis of achilles tendon rupture. the study was conducted in patients admitted to the clinic department of orthopedics and traumatology ii of cluj - napoca between 20112012. the patients studied, the following aspects were investigated : the use of the current diagnostic methods, mri or ultrasound, their role and contribution to diagnosis and to the choice of a method of treatment over another, the time of hospitalization, type of treatment, technical issues, post - operative management, the mean time to scarring, complications. the study included 23 patients, hospitalized during the period january 2011june 2012 ; 22 patients (95%) were men. the physical activity of the patients taken into this study was occasional, none of the patients practiced high performance sport in the past or at the time of study. the average age of the patients was 39, ranging between 25 and 61 years. in order to diagnose the lesion of achilles tendon, the objective clinical examination played the central role, using the thompson test (lateral pressure of the calf does not trigger the plantar flexion of the foot), the existence of a visible and tangible depression on the trajectory of the achilles tendon, the inability to stand on tiptoes on that foot, and even monopodal. among the imaging methods used in the process of diagnosing the lesions of achilles tendon, sonography and mri must be mentioned. they were used for the confirmation of the lesion and in order to establish its location and its extension. the treatment of the achilles tendon lesions included conservative or surgical treatment. the conservative treatment consisted in plaster immobilization, while surgical treatment aimed at reconstructing the achilles tendon using classical procedures, open or percutaneous techniques (tenolig material and technique - fig. we will describe the technique used for the percutaneous treatment of achilles tendon, according to the recommendations of the producer of the tenolig system. the surgical interventions were performed under rachianesthesia, the patient being positioned in ventral decubitus, without using a tourniquet at the level of the limb. i repaired the two parts of the broken tendon, the proximal and distal by palpation (fig. the entrance point is situated at about 6 cm proximal to the rupture area, postero - lateral and postero - medial. the exit point was located at about 45 cm distal to the tendon rupture, within the retromalleolar space. the introduction of the needle of the tenosynthesis material was made perpendicular on the tendon body, careful not to affect the sural nerve. the foot was held in an neutral position in order to ensure the suture system penetration correctly. after that, the foot was positioned in equinus in order to reduce the two fragments of the broken tendon. the sutures would be blocked by the provided buttons, thus maintaining the fragments of the tendon in contact. the patients were kept under post - operative follow - up for up to 6 months (medical examination at 4 weeks, 8 weeks, 4 months and 6 months), during which the following were assessed : the consolidation of the tendon lesion, complications related to the wound (infection, dehiscence) or mechanical complications (reruptures). the overall post operative functional status was evaluated, together with the recovery of the level of physical activity previous to the rupture of achilles tendon. the test used for of the patients studied, 82% (19 patients) presented a complete rupture of achilles tendon, while 18% (4 patients) were diagnosed with a partial rupture. in 4.3% of cases (1 patient), the achilles tendon rupture was bilateral. in 69% of the cases (16 patients), ultrasonography or mri were performed for the confirmation of the diagnosis, while in the rest of the cases, the diagnosis was exclusively based on clinical examination. the ultrasonography of the achilles tendon was used in 64% of the cases (15 patients) in order to establish the achilles tendon rupture diagnosis. in 27% of the patients (4 patients) evaluated by ultrasound, the diagnosis was of partial rupture of achilles tendon, while the clinical signs had pleaded for a complete rupture. the subsequent mri examination pointed to the near - complete nature of a rupture in one case. in terms of the chosen treatment, the conservative treatment was used in one patient, due to the associated conditions. among the patients who received surgical treatment (22 patients 23 interventions), 78.2% (18 patients) benefited from a classical reconstruction of the achilles tendon, while 21.8% of the patients (5 patients) were treated using the percutaneous technique. the choice of a method instead of the other was made based on each surgeon s experience, the extent of tendon damage (in a large defect the open procedure was performed), how soon after rupture the patient was diagnosed, wound appearance (for better cosmetic outcome the percutaneous repair was chosen), previous activity level (the open technique was preferred for the patients with higher physical activity level) and financial considerations (the percutaneous technique is more expensive). the average length of hospitalization was 7 days. the mean hospitalization time of the patients who benefited from a classic reconstruction was 7.53 days, while the mean hospitalization of the patients treated with the percutaneous technique was 6 days. 3) for a medium period of 7.78 weeks (varying between 48 weeks), the first three weeks with the foot at 120 degrees of plantar flexion, and then at 90 degrees. the consolidation of the lesion of achilles tendon was exclusively clinically appreciated, using the tests mentioned above. the complication rate was 8.6% in the case of surgically treated patients (2 cases) : wound dehiscence in the case of a patient treated by classic surgery and a secondary rupture in a patient operated with the percutaneous technique. no significant losses of the functional status were recorded, all the patients recovering the level of physical activity previous to the tendon lesion. the traumatic rupture of the achilles tendon mainly affects the male gender, the causes being not fully described in the medical literature as yet. in cross section, the achilles tendon is considerably smaller in women than in men, and therefore a smaller force is generated at this level, which could be an explanation for the decreased prevalence of achilles tendon lesions in women. in order to diagnose an achilles tendon lesion, the clinical examination plays a fundamental part, without any further laboratory explorations being necessary. nevertheless, the ultrasound confirmation of the rupture was required in about 2/3 of the patients included in this study. the sonography has the advantage of being a non - invasive technique, accessible and low cost. even though the achilles tendon is easily explored using ultrasonography, the major disadvantage of this investigation is the misinterpretation of a complete rupture of achilles tendon as being partial, with major implications on the choice of the right method of treatment. for the patients diagnosed with partial rupture of the achilles tendon by the performed ultrasound test, we have opted for surgical treatment based on the physical examination (palpable gap and a positive thompson test), because of the reduced risk of rerupture. the mri is superior to ultrasonography in terms of diagnosing the pathology of achilles tendon (fig. 4), it presents in a more accurate way the difference between a partial and a complete rupture, and it can also be successfully used in the degeneration tendinopathy diagnosis. the tridimensional ultrasonograpghy offers the possibility of an examination in real time, a superior differentiation between edema and hematoma, indicating more precisely the real area of the lesion, especially in the cases of repeated ruptures. among the methods proposed so far, the surgical treatment is the preferred option in most of the cases (fig. it is associated with better cicatrization of the tendinous lesion and significantly reduces the risk of rerupture in comparison with the non - surgical treatment, but has a higher rate of complications (dehiscence of the post operative wound, cutaneous or tendinous necrosis, infection etc.). at present, the surgical treatment includes alongside the open reconstruction of achilles tendon through the known methods, the reconstruction of achilles tendon through percutaneous techniques, minimally invasive, which are becoming more often used, while the range of indications widens. these procedures are simple, secure, permitting early mobillization, entailing a lower prevalence of post operative complications in comparison with the open techniques, the rate of rerupture, incriminated by some authors, being included here as well. our clinical study emphasized the reduction of the time spent in hospital by 21.4% in case of patients treated with the minimally invasive technique plus the decrease of the operative time, preservation of the vascularisation of the tendon, the possibility of early mobillization. the frequency of a sural nerve lesion is higher in the case of a technique imprecisely conducted. the percutaneous techniques have as main goal the joining of the two parts of the achilles tendon resulted from the rupture, avoiding the extensive approach at the level of the tendon, the exposure of the tendinous sheath and thus the devascularization of the tendon, allowing the immediate post - operative mobilization, with flexion - extension foot movements. the rapid mobilization allows the alignment of the collagen fibers and remakes the elasticity of the tendon. however, it is important to mention the fact that the post operative pain is significantly less in the case of percutaneous interventions. concerning the rate of the rerupture, the small control group of patients and the relatively short time of post - operative surveillance (6 months), does not allow us to formulate a clear conclusion. one patient presented a re - rupture of the achilles tendon in the percutaneous technique group which was associated with poor compliance to rehabilitation. we should mention the fact that the extent of the indications of the percutaneous achilles tendon repair in the case of elite sportsmen has proved to be a viable option, allowing them to undertake their high level sport activities quickly and safely. the post operative fixation in the functional orthesis has been associated with a decreased rate of post operative complications in comparison with the plaster immobilization, according to recent publications. our results demonstrate that the percutaneous techniques of surgical treatment are a viable alternative in cases of recent ruptures of achilles tendon, and the classical surgical treatment should be chosen in the case of late diagnosis of rupture (diagnosed after more than 8 days) and in the recurrent ones. the clinical examination is sufficient in case of recent achilles tendon rupture, and no other supplementary imaging investigations are usually required. ultrasonography can distinguish a near - complete rupture achilles tendon as partial, having an impact upon the therapeutic option. | aimsthe main aim of this article is an analysis of both advantages and disadvantages of the modern solutions of treatment percutaneous surgery, in comparison with the classic methods of treatments described in the surgery of achilles tendon.patients and methodthe study was conducted on 23 patients admitted to the orthopedics and traumatology clinic of cluj - napoca between january 2011june 2012. nineteen (19) patients were diagnosed with a complete rupture of the achilles tendon and 4 patients with a partial rupture. the diagnosis of traumatic achilles tendon ruptures was usually clinical, the ultrasound (common or 3d) and the mri confirmed the lesion and determined its location and extension. we analyzed the diagnostic methods, the elapsed time before surgery, the treatment options depending on lesion s location, technical difficulties, costs, postoperative care, the average healing time, complications.resultsthe ultrasound was performed in 65.2% of the patients (15 patients) for confirming the extension of the lesion and it served for pre - operative planning. in most of the cases, the classical methods of achilles tendon reconstruction were used (18 cases). the complications rate was about 8%. we diagnosed an iterative achilles tendon rupture (the patient was initially treated using the percutaneous methods) and a delay in cicatrisation.conclusionsthe percutaneous surgical techniques are a viable alternative for the acute ruptures of achilles tendon, the classic intervention has clear indications in lesions diagnosed late, in the recurrent tendon ruptures. |
residual and delayed sequelae include postpericardiotomy syndrome, fistulas, valvular dysfunction, ventricular aneurysms, and pseudoaneurysms.1 traumatic coronary artery - cameral fistulas (tcaf) are uncommon sequelae of trauma that require early surgical intervention to prevent complications.2 although the left coronary artery is the most frequently injured vessel of the heart, traumatic fistulas appear more often in the right coronary vessels, as the initial injury to the left coronary artery usually results in early death prior to hospitalization.3 we report the case of a patient with left anterior descending (lad) artery to right atrium fistula following cardiac penetrating trauma. a 17-year - old man presented to the emergency room with a stab wound to the heart about 45 minutes prior to arrival. at arrival, he was in a shock state and was pale, dyspnic, and agitated with blood pressure of 80/40 mmhg, heart rate of 130 beat / min, and respiration rate of 30/min. there was a 45 cm laceration located at the level of the 3 and 4 ribs in the left midclavicular line with active bleeding. he was transferred to the operating room immediately while his bleeding was controlled by finger pressure and initial resuscitation was administered. anterolateral thoracotomy and repair of the left ventricular rupture at the line of the lad were performed, but the patient suffered dyspnea two hours postoperatively and his electrocardiography (ecg) showed st - t segment elevation in the pericardial leads and st - t segment depression in the inferior leads. transesophageal echocardiography revealed severe left ventricular hypokinesia and apical akinesia with a left ventricular ejection fraction of 40% and possibility of the lad fistula. emergency coronary angiography, conducted approximately twelve hours postoperatively, showed an lad cut - off at mid part with poor distal run - off and fistula of the lad to the right ventricle (figures 1). on post - admission day 7, the patient underwent reoperation, during which he was placed on cardiopulmonary bypass and high - potassium blood cardioplegia was administered. the fistula of the lad to the right ventricle was repaired with prolene 5 - 0 and the left interior mammary artery was grafted to the lad, distal to the site of the suture ligation of the lad fistula. coronary arteriovenous fistulas were first described by krause in 1865.4 they commonly have a congenital origin but can be acquired as complications of surgical procedures and traumas. the venous side of the coronary arteriovenous fistula can be the coronary sinus, the great cardiac vein, the right atrium, or the right ventricle.5 most of the reported cases of accidental traumatic coronary artery fistulas were diagnosed several months to years after the initial operation when there was morbidity secondary to the fistula such as congestive heart failure and pulmonary hypertension. our case was diagnosed early postoperatively and underwent reoperation on post - admission day 7. early intervention in tcaf prevents the late complications of high flow left - to - right shunting, including the development of pulmonary artery hypertension and congestive heart failure.2 shimabukuro. reported the case of a patient with cardiac penetrating trauma who presented with congestive heart failure and tricuspid regurgitation due to coronary arteriovenous fistula 8 years after trauma. they suggested that early surgical repair be undertaken in cases of traumatic coronary artery fistula, even if the shunt is minimal and early symptoms are mild.6 bernard maitre. reported the unusual case of a patient with left ventricular aneurysm and coronary - pulmonary artery fistula detected 23 years after a thoracic wound ; the patient presented with severe hemoptysis and anemia.1 reubendra and associates reported the case of a patient with traumatic lad - to - pulmonary artery fistula that developed delayed pericardial tamponade and underwent emergency intervention ; they believe that survival is not unlikely with fistulas occurring in the mid - lad to right ventricle, as indicated by the significant number of reported cases. early repair seems indicated given the life - threatening sequelae such as delayed pericardial tamponade.7 depending on whether the drainage is into the left or right heart, coronary artery fistulas are classified into two major types. akhras. reported a right coronary artery - right atrial fistula about 40 years after shrapnel injury.8 alberto rangel and associates presented the case of a 17-year - old man who sustained a knife chest wound and secondarily developed a traumatic coronary arteriovenous fistula communicating the left main coronary artery to the pulmonary artery, associated with pulmonary valvular insufficiency and endocarditis.9 survivors of traumatic coronary artery fistulas have an excellent prognosis after successful closure of the fistula.10 we suggest that patients with traumatic coronary artery fistulas be considered for elective surgical repair to prevent the development of complications. most patients with traumatic coronary artery fistulas should undergo early surgical intervention to prevent the sequelae of a left - to - right shunt since survivors of traumatic coronary artery fistulas have an excellent prognosis after a successful closure of the fistula. | traumatic coronary artery - cameral fistulas (tcaf) are rare and may present secondary to penetrating injuries (80%) or iatrogenic traumas. early operative intervention remains the recommended treatment modality for accidental traumatic coronary artery fistulas. we report the case of a 17-year - old man who presented with left anterior descending coronary artery - right ventricle fistula following penetrating cardiac trauma, which was successfully repaired surgically. |
the epidemic of obesity continues unabated. according to the national health and nutrition examination survey (nhanes) 2011 - 2012, 8.4% of children aged 25 years are obese [1, 2 ]. despite reports of recent stabilization of overall obesity prevalence, rates of severe obesity (class 2 obesity, defined as bmi > 120% of 95th percentile) have increased by over 50% since 2000 [35 ], especially in the youngest children. childhood obesity tends to track into adulthood and portends a dramatic increases in diseases such as atherogenic heart disease, type 2 diabetes mellitus (t2 dm), dyslipidemia, sleep apnea, and early mortality [4, 79 ]. response to medical intervention of obesity is more effective in early childhood compared to adolescents and adults [10, 11 ]. hence, prevention and intervention for management of obesity in early childhood are optimal. in 2007, the american academy of pediatrics (aap) published expert committee recommendations on the prevention, assessment, and treatment of overweight and obese children. the committee emphasized assessment of body mass index (bmi) at every well - child visit as a method of identifying obesity and the first important step in the creation of a chronic care model with community involvement. however, several years after the guidelines, the rates of identification of overweight and obese status in children continue to be low. prior studies have shown poor documentation of obesity in pediatric patients using icd-9 codes [1316 ]. few studies have investigated the documentation of the obesity by providers in the progress notes. widespread availability of electronic health records (ehrs) and tools such as automatic bmi calculation and alerts for high bmi [1820 ] have improved the documentation of obesity in some settings. however, there is limited data on documentation in children under 6 years of age with severe early onset obesity, where the possibility of long - term adverse effects is the highest and early intervention is critical. this study seeks to identify the rates of clinical documentation of obesity in children under 6 years of age, using the ehrs at two large pediatric academic medical centers. we used structured data such as icd-9 codes and nonstructured data such as provider notes using natural language processing (nlp) to assess documentation. based on prior studies, we hypothesized that rates of documentation of obesity in the youngest age group, even among those with severe obesity, would be low. this is a retrospective cohort study utilizing ehr data for children with severe obesity between the ages of 15.99 years, from both outpatient and inpatient settings at cincinnati children 's hospital medical center (cchmc) and boston children 's hospital (bch) from 2007 to 2014. this study was conducted under the auspices of electronic medical records and genomics (emerge) network, a national consortium organized by the national human genome research institute (nhgri). institutional review board for research in human subjects approved the protocol at cchmc and bch, under a waiver of informed consent. cchmc has used epiccare (epic systems corporation, madison, wi) since 2010 and bch has used cerner solutions (cerner corporation, kansas city, mo) since 2006. data extraction was performed on the electronic patient records completed during routine clinical care at cchmc from january 2010 through june 2012 and from january 2007 through november 2014 at bch. a validated electronic algorithm was established to identify cases of severe obesity using structured and nonstructured data fields captured in the ehr during clinical care. bmi was calculated by the ehr systems from height (or length, if under the age of 2) and weight data recorded at the same visit by medical assistants and/or nurses during the course of routine clinical care. age- and gender - specific reference bmi percentiles were automatically calculated within the ehr using centers for disease control and prevention (cdc) 2000 growth charts for children older than 24 months of age and world health organization (who) 2006 growth charts for children 1223 months old. new evidence suggests that bmi is a more useful parameter to measure adiposity in children under 2 years of age, compared to weight - for - length. to minimize biologically implausible values, any height- or length - for - age measurement 60 kg / m was also eliminated as biologically implausible for children 75th percentile. if more than one recording was present for a given day, only the first recording for the day was included (figure 1). furthermore, if measurements were carried forward across several days of the same inpatient encounter, only the first day of that encounter was included in analysis. of note, the definition of bmi 99th percentile for severe obesity was used, as this is readily available at the point of care for the physician. the current ehr systems are not adapted to calculate or display 120% of 95th percentile in keeping with the current definition of severe obesity. known causes of obesity including endocrine (e.g., cushing 's syndrome), genetic (e.g., prader - willi syndrome), malignancy, and connective tissue disorders and diseases causing edema (e.g., renal failure) were excluded using icd-9 codes and written documentation (table 1). we excluded these patients because we felt that providers might not document obesity in these patients, not because it was not recognized, but because there were more pressing medical issues to address or because the cause of the obesity was not felt to be endogenous. prescription data was used to exclude patients on prolonged courses of steroids (longer than 14 consecutive days or three or more separate courses totaling more than 28 days in the six months prior to qualifying weight) or atypical antipsychotics. to validate the algorithm, a manual chart review of 200 charts was performed at each center by at least two physicians (pediatric endocrinology and emergency medicine). a systematic data collection of the chart review was maintained and an interrater reliability > 85% f measure and a positive predictive value of > 90% was achieved after the training phase. any disagreements were adjudicated by discussion. following identification of cases of severe obesity by the algorithm, the available clinical notes and diagnosis codes were extracted for all cases. natural language processing (nlp) was performed on the clinical notes using a regular expression search with descriptive obesity terms and phrases (table 2). a broad selection of weight- and obesity - related terms was used to maximize sensitivity. any encounter that had at least one term from the list in the notes or any of the listed diagnosis codes was considered to have documentation of obesity. additionally, a notation of an icd-9 code relevant to obesity in the problem list (table 2) was considered as positive documentation. an automatic notation of bmi value in the growth chart was not considered documentation, as the ehrs were not configured to provide an alert for a certain percentile of bmi. individuals were classified as having been ever documented if at least one severe obesity encounter showed documentation during the course of care. analyses were conducted using sas version 9.3 (sas institute, cary, nc). comparisons between centers, or within each center by obesity documentation status, were conducted using wilcoxon rank sum analysis or fisher 's exact test for continuous or categorical variables, respectively. we identified a total of 30,463 records for review from cchmc and bch of children between the ages of 15.99 years that met the inclusion criteria for severe obesity without a pathological etiology. of these, 891 records were eliminated for biologically implausible values of bmi > 60 kg / m or height - for - age 2 years of age (26%, table 4). consistent with this, those ever being documented had a later age at first bmi 99th percentile encounter than those never correctly documented, at both centers (both p < 0.001), and had more encounters (median 4, iqr 3, 6) compared to those who were not (median 3, iqr 2, 4) (table 4). the documentation was significantly higher in girls (268/921 = 29% documented) than in boys (289/1667 = 17% documented, p < 0.001). african american children were more likely to have a documentation of obesity (115/419 = 27% documented) compared to white children (237/1262 = 19% documented, p < 0.001), which was consistent at both institutions (table 4). documentation of individual encounters was lower (13.5% overall) than for each individual patient (21.5%, table 3). most of the children with documentation of obesity were class 2 obesity or higher. at the time of first documentation of obesity (median 48 months, table 5), the median bmi was 125% of 95th percentile (iqr 116, 140) overall, somewhat lower for bch (121% of 95th percentile (iqr 114, 133)) than at cchmc (133% of 95th percentile (iqr 121, 147), p < 0.001). documentation at the first encounter occurred 44% of the time overall and was more common for children 2 years old (51%) than children < 2 years old (23%, table 5). there was a median lag overall of 1.5 months (iqr 0, 13) after first encounter to documentation, which was somewhat longer at bch (5 months (iqr 0, 18)) compared to cchmc (0 months (iqr 0, 4), table 5). approximately half of all visits were documented for each child. the location of the encounter was also a significant factor in determining the documentation of obesity (table 5). children seen in the endocrine, nutrition, and obesity clinics had higher rates of documentation 70% of visits overall, compared to primary care (15% of visits) or other subspecialties (9% of visits). the data from this study identifies poor rates of clinical documentation of severe obesity in young children at two large pediatric academic hospitals utilizing a new, validated ehr algorithm that uses structured and unstructured data (through nlp) to identify chart documentation of obesity. rates of ever being recognized as obese in young children was 21.5% overall, varying by institution, and much less (13.5%) if evaluated at an encounter level. most children who were recognized to have some weight issue (based on a broad selection of weight - related terms) were not recognized in the clinical documentation as obesity or severe obesity, although they would qualify as severe obesity using bmi definition. poor documentation of obesity has been shown in previous studies : assessing icd-9 codes for obesity based on discharge diagnoses identified only 1.7% of children with obesity (mean age for those with obesity not documented, 11.4 4.9 years). chart documentation in a general pediatrics clinic showed rates of 34.1% for overweight or obese children of all ages, similar to the documentation rates in our study. a national study reviewing documentation and icd-9 codes in children 218 years old showed an 18% rate of identification of obesity. although these studies investigated identification in all age groups, there was little focus on the youngest age except for mention of poorer rates of documentation. we specifically studied the youngest children with severe obesity to leave out marginal cases and ensure that the degree of obesity was obvious to be acknowledged by the providers. at both institutions, the children who were identified appropriately had a significantly higher bmi than those not documented suggesting that the severity of the obesity played a role in the clinical documentation. as eating patterns are established early in life, this is a key age to recognize and address obesity. children who are overweight by kindergarten were found to have four times higher risk of progressing to an obese adolescence. as severe obesity continues into late childhood and adolescence, they develop an increased risk for dyslipidemia, hypertension, and hyperinsulinemia compared to those who are not obese and those with lesser degree of adiposity (bmi between 95 and 99th percentile) [4, 7, 8 ]. increasing bmi in children has also been associated with increased risk for other comorbidities and premature death as a young adult. in order to prevent these major complications, obese children must first be identified by medical providers at the youngest age possible for best outcomes. similar to previous studies, we identify poor rates of documentation despite evaluating both icd-9 codes and progress notes. however, we show that the age at first identification of severe obesity was over 36 months at both institutions indicating that children < 36 months old were more likely to be missed. possible explanations for this could be the current lack of clinical guidance for using standardized bmi curves for children less than 24 months or difficulty in approaching this topic at an age where the definitive trajectory of bmi may not yet have been established. it is to be noted that the documentation in children < 2 years old was much higher at cchmc (49% versus 14% at bch). this perhaps reflects the practice differences due to the presence of a weight management clinic focused on children < 6 years old at cchmc. a lack of clinical focus on endocrine or nutrition issues may account for the gap between these specialty encounters at both sites (bch : 69%, cchmc : 72%) and other subspecialty clinics (9% overall). however, even in clinics that should be addressing weight, such as nutrition and endocrine clinics, obesity was not documented at every encounter. the reasons for this are not clear : it could be that weight status was addressed and not documented. yet, it could be that even these types of providers can be distracted from weight status when addressing non - weight - related chief complaints. we show that males are less likely to be recognized at both institutions, despite a predominance of males in our study populations. societal norms perhaps play a role and the younger males are viewed as stocky as opposed to obese, which may explain their lack of documentation. interestingly at both institutions, caucasians were not documented as often as african americans. whether this was due to provider bias is not known but may be potentially explained by the heightened awareness of obesity and its complications in the minority populations [1, 2 ]. primary and tertiary medical providers must play an active role in obesity identification at all levels of care in these young children. for children without chronic illnesses, the most frequent encounters with medical professionals occur during the first two years of age. primary care providers need to pay attention to obesity development in young children even for sick visits, as these visits may represent their only encounters with some families. although primary care providers are the anchor for obesity management, tertiary care providers and subspecialists can play a valuable role in the identification of obesity, especially those likely to care for adiposity related complications. common childhood illnesses such as asthma, injuries, and joint pain and abdominal issues can be attributed to obesity even at young ages. furthermore, obesity in the critically ill patients can lead to acute respiratory distress syndrome, acute kidney injury, and other management difficulties [2729 ]. similarly, there is a higher rate of peri- and postoperative complications in obese individuals [2931 ]. the strengths of this study are the use of data from two large tertiary care children 's hospitals resulting in a large sample of young children. we were able to utilize nlp with a wide range of terms in addition to icd-9 codes to assess documentation of obesity with a high sensitivity. the documentation may also have been influenced by the poor reimbursement for the coding of obesity. however, we believe that the documentation of obesity is necessary for ongoing clinical care and should be made regardless of the billing practices. we were not able to obtain data on the clustering of the encounters by provider. it is possible that providers who use the captured methods had more positive records than those who did not. however, the concept of ever documentation that credits even a single documentation and analysis at the patient - level overcomes this limitation. however, the large sample size of this specialized age group may overcome this limitation. for the first time, this research identifies poor rates of clinically documenting obesity in young children with severe obesity (including those under the age of 2 years) at two large academic children 's hospitals utilizing a new, validated ehr algorithm that uses structured and unstructured data to identify appropriate chart documentation. given the importance of targeting this age group, these results show the need for improvement across all specialties in documentation of severe early onset obesity. with the increasing implementation of health information systems across the country, this offers a useful potential tool to enhance documentation in future, to avoid lifetime complications of excess weight and metabolic complications in the youngest children. | background and objectives. the prevalence of severe obesity in children has doubled in the past decade. the objective of this study is to identify the clinical documentation of obesity in young children with a bmi 99th percentile at two large tertiary care pediatric hospitals. methods. we used a standardized algorithm utilizing data from electronic health records to identify children with severe early onset obesity (bmi 99th percentile at age < 6 years). we extracted descriptive terms and icd-9 codes to evaluate documentation of obesity at boston children 's hospital and cincinnati children 's hospital and medical center between 2007 and 2014. results. a total of 9887 visit records of 2588 children with severe early onset obesity were identified. based on predefined criteria for documentation of obesity, 21.5% of children (13.5% of visits) had positive documentation, which varied by institution. documentation in children first seen under 2 years of age was lower than in older children (15% versus 26%). documentation was significantly higher in girls (29% versus 17%, p < 0.001), african american children (27% versus 19% in whites, p < 0.001), and the obesity focused specialty clinics (70% versus 15% in primary care and 9% in other subspecialty clinics, p < 0.001). conclusions. there is significant opportunity for improvement in documentation of obesity in young children, even years after the 2007 aap guidelines for management of obesity. |
taste sensation is important for animals and even exists in worms, including the model nematode, c. elegans. in the fruit fly, drosophila melanogaster, taste is first detected by neuronal cells which express a family of g - protein - coupled receptors (gpcrs). originate from the epithelium rather than neurons, receiving taste signals and transmitting them to adjacent neurons. taste cells are distributed either singly in the epithelium or form densely packed clusters, taste buds, which consist of up to 100 taste cells. in mammals, taste buds are distributed in the oral epithelium of the tongue, palate, and pharynx. taste receptors are located in the microvilli and sense taste substances in the oral cavity. there are five well - recognized taste sensations including sweet, bitter, umami, salty, and sour. detection of sweet and umami taste is especially important for recognition of food and nutrition. it is conceivable that recognition of a sweet sensation evolved to detect sugars, an important source of energy. sweet sensation is activated by various types of sweet compounds, including amino acids, peptides, proteins, carbohydrates, and other types of organic chemicals, some of which have no nutritional benefit. sweet sensation plays a critical role in animals in the recognition of food and detection of sugars in these foods. the molecular nature of the sweet taste receptor was revealed in the beginning of the 21st century by molecular cloning. the sweet taste receptor consists of a heterodimer of two gpcrs, t1r2, and t1r3 (fig. 1). t1r2 and t1r3, together with a related molecule t1r1, are members of the t1r family, which belongs to the class c gpcr. receptors in this family resemble, in many respects, those of the metabotropic glutamate receptor 1 (mglur1) and the calcium - sensing receptors. they have a large extracellular domain, termed the venous flytrap domain (vftd), in the n - terminal portion, and a cystein - rich domain, which connects the vftd and transmembrane (tm) domains. based on the 3-dimensional structure of mglur1, it is proposed that two cavities in the vftd accept sweet molecules, such as sugars, sweet amino acids, and a synthetic dipeptide, aspartame. binding of these molecules may induce conformational changes in the flytrap domain, leading to activation of the receptor. consistent with this notion, the amino acid - binding residues in the vftd of the t1r2-t1r3 are highly conserved compared with those of mglur1. however, the sweet taste receptor is activated by a variety of molecules with completely different structures. these include sugars, amino acids, peptides, proteins, and various other types of organic compounds. this implies that these molecules may bind to alternate portions of the receptor. in agreement with this concept, it has been proposed that sweet proteins interact with an external portion of the active form of the receptor. the cystein - rich region of t1r3 is another candidate site for the binding of sweet proteins. furthermore, the artificial sweetener cyclamate may bind to the c - terminal tm region of t1r3. collectively, the sweet taste receptor is activated by various compounds with diverse structures, with different types of compounds potentially binding to different portions of the receptor. binding of sweet substances to the sweet receptor t1r2-t1r3 activates trimeric g protein(s) and generates second messengers in taste cells. candidate g proteins include gustducin, a ' taste cell - specific ' trimeric g protein resembling transducin and gs gustducin has been shown to be involved in the signal transduction pathway activated by bitter taste receptors. given the fact that mice lacking the gustducin gene have impaired sweet sensation, it is thought that gustducin is also involved in the sweet receptor signaling system. with regard to the second messengers generated by activation of the sweet taste receptor, both cyclic amp (camp) and calcium may act as second messengers. using membrane fractions isolated from the anterior surface of the rat tongue, striem. this result suggests involvement of gs in the signaling pathway activated by the sweet taste receptor. also, sucralose elevates camp levels in taste buds through a calcium - independent mechanism. the signaling pathway downstream of camp production is still uncertain, and there is a controversy as to whether or not protein kinase a is involved in the downstream signaling. binding of a sweet tastant activates phospholipase c-2 (plc-2), presumably by a g protein - dependent mechanism. activation of plc-2 leads to generation of inositol (1, 4, 5)-trisphosphate (ins - p3) and diacylglycerol. ins - p3 mobilizes calcium from the endoplasmic reticulum (er) by activating the type iii ins - p3-receptor channel. one of the unique properties of sweet taste receptor signaling is that calcium released from the er activates the trpm5 channel in the plasma membrane. trpm5 is a calcium - activated cation channel, and activation of trpm5 results in sodium entry, which depolarizes the plasma membrane and thereby induces calcium entry through the voltage - gated calcium channel. consistent with this signaling cascade, knockout of the plc-2 or trpm5 gene reduces sweet taste sensation. molecular identification of the sweet taste receptor and its signaling effectors enabled us to investigate the expression of this receptor in various organs and tissues other than the gustatory system. it is now evident that the expression of the functional sweet taste receptor system is not restricted to the taste buds but is also present in other tissues, including cells in the gastrointestinal epithelium [18 - 20 ], endocrine cells of the pancreas, and glucose - sensing neurons in the brain. it is now thought that the sweet taste receptor has regulatory roles in these extra - gustatory cells. the expression of the signaling component of the sweet taste receptor in the extra - gustatory system was first described by hofer.. they found that -gustducin is expressed in rat stomach, duodenum, and pancreatic duct. although it is technically difficult to demonstrate coexpression of all components of the signaling system at the single cell level, these results suggest that the sweet taste receptor system is expressed in the gastrointestinal tract, especially the small intestine and colon. in accordance with these findings, dyer. noted expressions of t1r2, t1r3, and -gustducin in mouse duodenum and small intestine at the mrna and protein levels. the expressions of t1r2 and t1r3 are greater in the jejunum and duodenum than in the ileum, indicating that expression is more abundant in the upper small intestine. they also found that the enteroendocrine cell line stc-1 expresses t1r2, t1r3, and -gustducin and postulated that the sweet taste receptor may function as a luminal sugar sensor in the gut. expressions of the sweet taste receptor and its signaling components in humans have also been reported by bezencon.. these authors demonstrated that t1r2, t1r3, gustducin, plc-2, and trpm5 are expressed in the duodenum, jejunum, ileum, and colon. t1r1 and t1r3, components of the umami receptor, at least two types of cells in the epithelium of the gastrointestinal tract express sweet taste receptors. one is the brush cell, a putative chemosensory cell morphologically resembling taste cells with microvilli in the apical region. another type of cell expressing the sweet taste receptor is the enteroendocrine cell. indeed, enteroendocrine l - cells secreting glucagon - like peptide-1 (glp-1) and peptide yy (pyy) and k - cells secreting glucose - dependent insulinotropic peptide (gip) are positive for the sweet taste receptor. in the stomach, t1r3, a common subunit of the sweet and umami taste receptors, is expressed in brush cells and ghrelin - producing endocrine cells. intestinal neuroendocrine l - cells sense luminal glucose concentrations and secrete the incretin hormone glp-1, which regulates insulin secretion from pancreatic -cells. l - cells express t1r2 and t1r3 as well as gustducin, plc-2, and trpm5. when glucose is administered to the stomach, secretion of glp-1 this glucose - induced glp-1 secretion is attenuated in -gustducin knockout mice, and glucose - induced insulin secretion is delayed. likewise, when glucose is directly administered to the duodenum, secretion of glp-1 is induced. when duodenal tissue is incubated in vitro, a high concentration of glucose induces a 3.5-fold increase in glp-1-secretion. in duodenal tissue obtained from -gustducin knockout mice, a high concentration of glucose induces only a 2-fold increase in glp-1, significantly lower than that in normal mice. these results indicate that in vivo luminal glucose - induced glp-1 secretion is dependent on gustducin, and also there is a gustducin - independent action of glucose in duodenal cells. using ncl - h716 cells, a human enteroendocrine l - cell line, jang. demonstrated that sucrose, glucose, and sucralose, an artificial sweetener, stimulate glp-1 secretion, and that this stimulation is blocked by lactisole, an inhibitor of human t1r3. in humans, administration of glucose to the stomach or directly to the duodenum induces an elevation of plasma glp-1 concentration. this glp-1 response to glucose is markedly inhibited by lactisole, an inhibitor of the sweet taste receptor. this raises the possibility that the sweet taste receptor may also be involved in the regulation of glp-1 secretion in the stomach. as t1r2 is not detected in the stomach, a homodimer of t1r3 may function as a sweet taste receptor. taken together, secretion of glp-1 from enteroendocrine l - cells is induced by glucose, and this effect may be mediated, at least in part, by the sweet taste receptor. using mouse l - cells in primary culture, reimann. reported slightly different results. they isolated l - cells using a fluorescence - activated cell sorter from a transgenic mouse expressing a fluorescently tagged venus protein under the control of the proglucagon promoter. using these cells in culture, they found that the atp - sensitive potassium channel is functional in these cells, and that the sodium - glucose cotransporter (sglt1) is involved in glucose - induced glp-1 secretion. they also suggested that involvement of the sweet taste receptor is unlikely in glucose - induced glp-1 secretion. at present, the reason for the discrepancy is not certain. the l - cells obtained by a cell sorter may be a mixed population rather than a single population. in fact, in l - cells isolated from the colon, sucralose increases glp-1 secretion, whereas l - cells obtained from upper small intestine do not respond to sucralose. consequently, the property of l - cells obtained by a cell sorter may differ depending upon their original location along the intestinal tract. it is also possible that l - cells obtained from a certain portion of the intestine are not a single population. culture of l - cells is rather difficult, and cells cultured for a relatively long period may be slightly different from those in vivo., isolated l - cells survive in vitro only when cultured as a mixed population. cultured l - cells may have slightly different properties compared to l - cells in vivo. it is also possible that responsiveness changes as a function of time when cultured in vitro. in this regard, assessment of glp-1 secretion in knockout mice lacking t1r2 or t1r3 is needed to elucidate the definite role of the sweet taste receptor in glp-1 secretion. in addition to the regulation of incretins, the sweet taste receptor also controls glucose uptake from the intestine. glucose uptake from the intestinal lumen by absorptive enterocytes is mediated by two different types of glucose transporters, sglt-1 and the facilitative glucose transporter glut2. sglt-1 has an apparent km of 8 to 20 mm and is important at relatively low luminal concentrations of glucose. the expression of sglt-1 in brush - border of enterocytes is regulated by a glucose sensor facing the luminal membrane. margolskee. reported that glucose absorption and the expression of sglt-1 in enterocytes are regulated by dietary sugars and artificial sweeteners in normal mice. the effects of sugars and artificial sweeteners are attenuated in knockout mice lacking t1r3 or gustducin. they also found that t1r2, t1r3, and gustducin are expressed in enteroendocrine cells. given that the sweet taste receptor regulates secretion of glp-1 in enteroendocrine l - cells, they suggest that dietary sugars and artificial sweeteners act on the sweet taste receptor in l - cells, which stimulates glp-1 secretion and, in turn, upregulates sglt1 expression in enterocytes, thereby increasing glucose absorption. glut2 is a facilitative glucose transporter with higher capacity but lower km for glucose than sglt1. this implies that, at high concentrations of luminal glucose, transport through glut2 is more important than transport through sglt1. activation of this receptor leads to insertion of glut2 into the brush border membrane, which facilitates glucose uptake via this transporter. for insertion of glut2, activation of protein kinase c-ii caused by additionally, glucose uptake through sglt1 causes depolarization of the plasma membrane since sglt1 is a na - glucose cotransporter. resultant depolarization induces calcium entry via the voltage - gated calcium channel cav1.3, elevating cytoplasmic ca2 + concentration. taken together, the sweet taste receptor modulates the expressions and functions of sglt1 and glut2 and upregulates glucose transport in intestinal epithelium. pancreatic -cells produce and secrete insulin, a principal hormone regulating fuel metabolism in the body. thus, mrna for t1r2 and t1r3, as well as that for gustducin, is expressed in mouse pancreatic islets. immunohistochemistry using anti - t1r3 antibodies indicates that immunoreactivity of t1r3 is localized in insulin - producing -cells. when the sweet taste receptor in islet cells is activated by sucralose, insulin secretion is augmented. the effect of sucralose is observed in the presence of a low concentration of glucose (2.8 mm). at higher concentrations of glucose, the sweet taste receptor is also expressed in min6 cells, a glucose - responsive -cell line. in these cells, artificial sweeteners such as sucralose, saccharin and acesulfame - k all induce insulin secretion. again, the effects of sweeteners are observed in the presence of a low concentration of glucose and are much greater in the presence of high concentrations of glucose. it is now possible to monitor real - time changes in intracellular messengers in a single living cell. as shown in fig. 2, sucralose induces an immediate and sustained elevation of cytoplasmic ca concentration ([ca]c). the sucralose - induced elevation of [ca]c is inhibited by gurmarin, an antagonist of the sweet taste receptor. in addition, calcium response to sucralose is not affected by an inhibitor of gq, whereas the gq inhibitor completely blocks carbachol - induced calcium response. thus, results suggest that gq is not involved in the action of sucralose. when extracellular calcium is removed, the sucralose - induced elevation of [ca]c is markedly inhibited. a small initial peak is observed, but the sustained phase of [ca]c elevation is abolished. likewise, addition of nifedipine, an inhibitor of the l - type voltage - gated calcium channel abolishes a sustained elevation of [ca]c. these results indicate that sucralose increases [ca]c by causing release of calcium from an intracellular pool and also by stimulating calcium entry via voltage - gated calcium channels. note that sucralose - induced calcium entry is completely dependent on extracellular na, suggesting that this sweetener stimulates na entry, depolarizes the plasma membrane, and stimulates calcium entry via voltage - gated calcium channel. with regard to the effects of sucralose on camp, sucralose - induced elevation of [camp]c is not blocked by either removing extracellular calcium or by adding nifedipine. hence, sucralose - induced changes in [camp]c may not be secondary to the changes in [ca]c but are perhaps mediated by activation of gs. in addition to elevations in [ca]c and [camp]c, sucralose also rapidly activates pkc and induces phosphorylation of a pkc substrate (fig. collectively, the sweet taste receptor expressed in pancreatic -cells is unique in the sense that it activates both calcium and camp messenger systems. presumably, the sweet taste receptor increases [camp]c by activating gs and induces calcium signaling by activating a g protein different from gq, possibly gustducin. a schematic presentation of the signal transduction pathway activated by the sweet taste receptor in -cells is shown in fig. the physiologic significance of the sweet taste receptor in pancreatic -cells is unclear at present. unlike the sweet taste receptor in taste buds and gastrointestinal epithelium, the sweet taste receptor in -cells since -cells sensitively detect plasma glucose concentration, it would be interesting to clarify the role of the sweet taste receptor in the context of the glucose - sensing machinery of -cells. as -cells express the functional sweet receptor signaling system, and since this receptor is activated by glucose, the most straight - forward interpretation is that this receptor is part of the glucose - sensing machinery of -cells. a critical question is to what extent the sweet taste receptor is involved in glucose - sensing in -cells. in this regard, the sensitivity of this receptor to ambient glucose is not high, at least in a heterologous expression system. thus, it is uncertain whether or not the sweet receptor signaling system, as activated by ambient glucose, is reasonably detectable. this is an important issue and should be determined experimentally. given that the sweet taste receptor is activated by various compounds with completely different chemical structures, it is also possible that some endogenous agonists apart from glucose exist, either in the extracellular fluid or cytosol. such an endogenous compound, if any, would therefore modulate insulin secretion. this intriguing possibility should be examined experimentally. the sweet taste receptor expressed in -cells has a unique signaling system in that it activates both the calcium and camp signaling systems. since agents which increase camp production in -cells would protect these cells from various stresses and apoptosis, the sweet taste receptor may be a potential molecular target for developing novel therapeutic agents to treat diabetes. | the sweet taste receptor is expressed in taste cells located in taste buds of the tongue. this receptor senses sweet substances in the oral cavity, activates taste cells, and transmits the taste signals to adjacent neurons. the sweet taste receptor is a heterodimer of two g protein - coupled receptors, t1r2 and t1r3. recent studies have shown that this receptor is also expressed in the extragustatory system, including the gastrointestinal tract, pancreatic -cells, and glucose - responsive neurons in the brain. in the intestine, the sweet taste receptor regulates secretion of incretin hormones and glucose uptake from the lumen. in -cells, activation of the sweet taste receptor leads to stimulation of insulin secretion. collectively, the sweet taste receptor plays an important role in recognition and metabolism of energy sources in the body. |
antibodies have proved to be powerful tools in facilitating the elucidation of disease mechanisms and generating novel and effective therapeutics. however, the use of antibodies has been limited to outside the cell because of two major factors : antibodies containing disulfide bonds might be unstable in the reducing environment of cytosol, and antibodies are unable to cross the cell plasma membrane to reach the cytosol. there are numerous intracellular targets and protein protein interactions with large flat contact areas ; these are considered difficult to perturb by small molecules. we believe there would be great interest in the use antibody mimics to target the intracellular protein protein interactions, provided that they can be transported into the cytosol by a straightforward delivery platform. in recent years, certain robust, single - domain, cysteine - free scaffold proteins have emerged as antibody mimics. these include monobodies derived from the tenth type iii domain of human fibronectin (10fn3), affibodies derived from the immunoglobulin binding protein a, darpins based on ankyrin repeat modules, and the b1 domain of protein g (gb1). these antibody mimics have been engineered to bind extracellular receptors such as egfr, her2, vegfr and integrin, as well as various intracellular targets, including caspases, raf, erk, c - jun n - terminal kinase (jnk), abl - sh2, and c - jun. advances in directed evolution and molecular display technologies, such as phage display, yeast display, and ribosome display, make it possible to routinely generate a wide variety of high - affinity binders for specific protein targets. research effort is now focused on applying these antibody mimics inside the cell (intrabodies) to target cytosolic proteins. to achieve this, strategies are needed to allow facile and reliable delivery of these bioactive antibody mimics into the cytosol of various cell types. delivery methods based on lipid - derived compounds, polymeric nanoparticles, inorganic nanocarriers, supercharged proteins, and, most commonly, cell - penetrating peptides (cpps) such as the transactivator of transcription (tat) of hiv-1, oligoarginine, and penetratin peptide derived from the drosophila antennapedia, have been developed to deliver proteins of interest to the cytosol of mammalian cells. in most of these cases, high concentrations of these agents are required to achieve even modest effects, often because of inefficient cargo escape from the endosome. nature has evolved a variety of mechanisms to transport proteins across membranes into the cytosol of mammalian cells. one bacterial protein - transport nanomachine is protective antigen (pa ; 83 kda), a component of anthrax toxin. pa is a receptor - binding, pore - forming transporter that delivers the enzymatic moieties of the toxin from the external milieu to the cytosol of mammalian cells. pa binds to host - cell receptors and is cleaved by a furin - family protease to yield a 63 kda species (pa63) (figure 1 a ; step 1) that self - assembles to form ring - shaped heptamers and octamers. these oligomers then form complexes with the cargo proteins (kd1 nm) and are endocytosed., acidification triggers conformational change of the pa63 oligomers to form a transmembrane pore that unfolds and translocates the bound cargo proteins to the cytosol (figure 1 a ; step 5). pa63 oligomers recognize the n - terminal domain (lfn, 30 kda) of the toxin enzyme lethal factor (lf, 90 kda). studies have shown that cargo fused to the c terminus of lfn can be transported to the cytosol via pa ; most effort has focused on the delivery of peptides for vaccine development, enzymes such as -lactamase, and enzymatic domains from diphtheria toxin (dta), shiga toxin, pseudomonas exotoxin a (peiii), and rtx toxin (acd). more recently, the pa / lfn system was shown to deliver legionella pneumophila flagellin into macrophages. however, no study has investigated the ability of pa / lfn system to translocate antibody mimics for the perturbation of intracellular protein protein interactions. i d : 1q2n), gb1 (1pgb), darpin (adapted from 3zu7), and ha4 (3k2 m). c) variants of antibody mimics (stars) attached to the c terminus of lfn (lv) or lfn - dta (ldv). d) protein synthesis inhibition in cho - k1 cells treated with ldv14 in the presence of 20 nm pa for 30 min. the cells were then washed three times with pbs and incubated with 1 ci mlh - leucine for 1 h. subsequently, the cells were washed three times with pbs and scintillation fluid was directly added to each well, and h incorporation into the cellular proteome was measured to determine the level of ldv inhibition of protein synthesis (n=3). radioactive counts were normalized to those of cells treated with only pa (set to 1). : lfn - dta, : ldv1, : ldv2, : ldv3, : ldv4, : dta, : lv1+dta. here we used transpeptidase sortase (srta) to conjugate several commonly used antibody mimics to the c terminus of lfn and found that pa can mediate their transport into the cytosol of several different cell lines. we confirmed the refolding and binding of a tandem monobody to its protein target bcr - abl inside cells by co - immunoprecipitation. we observed inhibition of abl kinase activity and subsequent cell death caused by the pa - delivered monobody. we show that the pa system can deliver an affibody that binds hraf-1 to disrupt the mapk signaling pathway. our antibody mimics consisted of scaffolds widely used to generate highly specific and potent binders : affibody, protein gb1, darpin, and monobody (figure 1 b). these scaffolds are disulfide - free, thus avoiding possible interference with passage through the pa translocase and potential stability problems in the reducing environment of the cytosol. our chemoenzymatic bioconjugation route is based on srta, an evolved srta, and is shown in figure s1 in the supporting information. srta catalyzes the formation of covalent conjugates (designated lv, figure 1 c) between lfn containing the c - terminal lpxtg recognition motif and antibody mimics containing n - terminal oligoglycine. we also prepared a series of conjugates (designated ldv, figure 1 c) between lfn - dta and each antibody mimic, in order to measure pa - mediated translocation into the cytosol. in anthrax toxin translocation studies, the a chain of diphtheria toxin (dta), which catalyzes the adp - ribosylation of ef-2 and inhibits protein synthesis, has been frequently used as a straightforward measure the gold standard assay of pa - mediated translocation into the cytosol. therefore, lfn - dta variants (ldvs) allowed us to compare our findings with previous reports that used the same assay.[18a, c, 22 ] for each antibody mimic, after confirming translocation of the ldv, we also carried out studies with lvs that lack the toxic dta protein, thus avoiding interference with further characterization of antibody mimic function and delivery into the cytosol. each purified ldv (ldv14, figure s2) was added to cho - k1 cells in the presence of 20 nm pa. after 30 min, the cells were washed and incubated with medium supplemented with h - leucine. the efficiency of antibody mimic translocation was measured by the incorporation of h - leu in the cellular proteome, as the level of protein synthesis inhibition is determined by the amount of ldv - containing variant in the cytosol. despite their structural differences, all four variants (ldv14) translocated efficiently into the cytosol at levels comparable to that of the positive control, lfn - dta (figure 1 d). to confirm that the full - length protein was required for translocation to the cytosol, we performed control experiments by treating cells with dta plus pa, or dta together with lfn - affibody (lv1) plus pa ; we observed no protein synthesis inhibition in either case (figure 1 d). to investigate whether ldvs translocate to the cytosol by the same mechanism as for lfn - dta, we carried out a series of control experiments with ldv1 containing affibody as the cargo (figure 2 a). we investigated the role of lfn in ldv1 during the translocation process by using excess lfn (1 m) to outcompete ldv1 in binding to pa. we found that 1 m lfn significantly abolished translocation of ldv1, thus confirming the importance of lfn - mediated binding to pa (step 3, figure 1 a). we then studied the role of endocytosis in ldv1 translocation by incubating cells with pa and lfn - dta or ldv1 at 4 c (instead of 37 c) to arrest endocytosis. the translocation of both lfn - dta and ldv1 was abolished under these conditions, thus indicating that endocytosis is a necessary step in the translocation process (step 4, figure 1 a). to test the role of endosome acidification and active translocation (step 5, figure 1 a), we performed two additional control experiments. first, we treated cells with pa and lfn - dta or ldv1 in the presence of 200 nm bafilomycin a1, a specific inhibitor of vacuolar h - atpase, in order to block endosome acidification. we found that inhibition of endosome acidification abolished translocation of both lfn - dta and ldv1, thus confirming the importance of the ph gradient for translocation. an additional control experiment was performed with a pa mutant (pa[f427h ]) that binds and delivers the cargo to endosomes but arrests translocation through pa pore. we found that pa[f427h ] completely arrested translocation of ldv1, thus indicating that functional pa was required for ldv1 to reach the cytosol. these controls indicate that the translocation of ldv1 follows the same mechanism as that for lfn - dta. a) protein synthesis inhibition in cho - k1 cells treated with variants in the presence of 20 nm pa for 30 min. ba = addition of 200 nm bafilomycin a1 ; 4 c = incubation at 4 c (instead of 37 c) ; pa[f427h]=mutant pa instead of pa. : lfn - dta, : ldv1, : ldv1+lfn, : lfn - dta 4 c, : ldv1 4 c, : lfn - dta+ba, : lv1+ba, : ldv1+pa[f427h ]. b) cho - k1 cells were treated with 250 nm lv1 in the presence of 40 nm pa or pa[f427h ] for 12 h (conditions / modifications as above). total lysate and digitonin - extracted cytosolic proteins were prepared separately (300 000 cells per lane). after confirming the key steps responsible for the translocation of antibody mimics, we investigated the delivery of lvs into the cytosol by digitonin extraction and western blotting. digitonin is a weak, nonionic detergent, and at low concentrations selectively permeabilizes the plasma membrane, thereby releasing cytosolic components from cells while the nuclear envelope and other major membrane organelles remain intact. cho - k1 cells were incubated with lv1 and pa overnight (to allow multiple rounds of receptor - mediated endocytosis and translocation), washed, and trypsin digested to remove cell surface receptors and bound protein. in order to obtain the cytosolic fraction, cells were incubated with a buffer containing 50 g ml digitonin and 250 mm sucrose. immunoblot analysis with antibodies against rab5 (an early endosome marker) and erk1/2 (a cytosolic marker) was carried out to confirm that only the cytosolic fraction was present. minor amounts of rab5 were detected in the digitonin - extracted cytosolic fractions, thus indicating little contamination from early endosomes (figure 2 b). we observed a significant amount of lv1 in the cytosolic fraction, similar to that in total cell lysate obtained by lysis buffer containing 1 % np-40. no lv1 was observed when the cells were incubated at 4 c (endocytosis arrested), even in the total cell lysate. the absence of detectable lv1 at 4 c also validated that the wash and trypsin digestion protocol was sufficient to eliminate potential contamination from surface - bound protein. when the cells were treated with bafilomycin a1, a significant band for lv1 was observed in the total cell lysate, whereas this band was not detectable in the cytosolic fraction, thus indicating that lv1 was endocytosed but trapped in the non - acidified endosome. in addition, the absence of lv1 in the cytosolic fraction for cells treated with bafilomycin a1 served as further evidence that our digitonin extraction protocol was sufficient to separate the cytosolic proteins from the rest of the cell lysate, including endosomes. finally, when the cells were treated with lv1 and pa[f427h ] (instead of lv1 and pa), some lv1 was observed in the total cell lysate but none in the cytosolic fraction (figure 2 b). under these conditions, endocytosed lv1 is trapped in the endosome because of the non - functional pore (mutant pa), and subsequently sorted to the lysosome for degradation, thus leaving only a small amount of lv1 in the total cell lysate and none in the cytosolic fraction. these western blot results further corroborated the protein synthesis inhibition results with lfn - dta (vide supra, figure 1 d) and confirmed the translocation mechanism of the antibody mimic cargo. pa - mediated translocation of the other analogues (lv24) was also studied by digitonin extraction and western blotting (figure s3) ; bands at different molecular weights by anti - lf antibody confirmed the presence of these antibody mimic conjugates in the cytosol. to compare the efficiency of the pa / lfn system with the cpp system this construct showed at least 1000 times lower efficiency in protein synthesis inhibition than lfn - dta and pa (figure 3 a) ; this is consistent with a previous report and indicates that the pa / lfn system is more efficient in delivering dta to the cytosol. next, we compared the efficiencies of the pa / lfn and cpp systems in delivering antibody mimics. we conjugated oligoglycine - containing antibody mimics 14 to a tat peptide containing an ha tag and the lpstgg motif to generate tat - ha - antibody mimic constructs (figure s4). translocation was studied by digitonin extraction and western blot analysis with anti - ha antibody. we treated cells with 2.5 m tat - ha - antibody mimic constructs (tenfold higher than for lvs) for 4 h in serum - free medium and observed minor amounts of material in total - cell lysate (figure s5 a). then, when using the trypsin digestion and digitonin extraction protocol as described above, we detected no tat - ha - antibody mimic protein by anti - ha immunoblotting with the cytosolic fractions, whereas a significant amount of lv1 with ha tag (lv1-ha) was detected for cells treated with 250 nm lv1-ha and 40 nm pa for 4 h (figure 3 b). to test if a different incubation time would improve translocation by tat peptide, we treated cells with 2.5 m tat - ha - antibody mimics for 16 h ; again, no material was evident in the total cell lysate (figure s5 b). these results demonstrate the higher efficiency of the pa / lfn system in delivering antibody mimics over the tat peptide method. additionally, the amount of lv1-ha detected by anti - ha immunoblotting was similar to that of lv1 detected by anti - lf. the presence of a c - terminal ha - tag further validated the presence of full - length lv1-ha in the cytosol. tat peptide - mediated translocation of dta and antibody mimics. a) protein synthesis inhibition of cho - k1 cells treated with varying concentrations of tat - dta () for 30 min, in comparison to lfn - dta plus 20 nm pa () ; assay conditions as in figure 1. b) western blot of cytosolic fractions extracted by digitonin from cho - k1 treated with 2.5 m tat - ha-14 (see figure 1 b) for 4 h (4 c = incubation at 4 c instead of 37 c), in comparison to treatment with 250 nm lv1-ha and 40 nm pa for 16 h. the cytosolic fraction was extracted using digitonin (280 000 cells per lane). the corresponding immunoblots of total lysates from cells treated with tat - ha-14 for 4 h and 16 h are shown in figure s5. our next question was whether the delivered antibody mimics can refold and bind to their targets inside the cytosol. based on the protein synthesis inhibition assay, we confirmed that dta could correctly refold in the cytosolic environment after translocation. for the antibody mimics, we chose to study the tandem 10fn3 monobody (ha4 - 7c12), which binds with nanomolar affinity to the src homology 2 (sh2) domain of the oncoprotein bcr - abl.[2f ] first, we sortagged this tandem monobody to lfn - dta (ldv5) and used the protein synthesis inhibition assay to study the variant 's translocation efficiency in chronic myeloid leukemia (cml) k562 cells. the assay showed that pa translocated ldv5 as efficiently as the control (lfn - dta, figure s6). after confirming that the pa system efficiently translocated the tandem monobody ha4 - 7c12, we sortagged it to lfn (lv5, figure 4 a), which lacks the cytotoxic dta protein, and tested whether lv5 could bind to bcr - abl in k562 cells after translocation to the cytosol. to ensure that the binding affinity of the monobody was not affected by the presence of lfn, which (apart from initiating translocation) provides an important epitope for detection in the cytosol by western blotting, we first measured the binding affinity of lv5 for the abl sh2 domain by spr. we obtained a kd value of 12 nm, similar to that for the monobody alone (6 nm, figure s7). next, we investigated whether the delivered lv5 could refold and bind to abl kinase inside the cell. k562 cells were treated with lv5 and pa, and then subjected to immunoprecipitation with an anti - abl antibody linked to agarose beads. proteins eluted from the beads were subjected to immunoblot analysis with an anti - lf antibody. we observed a pull - down band corresponding to lv5 when cells were treated with lv5 and pa (figure 4 b), thus confirming that at least some of the monobody properly folded after reaching the cytosol and was bound to intracellular abl kinase. in control experiments, pa[f427h ] was used instead of pa, in order to arrest translocation from the endosome to cytosol. no band was observed, thus confirming that cytosolic access of lv5 was critical for the binding. finally, the cells were treated with the binding mutant lv5mut (ha4 : y87a ; 7c12 : y62e / f87k) instead of lv5. similar amounts of lv5 and lv5mut were delivered to the cell ; the pull - down band was absent for lv5mut (figure 4 b), thus indicating that the binding interaction only occurred when the lfn variant contained the functional binder. these results demonstrate that pa - delivered tandem monobody can refold and bind to its target inside the cell. a) construct of lv5 containing ha4 (pdb i d : 3k2 m), gs - rich linker, and 7c12 (pdb i d : 3t04). b) left : western blot of total cell lysate from k562 cells treated for 24 h with 50 nm lv5 or lv5mut in the presence of 20 nm pa or pa[f427h ]. right : the lysate was then subjected to co - immunoprecipitation (co - ip) with anti - abl agarose beads. cells treated with lv5mut / pa or lv5/pa[f427h ] served as negative controls for co - ip. our next goal was to investigate the possibility of using the delivered tandem monobody to perturb protein function and related signaling pathways in cancer cells. overexpression of ha4 - 7c12 in k562 cells has been reported to strongly inhibit kinase activity and induce apoptosis by disrupting a critical intramolecular sh2-kinase domain domain interaction.[2f ] despite the high affinity and specificity of the monobody in targeting the allosteric module in bcr - abl and its utility in fighting therapy resistance of bcr - abl mutants, hantschel. determined that intracellular delivery was the biggest hurdle to achieving its practical application.[2f, 27 ] in order to test if pa - mediated delivery could overcome this, we used pa to translocate lv5 into k562 cells. based on the linear relationship between the amount of protein loaded and the signal intensity of each band detected by anti - lf antibody (figure s8), we estimated a total of 1 ng lv5 delivered into 100 000 cells (figure s9) ; this is 10 fg (110 000 lv5 molecules) in each cell (cytosolic concentration 80 nm). although this concentration is above the kd for the abl sh2 domain, we did not expect strong inhibition of bcr - abl kinase, because of the high concentration of bcr - abl inside k562 cells. as detected by western blotting, monobody binding resulted in a modest reduction in activation loop (tyr412) phosphorylation of bcr - abl (figure s9). we then investigated the effect of this activity inhibition on inducing apoptosis of k562 cells. by tunel staining (detects dna fragmentation by labeling the termini), we observed apoptosis after k562 cells were treated with lv5 and pa (figure 5 a). this was not observed when pa[f427h ] or lv5mut was used. the proportion of apoptotic cells after delivery of lv5 was 20 % of that caused by the small molecule imatinib (1 m, figure s10), which inhibits kinase activity by binding close to the atp binding site of bcr - abl. the different inhibition mechanism and the much higher concentration of cytosolic imatinib might explain the difference in the proportion of apoptotic cells as compared to the lv5 and pa treatment. in addition, the extent of apoptosis by lv5 was also lower than that induced by overexpression of ha4 - 7c12.[2f ] the high concentration of monobody achieved by overexpression and selection for only transfection - positive cells met the challenge of requiring high local concentration to interfere with the intramolecular interactions between the sh2 domain and kinase domain in bcr - abl. the modest inhibition of kinase activity and induction of apoptosis by pa - mediated delivery of lv5 serves as a proof - of - concept that intracellularly delivered monobodies can perturb the activity of an oncoprotein. monitoring of apoptosis of k562 cells treated with lv5 and pa by the tunel assay. a) k562 cells treated with the indicated analogues (500 nm lv5 or lv5mut in the presence of 80 nm pa or pa[f427h ]) for three days and analyzed for apoptosis ; 1 m imatinib served as a positive control. representative dot plots from flow cytometry show terminal deoxynucleotidyl transferase (tdt) catalyzed brdutp incorporation into the dna strand breaks of apoptotic cells ; this is detected by alexa fluor 488-labeled anti - brdu antibody (fitc). b) quantification of tunel - positive cells : intensities normalized to k562 cells treated with imatinib (100 %) and non - treated cells (0 %). the dot - plots of imatinib - treated and untreated cells are shown in figure s10. data are averages of three independent experiments. we also investigated pa - mediated delivery of a binder based on affibody (abraf), which was evolved to bind to human raf-1 (hraf-1, kd=100 nm),[2c ] a protein kinase of central importance in the mapk / erk signaling pathway. although abraf has been shown to inhibit the ras / raf interaction in vitro, it has not been tested for inhibition of mapk signaling pathway in cells. we transfected human embryonic kidney 293 t (hek293 t) cells with abraf, and observed an average of 37 % reduction in phosphorylation levels of mapk (perk1/2, downstream of ras / raf pathway) upon epidermal growth factor (egf) activation figure 6 a), thus validating the cellular function of this binder in blocking the mapk signaling pathway. we then conjugated abraf to lfn (lv6), and found that pa delivered 240 nm lv6 (5 fg or 79 000 molecules per cell) in hek293 t cells (figure 6 b). we measured the phosphorylation level of mapk (perk1/2) upon egf activation in cells treated with lv6 and pa or pa[f427h ]. cells treated with lv6 and pa showed 25 % reduction of perk1/2 relative to egf - activated untreated cells (p<0.01, figure 6 c). pa[f427h ] (negative control) showed a similar level of perk1/2 as for egf - activated untreated cells (p=0.27, figure 6 c). this experiment is another example of using pa to translocate functional binders to disrupt protein perturbation of the mapk signaling pathway by pa - mediated delivery of an affibody (lv6) that targets raf. a) hek293 t cells were transfected with pcdna3-abraf for 24 h, starved overnight, treated with 5 ng ml egf for 7 min, lysed with buffer containing 1 % np-40, and subjected to anti - perk1/2 immunoblotting. the membrane was stripped and re - blotted with anti - erk1/2 antibody to serve as loading control. b) cytosolic fractions extracted by digitonin from hek293 t cells treated with 500 nm lv6 and 80 nm pa or pa[f427h ] in serum - free medium for 12 h (400 000 cells per lane). c) hek293 t cells treated with 500 nm lv6 and 80 nm pa or pa[f427h ] in serum - free medium for 12 h, then with 5 ng ml egf for 7 min, lysed, and analyzed as in (a). bar graph : quantification of phosphorylation of erk1/2 (perk1/2 ; n=3) ; bars correspond to the lanes above. perk1/2 bands normalized to that of erk1/2, and compared to cells treated with egf (set to 1). we have demonstrated the ability of pa / lfn to deliver antibody mimics to the cytosol of cells and also the possibility of using the delivered binders to perturb critical protein prior efforts with this system have mainly focused on the delivery of enzymes that can exert a strong biological effect at very low concentrations, as has been demonstrated here by the lfn - dta activity assays. however, for an antibody mimic binder to exert inhibitory effects on protein protein interactions inside cells, the concentration of the antibody mimic binder needs to reach a level above kd and close to the concentration of competing endogenous binding proteins. we aimed to answer the questions as to whether pa could both efficiently deliver the antibody mimics and transport sufficient amounts of cargo to perturb protein protein interactions. we first systematically investigated translocation mediated by pa of four different antibody mimics : all -helical (affibody, darpin), all -sheet (monobody), and -helical and -sheet proteins (gb1). we did not assume these scaffolds would translocate efficiently, because prior investigations have indicated that certain c - terminal modifications of lfn or lfn - dta can abrogate translocation through pa. in particular, imaging studies indicated that fluorescent proteins fused to the c terminus of lf significantly attenuate translocation. other studies have shown that dta with an artificial disulfide or dihydrofolate reductase (dhfr) complexed with methotrexate can arrest translocation when attached to the c terminus of lfn. using the dta - based protein synthesis inhibition assay, we found that all four antibody mimics translocated as efficiently as the positive control lfn - dta. we are unaware of any system that permits the facile delivery of antibody mimics ranging in structural diversity as shown here. our translocation studies began with ldvs and the protein synthesis inhibition assay, because this is commonly used to probe anthrax toxin entry into the cytosol and thus allowed us to compare our findings to other reports. however, in order to use antibody mimics to perturb protein protein interactions, we needed to eliminate the interference of dta. therefore, we also investigated the translocation of lfn - antibody mimics (lvs) in the presence of pa. to facilitate the analysis of lv delivery, we used a reliable western blot approach to assess full - length cargo in the cytosolic fraction (obtained by using digitonin to permeabilize the plasma membrane). for all experiments, we confirmed successful extraction of cytosolic proteins from the rest of the cellular components by staining to check for the presence of erk1/2 and absence of rab5. the successful extraction of cytosolic proteins was further validated by the absence of lv1 in the digitonin - extracted fractions from cells treated with bafilomycin a1 or pa[f427h ]. additionally, we observed no lv1 in the total cell lysate under 4 c treatment conditions, in order to validate the effectiveness of trypsin digestion in removing surface - bound lv (leaving only intracellular proteins for detection). we detected the conjugates of antibody mimics to lfn by the anti - lf antibody, and confirmed the presence of the antibody mimics attached to lfn by their different molecular weights. further evidence that full - length material translocated into the cytosol was obtained by detection of lv1-ha with the c - terminal ha - tag, which gave a similar amount of material detected as that by the anti - lf antibody. we also used digitonin extraction and western blot to gauge the amount of material delivered into the cell cytosol. based on the linear relationship between the amount of loaded protein and the signal intensity on the anti - lf immunoblot, we were able to estimate the amount of material delivered, by comparing the immunoblot signal to that of a known amount of protein loaded on the same blot. as the antibody mimics do not interfere with translocation through the pa pore, the amount delivered to the cytosol is dependent on the number of anthrax receptors on cells ; these are present on most human cells (2000 50 000 per cell, higher in certain cancer cells). theoretically, one round of translocation for cells harboring 50 000 receptors would give 20 000 molecules in one cell, if seven receptors deliver three copies of lfn - cargo variant. thus we estimated, by western blot quantification, that after multiple rounds of translocation we achieved mid - nanomolar concentrations of cargo in the cytosol. we next studied whether a functional antibody mimic binder can properly refold after translocation, as translocation through the pa pore requires protein unfolding. based on co - immunoprecipitation of the monobody with the anti - abl antibody, we confirmed that the monobody correctly refolds in the cytosolic environment after translocation. the efficiency of refolding and the portion of functional protein is presumed to depend on the cytosolic stability and degradation rates of the variants. for the binders based on monobody and affibody, we observed inhibition of bcr - abl kinase and disruption of the mapk signaling pathway, respectively, thus providing evidence that the antibody mimics properly fold and function after translocation into the cytosol. as the amount of tandem monobody delivered was not significantly higher than the kd for the bcr - abl target, the extent of apoptosis caused by lv5 was not as great as that achieved by overexpression of the tandem monobody (where its cytosolic concentration reaches a much higher level). the lower biological effect of lv5 was probably due to the presence of high concentration of endogeneous abl kinase, as well as the difficulty in interfering with the intramolecular domain in contrast, even though the delivered amount of affibody binder for hraf-1 was not much higher than the kd for its target, the inhibition of the mapk signaling pathway by lv6 was close to that achieved by overexpression of the affibody binder. this could be attributable to the lower target concentration and the higher efficiency in disrupting ras / raf intermolecular interactions, compared to the bcr - abl target. the delivery of more material and/or increased potency of the cargo for its target are additional challenges in biomolecular delivery. the high adaptability and promiscuity of the pa transporter enabled delivery without the need for protein engineering or screening, unlike delivery methods such as cpps where a number of sequences or linkers need to be screened for efficient delivery of each cargo. in the case of cpps, the stability and identity of the peptide transduction sequence can lead to significant differences in delivery efficiency, and strong adherence to the cell surface and endosomal membranes often blocks cargo escape into the cytosol. for example, it was reported that cpp fusions to dta were not able to achieve delivery into the cell, even at concentrations a thousand times higher than that required for pa - mediated translocation ; we confirmed this result. we also investigated the efficiency of the tat peptide in the delivery of the antibody mimics and found that, even after treatment of the same cell lines with tenfold more material, the tat peptide was not able to deliver any of the four antibody mimics. additionally, because of highly inefficient endosomal escape for delivery into the cytosol, most delivery methods use high concentrations of components, and this can give rise to cellular toxicity. we studied whether the delivery components pa and lfn (by themselves or in combination) are toxic to cells. under the conditions tested, we did not see any toxicity, based on a tunel apoptosis assay (figure 5) or an mts assay (figure s11). in summary, we report for the first time pa - mediated delivery of antibody mimics into the cytosol and successful disruption of critical protein, we found that an sh2-binding tandem monobody can be delivered and functioned as an inhibitor of the oncoprotein bcr - abl inside cancer cells. we also demonstrated inhibition of the ras / raf interaction by an affibody thereby blocking the mapk signaling pathway, which plays a central role in the control of cell proliferation, survival, and growth. the delivery of antibody mimics to the cytosol of cells as indicated by dta activity and western blotting, together with the observation of functional tandem monobody binder to the oncoprotein bcr - abl and affibody binder to hraf-1, supports our belief that the pa - mediated protein delivery system (designed by nature) will significantly expand the biomolecular delivery toolbox. our future efforts will focus on increasing the amount of material delivered and the delivery of more potent antibody mimics. this platform provides new possibilities to apply modern intrabody technology to disrupt processes inside cells. materials : all reagents were purchased from sigma aldrich and life technologies except where otherwise indicated. the following primary and secondary antibodies were used : goat anti - lf (bd-17 ; santa cruz biotechnology, dallas, tx), rabbit anti - ha (sigma aldrich), rabbit anti - abl (agarose conjugate, k-12 ; santa cruz biotechnology), rabbit anti - pabl (ptyr412 ; cell signaling technology, danvers, ma), rabbit anti - erk1/2 and rabbit anti - phospho - erk1/2 (thr202/tyr204, cell signaling), rabbit anti - rab5 (cell signaling), goat anti - rabbit (irdye 800cw ; li - cor biosciences, lincoln, ne), donkey anti - goat (irdye 680lt ; li - cor biosciences). additional methods including expression and purification of the proteins used in our studies are described in detail in the supporting information. one - pot sortagging reaction using staphylococcus aureus srta : srta-mediated ligation was used to ligate proteins to his6-sumo - lfn - dta - lpstgg - his5 or his6-sumo - lfn - lpstgg - his6 as previously described (figure s1).[21b ] lfn - dta - lpstgg - his5 or lfn - lpstgg - his6 (50 m), srta (5 m), and g5-protein (100500 m) were incubated with ni - nta beads in sortase buffer (trishcl (50 mm, ph 7.5), cacl2 (10 mm), nacl (150 mm)) for 30 min at rt rocking. ni - nta beads that have bound unreacted starting material, srta-his6, his6-sumo, and gg - his6 were centrifuged (16 000 g, 4 c) to minimize the formation of hydrolyzed side product. the supernatant (containing sortagged product) was collected. the supernatant and the three washes were subjected to gel filtration to separate excess oligoglycine protein reactants. sds - page and lcms were used to assess the purity of the products (figure s2, table s2, and lcms appendix ; isolated yields in table s3 ; protein variants are listed in table s5). protein synthesis inhibition assay : cho - k1 cells were maintained in f-12k medium supplemented with fbs (10 %, v / v) at 37 c with 5 % co2. the cells were plated in a 96-well plate (30 000 per well) 16 h prior to the assay. ldvs were prepared in tenfold serial dilutions followed by the addition of protective antigen (pa83 ; 20 nm). the samples were added to cho - k1 cells for 30 min at 37 c and 5 % co2. the cells were washed three times with pbs, then incubated with leucine - free f-12k medium (100 l) supplemented with h - leucine (1 ci ml, perkinelmer) for 1 h at 37 c with 5 % co2. the cells were washed three times with pbs and suspended in scintillation fluid (150 l). h - leu incorporation into cellular proteins was measured to determine the inhibition of protein synthesis by lfn - dta. the scintillation counts from cells treated with pa only (control) were used for normalization. the data were fitted by using originlab software (northhamptown, ma) with a sigmoidal boltzmann fit using equation (1) : where x0 is the log ec50 value (table s4). uptake of lv and tat - ha-14 in cho - k1 cells : cho - k1 cells were plated in 12-well plate 16 h prior to treatment. for lvs, cells were treated with lv (250 nm) in the presence of pa (40 nm) in f-12k with fbs overnight at 37 c with 5 % co2. for tat - ha - antibody mimic constructs, cells were treated with tat - ha-14 (2.5 m) in f-12k without fbs for 4 h at 37 c and 5 % co2. translocation controls included pa[f427h ] (instead of pa), addition of bafilomycin a1 (200 nm), and incubation at 4 c instead of 37 c. cytosolic protein extraction and whole - cell lysate preparation : after uptake of the antibody mimics, cells were washed with pbs, detached and digested with trypsin - edta (0.25 %) for 5 min at 37 c to remove surface - bound protein, then washed twice with pbs. the cell pellets were then subjected to cytosolic extraction or whole - cell lysate preparation. for cytosolic protein extraction, cells (10) were suspended in buffer (100 l : digitonin (50 g ml) in nacl (75 mm), nah2po4 (1 mm), na2hpo4 (8 mm), sucrose (250 mm)) supplemented with protease inhibitor cocktail (roche) for 10 min on ice, then centrifuged (16 000 g, 5 min). for whole - cell lysate, cells were lysed in ip lysis buffer (trishcl (25 mm, ph 7.5), nacl (150 mm), np-40 (1 %, v / v)) supplemented with protease inhibitor cocktail on ice for 30 min, then centrifuged (16 000 g, 10 min). western blot : transfer was done with a te 70 semi - dry transfer unit (ge healthcare) and nitrocellulose membranes (whatman) in trishcl (48 mm) containing glycine (39 mm), sds (0.0375 %, v / v), and methanol (20 %, v / v). the membrane was blocked (rt, 2 h) with blocking buffer (li - cor) and then incubated with goat anti - lf or rabbit anti - ha antibody in li - cor blocking buffer overnight at 4 c. the membranes were washed with tbst (trishcl (50 mm), nacl (150 mm), tween 20 (0.1 %, v / v)) then blotted with secondary antibody conjugated to irdye (li - cor) and imaged with an odyssey infrared imaging system (li - cor). the western blot images were analyzed and quantified with the image studio lite program (li - cor). co - immunoprecipitation of lv5 with abl kinase : k562 cells were treated with pa or pa[f427h ] (40 nm) and lfn - antibody mimic (50 nm) for 24 h. cells (1110) were trypsinized, washed with pbs, frozen at 80 c, and then lysed in ip lysis buffer (500 l) supplemented with protease inhibitor cocktail (roche) on ice for 30 min. after centrifugation (16 000 g, 15 min), the lysate (450 l) was incubated with 12.5 g anti - abl agarose beads for 4 h. the resulting immune complexes were washed three times with lysis buffer and once with lysis buffer without np-40. the bound proteins were eluted with sds (0.2 %) and tween 20 (0.1 %) and subjected to sds - page separation. tunel assay with lv5 in k562 cells : k562 cells were plated in a 24-well plate (150 000 cells per ml in each well) and treated with lfn - antibody mimic (500 nm) and pa or pa[f427h ] (60 nm) in serum - free medium for one day. fbs (10 %) was added, and the cells were incubated for two days (positive control : imatinib (1 m)). the cells were fixed with paraformaldehyde (3.2 %), then treated with methanol (90 %) on ice or stored at 20 c, followed by tunel staining by using the apo - brdu tunel assay kit from life technologies. transfection of hek293 t with pcdna3-abraf : hek293 t cells were plated in a 24-well plate overnight to reach 90 % confluency. the cells were then transfected with plasmid pcdna3-abraf - gfp (see the supporting information for plasmid details) by using lipofectamine 2000 (life technologies). the medium was changed after 5 h. the total transfection time was 24 h. for perk1/2 analysis, the cells were starved for 12 h then treated with egf (5 ng ml) for 7 min, washed with cold pbs, and lysed in - plate with ip lysis buffer containing np-40 (1 %), protease inhibitor cocktail (roche), and phosphostop (roche). the lysate was subjected to sds - page separation, transferred to nitrocellulose membrane, and blocked with bsa (5 %), na3vo4, and naf. the immunoblotting with anti - perk1/2 antibody (cell signaling) was performed overnight in bsa (3 %) with na3vo4 and naf. after stripping the membrane with restore plus western blot stripping buffer (thermo scientific), the membrane was blocked again and immunoblotted with anti - erk1/2 antibody (cell signaling). t cells : hek293 t cells were plated in a 24-well plate and left overnight to reach 80 % confluency. the cells were then treated with pa (80 nm) and lv6 (500 nm) in serum - free medium for 12 h. for detection of cytosolic lv6, the cells were detached with trypsin, washed with pbs, and suspended in digitonin (100 g ml) for 10 min (as above). for detection of perk1/2, the cells were treated with egf (5 ng ml) for 7 min, washed with cold pbs, lysed in - plate with ip lysis buffer containing np-40 (1 %), protease inhibitor cocktail (roche), and phosphostop (roche). as a service to our authors and readers, this journal provides supporting information supplied by the authors. such materials are peer reviewed and may be re - organized for online delivery, but are not copy - edited or typeset. technical support issues arising from supporting information (other than missing files) should be addressed to the authors | antibody mimics have significant scientific and therapeutic utility for the disruption of protein protein interactions inside cells ; however, their delivery to the cell cytosol remains a major challenge. here we show that protective antigen (pa), a component of anthrax toxin, efficiently transports commonly used antibody mimics to the cytosol of mammalian cells when conjugated to the n - terminal domain of lf (lfn). in contrast, a cell - penetrating peptide (cpp) was not able to deliver any of these antibody mimics into the cell cytosol. the refolding and binding of a transported tandem monobody to bcr - abl (its protein target) in chronic myeloid leukemia cells were confirmed by co - immunoprecipitation. we also observed inhibition of bcr - abl kinase activity and induction of apoptosis caused by the monobody. in a separate case, we show disruption of key interactions in the mapk signaling pathway after pa - mediated delivery of an affibody binder that targets hraf-1. we show for the first time that pa can deliver bioactive antibody mimics to disrupt intracellular protein protein interactions. this technology adds a useful tool to expand the applications of these modern agents to the intracellular milieu. |
pericardial masses usually do not cause any symptoms, and come to medical attention as an incidental finding on chest imaging modalities. this is a report on a case of an unusual symptomatic pericardial mass - like lesion in a patient with an secundum atrial septal defect (asd). as a result of the compression of the right side of the heart, severe right to left shunting of blood developed through the asd. the patient presented with sub - acute hypoxia, exertional dyspnea and reactive erythrocytosis similar to those in patients with eisenmenger 's syndrome. a 46-year - old woman who had undergone peripheral stem cell transplantation for acute undifferentiated leukemia 1.5 years ago was admitted for operation of graft - versus - host disease of the eye. prior to the operation, markedly decreased sao2 and pao2 were noted and hence a comprehensive re - evaluation of her medical problems was performed. the patient 's echocardiogram before undergoing the peripheral stem cell transplantation had revealed a small secundum asd with left - to - right shunt accompanied with mild right atrial enlargement and mild tricuspid regurgitation (fig. the surgery for asd had been planned after completion of the treatment for leukemia. when the patient was admitted for the eye operation, she presented with severe hypoxia (sao2 79.8%, qp / qs 0.87) and subacute dyspnea on exertion. on physical examination, mild cyanosis of the lips and tachycardia were noted and no other specific findings could be found. the follow - up echocardiogram 1.5 years after the initial echocardiogram revealed a huge pericardial mass which was compressing the right atrium and ventricle almost completely (fig. using contrast echocardiography, the extra - cardiac mass and compression of the right side of the heart due to it could be clearly delineated (fig. no flow was seen between the mass and the heart or the vascular structures, and the direction of the shunt flow through the asd had changed from left - to - right to right - to - left due to increased intra - cardiac pressure in the right side of the heart as a result of the external compression (fig. chest ct and mri of the heart showed a loculated cystic mediastinal mass with hemorrhage measuring 5.58 cm compressing the right atrium and ventricle. axial t2 weighted mr image showed a dark fluid - fluid level within the dependent portion of the mass (fig. the cyst was firmly adherent to the anterior epicardium of the right side of the heart and was filled with brownish fluid resembling the fluid inside an old hematoma. after the removal of this cystic mass, there was normalization of the right ventricular size and no remnant shunt flow was seen on follow - up echocardiography. on follow - up arterial blood gas analysis, sao2 and pao2 were normalized and the patient did not have any signs and symptoms of congestive heart failure such as dyspnea or cyanosis. this is an unusual case of a rare complication of a pericardial cystic mass - which was initially thought to be a primary pericardial cyst or a thymic cyst that had developed following chemotherapy - where severe hypoxia was developed in a subject with asd. other less common causes include benign thymic, bronchogenic, enteric and thoracic duct cyst as well as malignant cysts of thyroid, parathyroid, lymphoma, thymomas, teratoma and seminoma.1)2) there has been a report on development of a benign thymic cyst in the anterior mediastinum following chemotherapy for non - hodgkin 's lymphoma.3) a rare cause of a pericardial cystic mass is an acute or chronic cystic hematoma as in the case presented here. pericar - dial cystic masses are usually asymptomatic and found incidentally on routine chest x - rays. the clinical presentation described includes chest pain, tachycardia, persistent cough, dyspnea, cardiac arrhythmia and lower respiratory tract infection.4 - 9) the symptoms can result from the pressure of the mass on the adjacent organs.11) the pericardial masses have also been associated with multiple complications including right ventricular outflow tract obstruction, pulmonary stenosis related to extrinsic compression, torsion of a vascular pedi - cle, subsequent development of ischemia - related lesion of the cyst, spontaneous internal hemorrhage and tamponade, partial erosion into the superior vena cava or the anterior wall of the right ventricle. these masses can lead to constrictive pericarditis and congestive heart failure, but it is unknown whether a particular size or position of the cyst corresponds with a higher rate of complications.5)6)11 - 13) in the present case, the pericardial cystic mass became large enough to compress the right side of the heart and cause a rare complication due to the reversal of the direction of the shunt flow through the asd. the direction and magnitude of the shunt through asds are determined by the size of the defect and the relative compliance of the ventricles. as a rule, left atrial blood is shunted to the right atrium in the early phase of the disease. most patients with asd will have impaired exercise tolerance and exertional dyspnea, but these symptoms may be well compensated for years. if the right ventricle fails or its compliance declines, the left - to - right shunting diminishes in magnitude and right - to - left shunting may occur.14) in the present case, the change in direction of the shunt flow developed due to increased pressure in the right side of the heart due to extra - cardiac compression as a result of increased size of the pericardial cystic mass. as a result, persistent hypoxia and cyanosis developed, which could be corrected by the resection of the cystic mass. pericardial organizing hematoma can be subject to internal hemorrhage and consequent growth in size as seen in the present case. as a result, our case presented with reversal in the direction of the shunt flow through the asd from left - to - right to right - to - left. but even in other cases without any accompanying abnormalities of the heart, the heart can be compressed due to external pressure resulting in a decrease in the cardiac output and deterioration of the patient 's health. hence, the knowledge of such likely complications, especially in cases of hematologic malignancies such as acute leukemia, is crucial for early detection and curative treatment. | we report a case of a 46-year - old woman who presented with subacute exertional dyspnea and severe hypoxia. a large cystic mass compressing the right side of the heart along with right - to - left atrial shunt flow through an alleged atrial septal defect (asd) were detected on echocardiography. ct scan of the chest and mri of the heart revealed a loculated cystic mediastinal mass with hemorrhage measuring 5.58 cm compressing the right atrium and ventricle. the patient underwent cyst resection and primary closure of the asd. this report illustrates a case of an unusual symptomatic pericardial mass compressing the right atrium and ventricle in a patient with an secundum asd. |
the internet has become an important tool for social interaction, information, and entertainment. however, as the internet has moved into homes, schools, internet cafes, and businesses, there has been a rapidly growing public awareness of the potential adverse effects arising from excessive, mal - adaptive or addictive internet usage which is a condition also known by terms such as problematic internet use (piu), internet addiction, internet dependence, and pathological internet use. particularly among adolescents, the internet is observed to be increasingly adopted as a readily accessible means for information retrieval, entertainment, and socialization. as adolescents allocate ever increasing time periods for internet use, the risk for developing mal - adaptive internet use (miu), including potential piu and piu, is inherent. beard and wolf defined piu as use of the internet that creates psychological, social, school, and/or work difficulties in a person 's life. the proposed criteria for piu initially included : (1) an uncontrollable use of the internet, (2) internet use which is markedly distressing, time consuming or resulting in social, occupational, or financial difficulties, and (3) internet use not solely present during hypomanic or manic clinical episodes. hence, piu is conceptualized as an individual 's inability to control his / her use of the internet, thus causing marked distress and/or functional impairment. worldwide, the prevalence of piu among adolescents and young adults has been observed to range between 0.9% and 38%. the prevalence has been reported to be between 1% and 9%, in the middle east the prevalence is between 1% and 12%, and in asia the prevalence has been reported to be between 2% and 18%. as one of the common mental health problems among chinese adolescents, piu is currently becoming more and more serious. recently, numerous studies had shown indulging in the use of the internet is associated with a variety of problems. high - risk internet users have inappropriate dietary behavior and poor diet quality, which could result in stunted growth and development. piu was also associated with other potential addictive personal habits of smoking, drinking alcohol or coffee, and taking drugs. internet use in adolescents was associated with more severe psychiatric symptoms, and interpersonal problems. the primary objective of the present study is to assess the prevalence of piu among high school students in el - minia governorate. the secondary objective is to investigate the potential risk factors for piu among high school students in el - minia governorate. the primary objective of the present study is to assess the prevalence of piu among high school students in el - minia governorate. the secondary objective is to investigate the potential risk factors for piu among high school students in el - minia governorate. this study was conducted during january - march 2012, at el - minia governorate. this governorate is one of the upper egypt governorates and is 240 km to the south of cairo. it is a cross - sectional descriptive study to assess the prevalence and determinants of piu among adolescent students at different high schools at el - minia governorate. in el - minia governorate four schools were randomly selected to cover the whole sample size (two boys schools and two girls schools). the sample size was 574 calculated using epi info 2000 entering average estimates of piu 3% based on a pilot study that was carried out on 50 high school students who were not included in the main study and the total number of high school students as 12,283 and the confidence level at 99.99%. to regard against non responsiveness, 620 students were contacted and of these, 605 agreed to participate in the study. our questionnaire was filled in within a 20 - 30 min session in the classroom in the presence of the teachers to minimize any potential information bias. the questionnaire started with demographic data about each participant, followed by family, dietary and health - related data. the young 's internet addiction test (yiat) was applied in order to assess piu. the yiat consists of 20 items for the evaluation of the degree of preoccupation, compulsive use, behavioral problems, emotional changes, and diminished functionality associated with internet use. each item is scored from 1 to 5, with 1 representing not at all and 5 representing always. hence, possible total scores range from 20 to 100. the following cut - off points were applied to the total yiat score (1) normal internet use : scores 20 - 49 ; (2) potential piu : scores 50 - 79 ; (3) pius : scores 80 - 100. official permissions were obtained from relevant authorities to proceed with the study. prior to embarking on the study, ethical approval was obtained from the scientific research ethics committee of el - minia university, faculty of medicine. official permission was obtained from the administration of secondary education and from the manager of each school before data collection. the purpose of the study was explained to all the participants and was ensured strict confidentiality and anonymity before proceeding in the interview. all data were analyzed by using the statistical package for social sciences (spss-16) software. chi - square test(x), fisher 's exact test and one - way analysis of variance (anova) were used whenever, applicable. multinomial logistic regression analyses were also applied to calculate the odds ratios (or) and 95% ci of the determinants of internet addiction among students. this study was conducted during january - march 2012, at el - minia governorate. this governorate is one of the upper egypt governorates and is 240 km to the south of cairo. it is a cross - sectional descriptive study to assess the prevalence and determinants of piu among adolescent students at different high schools at el - minia governorate. in el - minia governorate, there are 85 different high schools. from these schools, four schools were randomly selected to cover the whole sample size (two boys schools and two girls schools). the sample size was 574 calculated using epi info 2000 entering average estimates of piu 3% based on a pilot study that was carried out on 50 high school students who were not included in the main study and the total number of high school students as 12,283 and the confidence level at 99.99%. to regard against non responsiveness, 620 students were contacted and of these, 605 agreed to participate in the study. our questionnaire was filled in within a 20 - 30 min session in the classroom in the presence of the teachers to minimize any potential information bias. the questionnaire started with demographic data about each participant, followed by family, dietary and health - related data. the young 's internet addiction test (yiat) was applied in order to assess piu. the yiat consists of 20 items for the evaluation of the degree of preoccupation, compulsive use, behavioral problems, emotional changes, and diminished functionality associated with internet use. each item is scored from 1 to 5, with 1 representing not at all and 5 representing always. the following cut - off points were applied to the total yiat score (1) normal internet use : scores 20 - 49 ; (2) potential piu : scores 50 - 79 ; (3) pius : scores 80 - 100. official permissions were obtained from relevant authorities to proceed with the study. prior to embarking on the study, ethical approval was obtained from the scientific research ethics committee of el - minia university, faculty of medicine. official permission was obtained from the administration of secondary education and from the manager of each school before data collection. the purpose of the study was explained to all the participants and was ensured strict confidentiality and anonymity before proceeding in the interview. all data were analyzed by using the statistical package for social sciences (spss-16) software. chi - square test(x), fisher 's exact test and one - way analysis of variance (anova) were used whenever, applicable. multinomial logistic regression analyses were also applied to calculate the odds ratios (or) and 95% ci of the determinants of internet addiction among students. among the study population (n = 605), there were 396 (65.5%) male students and 209 (34.5%) female students. the mean age standard deviation (sd) of adolescents with piu did not significantly differ from that of their normal internet user counterparts (16.9 0.3 years vs. 16.49 0.8 years, f = 2.4, p = 0.09). approximately 2.6% (16) were identified as pius and males comprised 87.5% among them, while 110 (18.2%) were identified as potential pius and most of them were males (70%). mostly of pius their fathers have professional work (93.7%) and their mothers were housewives (68.7%). beginning age of internet use was earlier among piu students than normal internet users (12.2 1.9 vs. 13.25 1.9, f = 3.5, p = 0.03). with respect to the locations of internet access, most of the participants owned and frequently used computers in their homes and adolescents with piu were significantly more likely to access the internet via their own home portal as compared to normal internet users [table 1 ]. socio - demographic characteristics based on level of internet addiction among the students as shown in table 2, piu was significantly associated with a series of variables : low social friends (62.8% vs. 19.8%, x = 40.6, p = 0.001), bad family relations (43.8% vs. 20.3%, x = 5.2, p = 0.07), irregular bedtime (62.5% vs. 2.5%, fisher 's exact test = 189, p = 0.0001), and bad personal hygiene (50% vs. 16.7%, x = 26.7, p = 0.0001). moreover, the proportion of adolescents with piu reporting excellent academic performance was lower than that among normal internet users (6.5% vs. 20.9%, x = 16.2, p = 0.03). lifestyle patterns based on the level of internet addiction among the students most of pius answered that their dietary habits had been changed to have small meal sizes, a poor appetite, and faster eating speeds than normal internet users (x = 43.4, p = 0.001, x = 32.6, p = 0.001, and x = 13.01, p = 0.01, respectively). pius had a higher percentage of skipping breakfast (62.5% vs. 33.4%, x = 6.6, p = 0.03) as shown in table 3. dietary habits based on the level of internet addiction among the students table 4 showed the percentage of some physical and emotional symptoms among adolescents with piu, potential piu, and normal internet use. compared with normal internet use, adolescents with piu were more likely to suffer from physical symptoms ; weight gain (31.2% vs. 15.9%, x = 8.5, p = 0.01), joint stiffness (12.5% vs. 2.9%, x = 6.3, p = 0.04), lack of physical energy (43.7% vs. 24.6%, x = 14.9, p = 0.001), back pain (62.5% vs. 39.5%, x = 5.7, p = 0.05), eye strain (62.5% vs. 34.03%, x = 18.6, p = 0.0001), and emotional symptoms ; feeling sad (25% vs. 5.6%, x = 22.1, p = 0.001), feeling excited (68.7% vs. 12.1%, x = 85.1, p = 0.001), euphoric (18.7% vs. 5.4%, x = 17.7, p = 0.001), and anxious (6.25% vs. 8.03%, x = 9.17, p = 0.01). physical and emotional health problems caused by internet use among students determinants of potential piu and piu : the multinomial logistic regression analysis [table 5 ] indicated that father 's professional work, poor family relation, male gender, and limited social friends were independently associated with potential piu and piu. the internet is extremely an important social and communications tool, and is changing our daily lives at home and at work. there is no an epidemiological study on piu neither in general nor in adolescence in egypt. in light of these findings, this study was conducted in order to assess the prevalence of piu among high school students and to determine the personal, clinical, family, and social characteristics of piu among adolescents. three types of internet users were identified in this study : normal, potential pius, and pius. the prevalence of piu among adolescents was 2.6%, which is closely related to that reported by some other studies of internet use among students worldwide ; such as piu was 1% in greece, 4% in south korea, 3.1% in finland, 4.2% in lebanon, and 4.6% in australia. its lower percentage may be attributed to the limited access of computer / internet access among urban egyptian youth. however, marked international variances regarding the prevalence rates of piu may also be attributed to a measurement bias incurred by a lack of international consistency regarding both the definition and assessment of piu and to different samples and social contexts. furthermore, among the study population examined about 18.2% of adolescents were identified with potential piu which is slightly lower than what found in another study ; that approximately one - fifth (19.4%) of adolescents were identified with potential piu. in contrast, another study contended that females were more prone to piu than males. however, one study found no gender differences in relation to internet addiction (ia). this study supports the general literature that males tend to be more subject to piu and the explanation for this may be that males are more likely to play online games, engage in cybersex, and gamble online. according to korean scientists, the causes of piu do not have only habitual bases, but also demographic and socioeconomic. the present study partly confirmed this by the finding that parents work and students who have higher number of siblings were more in danger of piu. this study shows that piu adolescents at were much more likely to have interpersonal problems, as piu was significantly higher among adolescents had poor social friends and family relations. researchers state that widespread use of the internet among the adolescents makes them feel alone, causes problematic behaviors, and leads to poor family and friends relationships. high parent - adolescent conflict predicted piu in adolescents ; as adolescents with a higher conflict level with their parents refused to obey the supervision of their parents, including the rules set for internet use. high - risk internet users reported more irregular sleep patterns and more episodes of sleep disturbance than no risk internet users. this is consistent with a previous study of korean adolescents that showed that piu was associated with insomnia, apnea, and nightmare. late night use of the internet can cause sleep deprivation and fatigue, which can adversely affect academic performance and can result in reversed sleep pattern and poor academic performance. in this study this finding seems reasonable since piu stay up late at night and may get up too late for breakfast. the high frequency of snaking could be related to skipping meals, more frequent snacking was observed in pius than normal internet users. moreover, the favorite snacks of our participants were fast food, which are nutritionally poor foods with high calories provided by fats and simple sugars but with few other nutrients such as vitamins and minerals. this is matched with a study found that piu had the lowest meal regularity, reflected by a higher rate of snaking than in normal internet users. children and adolescents carry a much higher risk for negative effects of piu than adults due to incomplete developmental processes. our study found adolescents with piu were more likely to suffer from physical symptoms, such as lack of physical energy (students may look overly tired or sleep in class because of all - night internet sessions), change of sleep pattern, back strain, and eye strain from the long periods of sedentary computer use. pius may be depressed, withdrawn, or anxious as a result of both the physical and psychological toll of the piu. frequently and increasingly pius cut themselves off from their family, friends, and social activities and choose to spend most of their time alone. this study shows that father 's professional work, and poor family relation were the most contributing risk factor of piu, these results may suggest a relationship between a poor social support system and piu. first, this study was a cross - sectional study, therefore, we could not confirm causal associations between piu and its consequences. second, the questionnaire was self - reported and is subject to recall or report bias. third, since the survey was administered during class time, it is possible that some students, especially those had piu, were absent from class when the questionnaire was administered. therefore, the survey may have under - represented piu by failing to record the responses of those who are so consumed by the internet that they rarely leave their rooms, thus leading to an underestimation of the prevalence of piu. future studies should attempt to determine implementation of preventive measures, and the development of treatment approaches for piu. adolescents with piu had an augmented likelihood to report poor social and familial relationships, and enhanced risk for some physical and emotional health problems. school counselors and teachers also need to be aware of the prevalence and the problematic behaviors associated with excessive internet use for early prevention. it is also necessary to make the youth and their parents aware of the dangers of piu and pay attention to consequences connected with it. school counselors and teachers also need to be aware of the prevalence and the problematic behaviors associated with excessive internet use for early prevention. it is also necessary to make the youth and their parents aware of the dangers of piu and pay attention to consequences connected with it. | background : problematic internet use (piu) is a growing problem in egyptian adolescents. this study was designed to assess the prevalence of piu among high school students in el - minia governorate and to determine the personal, clinical, and social characteristics of them.methods:a cross - sectional study was applied among a random sample of high school students in el - minia governorate. piu was assessed by the 20-item young internet addiction test (yiat). information was also collected on demographics, dietary, and health - related factors. statistical analysis used : statistical package for social sciences (spss-16) software was used. chi - square test (x2), fisher 's exact test, and one - way analysis of variance (anova) were used whenever, applicable. multinomial logistic regression analyses were also applied in order to calculate the odds ratios (or).results : of the 605 students, 16 (2.6%) were problematic internet users (pius), 110 (18.2%) were potential (pius). adolescents with piu were associated with male gender, poor friends relations, bad family relations, irregular bedtime, and bad personal hygiene. pius were more likely to suffer from physical symptoms ; weight gain, joint stiffness, lack of physical energy, and emotional symptoms.conclusions:the prevalence of piu reported in this study is low, however, the potential pius was high and preventative measures are recommended. |
narcissistic personality disorder (npd) has its roots in nearly a century of psychoanalytic studies. kernberg 's and kohut 's groundbreaking efforts to organize psychoanalytic theory and clinical studies into comprehensive descriptions and treatment strategies moved npd towards recognition as a separate personality disorder. in the diagnostic and statistical manual of mental disorders (dsm)-iv, npd has been characterized as a pervasive pattern of grandiosity, need for admiration, and lack of empathy, with interpersonal entitlement, exploitativeness, arrogance, and envy. other notable phenotypic characteristics include interpersonal distancing and avoidance, insecurity and vulnerability, hypersensitivity, aggressivity, and proneness to shame. the transformation of npd into a dsm diagnostic category in 1980 required significant adjustments and narrowing of extensive clinical observations. several components and characteristics of narcissistic personalitypathology that were central in the psychoanalytic conceptualization of narcissism and npd were left aside in the final choice and formulation of the diagnostic trait criteria. one such characteristic relates to the process and feeling of fear, frequently acknowledged in psychoanalytic studies as a significant part of narcissistic pathology. he also observed the shocking reaction when individuals face the discrepancy between an endorsed or ideal view of the self and a drastically contrasting realization. rothstein associated such fear of falling short of ideals with the loss of perfection and accompanying humiliation, an important aspect of narcissistic personality functioning. fiscalini emphasized fear of autonomy in narcissistic interpersonal relations, and kohut pointed to fear associated with rejection, isolation, and loss of contact with reality, and loss of admiration, equilibrium, and important objects. recently, horowitz highlighted fear in the context of wishes and defenses, and kernberg has referred to the unfolding of underlying fear in treatment of people with npd, including fear of dependency and destroying the relationship with the analyst, fear of retaliation, of one 's own aggression and destructiveness, and fear of death. maldonado identified the narcissistic intrapsychic trauma caused by the loss of a bond with a good object associated with ideals and meaning. such a trauma threatens the individual 's sense of continuity, coherence, stability, and wellbeing. in the delicate balance between repairing such traumas and working through conflicts, reactivations of fear an additional limitation in dsm is the absence of diagnostically specified levels of personality functioning. consequently, pathological narcissism and npd often co - occur with consistent or intermittent areas and periods of high functioning, including areas or periods of real competence and qualities, as well as cognitive, emotional, and interpersonal capabilities, and social skills. in clinical and social psychological reports, identification of narcissistic character pathology takes into consideration the functional aspects of shifts between selfenhancement and self -deflation, with intermittent periods and areas of competent functioning. these dimensions, which capture the individual 's intentions, choices and strivings, purpose and goals, causal influence, and prediction and problem - solving skills, are especially useful for defining narcissistic self- and self - esteem regulation. decision - making, a central component in self -regulatory and self - directing efforts, has gained attention in psychoanalytic studies, and recently also in social psychological studies of narcissism. in order to advance our understanding of the different components operating in pathological narcissism and npd it is necessary to further connect and integrate the psychoanalytic and clinical, as well as the social psychological, conceptualization of the disorder. one unifying approach may be to examine the neural underpinnings in narcissism as a way to refine its phenotype. research on empathy and empathic functioning has alreadyproven such a link to be most constructive and informative for npd, contributing to a significant change in identifying empathy, not as absent or present, but as a multifactorial and fluctuating capability. this research has also influenced the discussion of the dsm-5 personality disorder section, suggesting that empathy is an ability with inconsistencies and impairments, multiple components, a functional range, and a regulatory role. the aim of this paper is to further identify possible links between the psychoanalytic perspective on pathological narcissism and npd, and neuroscientific research on narcissism and related pathologies. in this review, we will focus primarily on fear, as it has been considered a central and even a motivating factor in narcissistic personality functioning in psychoanalytic and clinical studies. further, we will explore the impact that fear may have on decision - making. understanding the processes and neurological underpinnings of fear and decision - making can potentially influence both the diagnosis and treatment of npd. fear is generally considered to be an emotional state, a psychological and psychophysiological response to perceived or anticipated threats or danger. fear can often serve as an adaptive alert and survival mechanism. as such, it represents an ability to recognize danger and an urge to either confront or to avoid or escape, but fear can also in extreme situations cause paralysis and inability to protect oneself. fear differs from anxiety as it is a response to real threats, a frightening object, event, or experience, while anxiety is considered an anticipatory warning signal, related to the expectation of unreal or imagined danger, including intrapsychic, unconscious conflicts and erotic feelings. from a psychoanalytic perspective, fear can be triggered by concrete external events as well as by internal subjective or emotional experiences. fear of not measuring up and falling short can be triggered in specific situations, ie, in the context of evaluation, performance, or exposure. such fear differs from the more complex or ambiguous fear that in the same way can threaten self - esteem, ie, fear of being overwhelmed, and facing success or relationships and intimacy, feelings of shame or guilt, and experiencing loss of control. the subjective meaning ascribed to the experience of external life events, such as changes, gains and losses, challenges, or discouragements, can evoke sudden unexpected fear. intense overwhelming affect, independently of whether the cause is external or internal, can also in itself be terrifying as it may challenge the individual 's sense of internal control. fear can also become maladaptive or pathological, as such feelings, generated from an initial fear - provoking event, persist and have a negative effect on day - to - day behavior. fear of dark and negative self - experiences or of intolerable aspects of identity, in particular, can drive protective self - aggrandizement as well as destructive suicidal behavior enforced by overwhelming feelings of despair. experiences in the present are linked to disorganized and fragmented memories of earlier mortifying or traumatic experiences. sensory and emotional experiences associated with such early trauma also contribute to the subjective perception and interpretation of a present event as traumatic, ie, retraumatizing. a number of social psychological and personalityfocused studies related to narcissistic functioning further indicate that fear and fear avoidance, especially of failure, are important motivating factors, a self -regulatory strategy driven by specific achievement motives, namely, fear of failure similarly, fear of failure and accompanying shame can motivate procrastination or avoidance of commitment and performance. on the other hand, defensive behavior in response to exposure to failure and accompanying fear of failure is considered to be deeply ingrained, with automatic efforts to avoid failure. in general, these studies indicate that people who are afraid of failing can be motivated or even susceptible to either invest greater efforts in a task after being exposed to failure information, or to completely avoid such efforts. fear related to self - esteem regulation and risk of falling short can underlie and motivate a range of behavior in narcissistic personality disorder. high achievements can be motivated by fear of incompetence and failure ; selfenhancement by fear of worthlessness and inferiority ; perfectionism by fear of shame and self - criticism ; pursuit of special affiliations by fear of losing status or influence ; interpersonal ignorance and distancing by fear of humiliation, or being overpowered and lose control ; and avoidance by fear of shame and exposure. these studies and observations raise several questions about the interaction between identifying, processing, and controlling fear from the perspective of narcissistic self - regulation. so far, studies have shown that people with high narcissism but not meeting criteria for npd present with higher degree of alexithymia, ie, difficulties assessing own and other 's emotions. however, it is possible that by exploring neuroscientific evidence in narcissism and related pathologies, researchers and clinicians may begin to clarify and differentiate specific fear susceptibility and fear processing in people with npd. over the last several decades there has been significant growth in the understanding of the neurobiological basis of fear. at the center of the fear circuitry additional regions (eg, nucleus accumbens, hippocampus, some prefrontal regions, etc) form a neural network involved in the perception of threat, fear learning, and fear expression. these areas individually mediate symptoms of fear and collectively act to produce an integrated fear response. our nuanced understanding of this complex neural network results from imaging (eg, during fear conditioning studies), physiological (eg, skin conductance, eye - blink response), and psychopharmacological studies that not only enhance the mechanistic understanding of fear but also highlight the role of fearrelated dysfunction in the generation and maintenance of various forms of psychopathology. failure to properly regulate fear responses is central to specific phobia, post - traumatic stress disorder, generalized anxiety, and some axis ii disorders (ie, fear of separation and loss of support in dependent personality disorder (dpd) of abandonment in borderline personality disorder (bpd), and of criticism, disapproval, and rejection in avoidant personality disorder (apd). while some disorders are largely associated with hyperviglance and an over - reactive fear response (eg, anxiety disorders and bpd), others are related to deficient fear reactivity (eg, psychopathy). studies on the relationship between fear and narcissism have been sparse, both at a phenotypic and mechanism level. one study of individuals with narcissistic traits, as measured by the narcissism personality inventory (npi) reported that they display diminished electrodermal reactivity to aversive stimuli, indicating weak responses to punishment or aversive cues. despite the limited research directly examining fear and narcissism, there are studies of other related conditions with relevance to pathological narcissism that highlight the importance of fear in the expression of psychopathology. specifically, the role of fear in psychopathy - related disinhibition has been the focus of studies for decades. npd and psychopathy are considered to be overlapping constructs, both expressing symptoms of grandiosity, compromised empathic functioning, and callousness. psychopathic individuals generally display an inability to form genuine relationships ; limited (ie, grandiose) affective processing, especially with respect to anticipatory anxiety and remorse ; an impulsive behavioral style involving a general failure to evaluate anticipated actions and inhibit the inappropriate ones ; and a chronic antisocial lifestyle that entails great costs to society as well as for the affected individual. while both affective and behavioral characteristics are important elements of psychopathy, the affective deficits have traditionally been considered to be the root cause of the psychopath 's problems. affective deficits in psychopathy have most often been understood in the context of the low - fear model. consistent with this model, psychopaths display poor fear conditioning, minimal electrodermal response in anticipation of aversive events, and a lack of startle potentiation while viewing unpleasant versus neutral pictures. however, other studies examining startle potentiation (eg, fear - potentiated startle and emotionmodulated startle) demonstrate that the psychopathy - related fear deficit is not absolute, but rather conditional depending on contextual variables. neuroimaging evidence suggests that psychopaths display reduced amygdala activation than controls during aversive conditioning, moral decision - making, social cooperation, and reduced memory for emotionally salient words. other research indicates that the amygdala is hyper - reactive when psychopaths view certain emotionally salient scenes. thus, existing research does not indicate the presence of a reliable fear deficit in psychopathic individuals, though such deficits may be revealed under specific circumstances. one explanation for the inconsistent nature of psychopathy - related fear deficits may involve an abnormality in attentional processes. developments in neuroscience indicate that the function of the amygdala is more complex than just fear processing, and likely plays a significant role in attention and in detecting relevance. with regard to psychopathy, according to the response modulation hypothesis, attention plays a crucial role in moderating fear and self - regulatory deficits. response modulation involves the temporary suspension of a dominant response set and a brief concurrent shift of attention from the organization and implementation of goal - directed responding to its evaluation an individual is prone to ignore crucial contextual information needed to evaluate his or her behavior and exercise adaptive self - regulation. consequently, psychopaths are oblivious to potentially meaningful peripheral information because they fail to reallocate attention while engaged in goal - directed behavior. this difficulty balancing demands to process goal - directed and peripheral information creates a bias whereby psychopaths are unresponsive to information unless it is a central aspect of their goal - directed focus of attention. an important implication of the response modulation hypothesis is that the emotion deficit of psychopathic individuals varies as a function of attentional focus. a recent experiment by newman involving fearpotentiated startle (fps) provides striking support for this hypothesis. of note, existing evidence suggests that fps is generated via the amygdala. the task used in this study required participants to view and categorize letter stimuli that could also be used to predict the administration of electric shocks. instructions engaged either a goal - directed focus on threat - relevant information (ie, the color that predicted electric shocks) or an alternative, threat - irrelevant dimension of the letter stimuli (ie, upper / lower case of the letter or its match / mismatch in a 2-back task). the results provided no evidence of a psychopathy - related deficit in fps under conditions that focused attention on the threat - relevant dimension. however, psychopathy scores were significantly and inversely related to fps under conditions that required participants to focus on a threat - irrelevant dimension of stimuli (ie, when threat cues were peripheral). in a follow - up study, baskin - sommers and colleagues specified this attentional - mediated abnormality in a new sample of offenders by measuring fps in four conditions that crossed attentional focus (threat versus alternative focus) with early versus late presentation of goal - relevant cues. first, the authors replicated the key findings reported by newman : that psychopaths ' deficit in fps was virtually nonexistent under conditions that focused attention on the threat - relevant dimension of the experimental stimuli (ie, threat - focus conditions), but was pronounced when threat - relevant cues were peripheral to their primary focus of attention (ie, alternative - focus conditions). more specifically, the psychopathic deficit in fps was only apparent in the early alternative focus condition, in which threat cues were presented after the alternative goal - directed focus was already established. these results confirm the idea that attention moderates the fearlessness of psychopathic individuals and, moreover, implicate an early attention bottleneck as a proximal mechanism for deficient response modulation in psychopathy (see ref 71 for discussion of the bottleneck). additionally, larson and colleagues (unpublished data) recently completed an imaging study using this paradigm with an independent sample of inmates. results indicated that decreased amygdala activation in psychopathic offenders occurred only during the early alternative focus condition. under this condition, psychopaths also exhibited greater activation in selective attention regions of the lateral prefrontal cortex (lpfc) than nonpsychopaths, and this increased lpfc activation was associated with decreased amygdala activation. in contrast, when explicitly attending to threat, amygdala activation in psychopaths did not differ from nonpsychopaths. this pattern of amygdala activation closely parallels results for fps and, moreover, highlights the potential role of lpfc in mediating the failure of psychopathic individuals to process emotion and other important information when it is peripheral to the primary focus of goal - directed attention. overall, it appears that psychopathic individuals do ignore fear - related information, but only in the service of focusing on a specific goal. for example, such an inflexible focus on personal goals may underlie the selfcentered, callous traits associated with psychopathy and may leave psychopathic individuals oblivious to the potentially devastating consequences of their behavior. while one relationship between fear and psychopathology is related to deficient fear processing, another relationship between fear and psychopathology is related to over - reactivity to fear. specifically, research on other forms of externalizing psychopathology, like borderline personality disorder, report increased fps during instructed fear conditioning and increased amygdala activity while viewing emotional slides. similarly, studies of trait externalizing demonstrated significant increases in fps, amygdala, and emotion - related prefrontal cortex activity during fear conditioning. thus, these individuals appear unable to regulate their reaction to fear, essentially becoming consumed by its presence, ultimately resulting in a cascade of emotion - driven disinhibited behavior. although this neuroscientific overview applies to near neighbor psychopathologies, several findings introduce possible links to fear processing in pathological narcissism and npd. similar to people with psychopathy, focused attention on goals, such as ambitions, competition, and aspirations, and even on risk - taking efforts, may, for some people with pathological narcissism and npd, enable ignorance of fear and serve as a fear modulator. the narcissistic individual 's awareness is then directed away from potential triggers of feelings of fear and towards more securing or rewarding self - enhancing experiences. on the other hand, given the psychoanalytic observations of profound fear in npd, and the recognition of the thin - skinned and vulnerable narcissistic personality types, the question is whether some people with pathological narcissism and npd indeed are hypersensitive or over - reactive to fear, or can have impaired capability to tolerate and/or process feelings of fear. it is also possible that when people with pathological narcissism or npd have to face fear without the possibilities of engaging in avoiding, goal - directed, or self - enhancing strategies, the experience becomes overwhelming and consuming, forcing drastic decisions with seemingly immediate short - term gains. one avenue for understanding the role of fear in narcissism is to examine its impact on functionality, in processes such as decision - making. over the last several decades there has been significant growth in the understanding of the neurobiological basis of fear. at the center of the fear circuitry additional regions (eg, nucleus accumbens, hippocampus, some prefrontal regions, etc) form a neural network involved in the perception of threat, fear learning, and fear expression. these areas individually mediate symptoms of fear and collectively act to produce an integrated fear response. our nuanced understanding of this complex neural network results from imaging (eg, during fear conditioning studies), physiological (eg, skin conductance, eye - blink response), and psychopharmacological studies that not only enhance the mechanistic understanding of fear but also highlight the role of fearrelated dysfunction in the generation and maintenance of various forms of psychopathology. failure to properly regulate fear responses is central to specific phobia, post - traumatic stress disorder, generalized anxiety, and some axis ii disorders (ie, fear of separation and loss of support in dependent personality disorder (dpd) of abandonment in borderline personality disorder (bpd), and of criticism, disapproval, and rejection in avoidant personality disorder (apd). while some disorders are largely associated with hyperviglance and an over - reactive fear response (eg, anxiety disorders and bpd), others are related to deficient fear reactivity (eg, psychopathy). studies on the relationship between fear and narcissism have been sparse, both at a phenotypic and mechanism level. one study of individuals with narcissistic traits, as measured by the narcissism personality inventory (npi) reported that they display diminished electrodermal reactivity to aversive stimuli, indicating weak responses to punishment or aversive cues. despite the limited research directly examining fear and narcissism, there are studies of other related conditions with relevance to pathological narcissism that highlight the importance of fear in the expression of psychopathology. specifically, the role of fear in psychopathy - related disinhibition has been the focus of studies for decades. npd and psychopathy are considered to be overlapping constructs, both expressing symptoms of grandiosity, compromised empathic functioning, and callousness. psychopathic individuals generally display an inability to form genuine relationships ; limited (ie, grandiose) affective processing, especially with respect to anticipatory anxiety and remorse ; an impulsive behavioral style involving a general failure to evaluate anticipated actions and inhibit the inappropriate ones ; and a chronic antisocial lifestyle that entails great costs to society as well as for the affected individual. while both affective and behavioral characteristics are important elements of psychopathy, the affective deficits have traditionally been considered to be the root cause of the psychopath 's problems. affective deficits in psychopathy have most often been understood in the context of the low - fear model. consistent with this model, psychopaths display poor fear conditioning, minimal electrodermal response in anticipation of aversive events, and a lack of startle potentiation while viewing unpleasant versus neutral pictures. however, other studies examining startle potentiation (eg, fear - potentiated startle and emotionmodulated startle) demonstrate that the psychopathy - related fear deficit is not absolute, but rather conditional depending on contextual variables. neuroimaging evidence suggests that psychopaths display reduced amygdala activation than controls during aversive conditioning, moral decision - making, social cooperation, and reduced memory for emotionally salient words.. other research indicates that the amygdala is hyper - reactive when psychopaths view certain emotionally salient scenes. thus, existing research does not indicate the presence of a reliable fear deficit in psychopathic individuals, though such deficits may be revealed under specific circumstances. one explanation for the inconsistent nature of psychopathy - related fear deficits may involve an abnormality in attentional processes. developments in neuroscience indicate that the function of the amygdala is more complex than just fear processing, and likely plays a significant role in attention and in detecting relevance. with regard to psychopathy, according to the response modulation hypothesis, attention plays a crucial role in moderating fear and self - regulatory deficits. response modulation involves the temporary suspension of a dominant response set and a brief concurrent shift of attention from the organization and implementation of goal - directed responding to its evaluation (p 717). in the absence of normal response modulation, an individual is prone to ignore crucial contextual information needed to evaluate his or her behavior and exercise adaptive self - regulation. consequently, psychopaths are oblivious to potentially meaningful peripheral information because they fail to reallocate attention while engaged in goal - directed behavior. this difficulty balancing demands to process goal - directed and peripheral information creates a bias whereby psychopaths are unresponsive to information unless it is a central aspect of their goal - directed focus of attention. an important implication of the response modulation hypothesis is that the emotion deficit of psychopathic individuals varies as a function of attentional focus. a recent experiment by newman involving fearpotentiated startle (fps) provides striking support for this hypothesis. of note, existing evidence suggests that fps is generated via the amygdala. the task used in this study required participants to view and categorize letter stimuli that could also be used to predict the administration of electric shocks. instructions engaged either a goal - directed focus on threat - relevant information (ie, the color that predicted electric shocks) or an alternative, threat - irrelevant dimension of the letter stimuli (ie, upper / lower case of the letter or its match / mismatch in a 2-back task). the results provided no evidence of a psychopathy - related deficit in fps under conditions that focused attention on the threat - relevant dimension. however, psychopathy scores were significantly and inversely related to fps under conditions that required participants to focus on a threat - irrelevant dimension of stimuli (ie, when threat cues were peripheral). in a follow - up study, baskin - sommers and colleagues specified this attentional - mediated abnormality in a new sample of offenders by measuring fps in four conditions that crossed attentional focus (threat versus alternative focus) with early versus late presentation of goal - relevant cues. first, the authors replicated the key findings reported by newman : that psychopaths ' deficit in fps was virtually nonexistent under conditions that focused attention on the threat - relevant dimension of the experimental stimuli (ie, threat - focus conditions), but was pronounced when threat - relevant cues were peripheral to their primary focus of attention (ie, alternative - focus conditions). more specifically, the psychopathic deficit in fps was only apparent in the early alternative focus condition, in which threat cues were presented after the alternative goal - directed focus was already established. these results confirm the idea that attention moderates the fearlessness of psychopathic individuals and, moreover, implicate an early attention bottleneck as a proximal mechanism for deficient response modulation in psychopathy (see ref 71 for discussion of the bottleneck). additionally, larson and colleagues (unpublished data) recently completed an imaging study using this paradigm with an independent sample of inmates. results indicated that decreased amygdala activation in psychopathic offenders occurred only during the early alternative focus condition. under this condition, psychopaths also exhibited greater activation in selective attention regions of the lateral prefrontal cortex (lpfc) than nonpsychopaths, and this increased lpfc activation was associated with decreased amygdala activation. in contrast, when explicitly attending to threat, amygdala activation in psychopaths did not differ from nonpsychopaths. this pattern of amygdala activation closely parallels results for fps and, moreover, highlights the potential role of lpfc in mediating the failure of psychopathic individuals to process emotion and other important information when it is peripheral to the primary focus of goal - directed attention. overall, it appears that psychopathic individuals do ignore fear - related information, but only in the service of focusing on a specific goal. for example, such an inflexible focus on personal goals may underlie the selfcentered, callous traits associated with psychopathy and may leave psychopathic individuals oblivious to the potentially devastating consequences of their behavior. while one relationship between fear and psychopathology is related to deficient fear processing, another relationship between fear and psychopathology is related to over - reactivity to fear. specifically, research on other forms of externalizing psychopathology, like borderline personality disorder, report increased fps during instructed fear conditioning and increased amygdala activity while viewing emotional slides. similarly, studies of trait externalizing demonstrated significant increases in fps, amygdala, and emotion - related prefrontal cortex activity during fear conditioning. thus, these individuals appear unable to regulate their reaction to fear, essentially becoming consumed by its presence, ultimately resulting in a cascade of emotion - driven disinhibited behavior. although this neuroscientific overview applies to near neighbor psychopathologies, several findings introduce possible links to fear processing in pathological narcissism and npd. similar to people with psychopathy, focused attention on goals, such as ambitions, competition, and aspirations, and even on risk - taking efforts, may, for some people with pathological narcissism and npd, enable ignorance of fear and serve as a fear modulator. the narcissistic individual 's awareness is then directed away from potential triggers of feelings of fear and towards more securing or rewarding self - enhancing experiences. on the other hand, given the psychoanalytic observations of profound fear in npd, and the recognition of the thin - skinned and vulnerable narcissistic personality types, the question is whether some people with pathological narcissism and npd indeed are hypersensitive or over - reactive to fear, or can have impaired capability to tolerate and/or process feelings of fear. it is also possible that when people with pathological narcissism or npd have to face fear without the possibilities of engaging in avoiding, goal - directed, or self - enhancing strategies, the experience becomes overwhelming and consuming, forcing drastic decisions with seemingly immediate short - term gains. one avenue for understanding the role of fear in narcissism is to examine its impact on functionality, in processes such as decision - making. psychoanalytic studies have primarily attended to the intrapsychic aspects of decision making. identified as a secondary ego process linked between motivation and action, the unconscious courses involved in decision - making have nevertheless been a prime focus of interest. used to institute defenses and reach resolutions or compromises, decision - making is also influenced by physiological factors, and part of symptom formation and psychopathology. fear can influence decision - making by engulfing either an individual 's sense of agency or sense of identity, or both. the former can affect competence, while the latter can cause self - confusion and uncertainty about who one really is. although not currently acknowledged as a diagnostic or clinical indicator of npd, nevertheless, remarkable lapses in some narcissistic individuals ' decisions can force them into unbearable situations and life crises that call for urgent need of intensive treatment. in clinical settings, therapists can face a paradoxical discrepancy between such patients ' consistent self - control and proactive competence, and their sudden disparate decision strategies that seem ruled by immediate short - term gain and misjudgment, or by ignorance of salient negative or even destructive consequences, especially in interpersonal or professional / financial areas. usually referred to either as narcissistic crises or trauma motivated by urgent, defensive push for protection and enhancement of self - esteem, or by avoidance of perceived inevitable ultimatums, many of the roots and underpinnings for such decision - making are still relatively unknown. as with fear, there is an important normal aspect of decision - making, especially its role in self - esteem regulation and sense of control, that contributes to an organizing perception of being in charge of cause - effect, input - outcome, and action - result. in particular, efforts to optimize reward, self - enhancement, and self - promotion have proved important. decision - making as part of an agency model for narcissistic personality functioning has been studied in social psychology in the context of approach avoidance motivation, specifically in relationships and in financial and business decisions. narcissistic factors accompanying and guiding decision - making can include arrogance, overconfidence and overestimation, visibility, or impulsivity and risk taking. contrary to lapses in decision making, some people with pathological narcissism or npd can present with more consistent patterns of selfpromoting decision making, involving risk - taking, and disregard or ignorance of both their own and others ' feelings and wellbeing. on the other hand, it is also possible that dysregulated feelings of fear can impact the decisionmaking patterns of these individuals. in recent years, there has been a surge of research on decision making from a neuroscience perspective. though there are a number of decision - making models, in this review we focus on a particular neurobiological theory of decision making that highlights the interaction between experience and emotion : the somatic marker hypothesis. antonio damasio 's somatic marker hypothesis posits that physiological processes, such as emotion, act as signals to influence behavior. more specifically, for each experience an association between that situation and the corresponding somatic states (ie, emotions) is made. the recurrence of a particular situation triggers the reactivation of emotion - influenced neural patterns, which biases decision - making toward choices that maximize reward and minimize punishment. damasio and others propose that the orbitofrontal cortex, specifically the ventromedial prefrontal cortex (vmpfc), is central to decision - making. patients with lesions to the vmpfc display deficits in learning from previous experiences, poor decision - making, flat affect, and impairments in their ability to react to emotional situations. this pattern of impairment led damasio to hypothesize that the primary dysfunction of patients with vmpfc damage was an inability to use emotions to aid in decision - making (eg, in personal, monetary, and moral domains). to test this hypothesis in an experimental context, this task consists of four decks of cards, each associated with varying levels of reward and punishment (two decks are low reward / low punishment [advantageous ] ; two decks are high reward / high punishment [disadvantageous ]). in general, participants sample both the advantageous and disadvantageous decks equally, but after experiencing a number of high punishments, they shift predominantly to advantageous decks. in contrast, subjects with vmpfc damage tend to continue choosing from the disadvantageous decks. moreover, vmpfc lesion patients did not display anticipatory emotional responses (eg, skin conductance), indicating a deficit in anticipating the emotional impact of future rewards and punishments (see ref 84 for review). finally, individuals with lesions to the amygdala also display impairments, similar to vmpfc patients, in performance on the iowa gambling task. however, unlike the vmpfc patients, those with amygdala lesions display impairments in registering the emotional impact of rewards and punishments, rather than the anticipation of this feedback. ultimately, emotional states are elicited during decision - making and are represented in the brain through both cortical (eg, insular cortex ; vmpfc) and subcortical pathways (eg, mesolimbic dopamine system ; amygdala). taken together, this model provides a basis for understanding how basic motivational and emotional processes are related to complex decision - making processes in a variety of contexts. increasingly, the principles gleaned from observing decision - making deficits in patients with lesions are being applied to understanding a diverse range of pathologies in which deficits in decision - making are evident and where emotions can play a critical role. individuals with npd are characterized by a sense of entitlement (ie, self - serving bias), taking advantage of others for personal gain, and hypersensitivity to criticism / punishment. however, it is possible that decision - making in such context is a consequence of weak somatic markers due to the underlying defect in emotional reactivity (see above section on fear). in a study by krusemark undergraduates who scored high on a questionnaire measure of narcissism displayed reduced brain activity for self - serving attributions following success (positive) feedback. more specifically, these individuals had decreased activity in bilateral occipital cortex, bilateral temporal cortex, left posterior parietal cortex, right dorsomedial prefrontal cortex, and bilateral vmpfc (see also ref 46 for similar neural patterns related to narcissism). as noted above, deficits in vmpfc are associated with poor decisionmaking, possibly because of the inability to integrate affective information from external stimuli. based on the evidence that individuals with high narcissism displayed reduced physiological responses to arousing cues, the results reported by krusemark and colleagues may suggest that they make more self - serving attributions following success because of weak stimulus registration, integration, and affective reactivity. the association between impaired decision - making and vmpfc activity has also been made with psychopathy. a number of laboratory paradigms demonstrate vmpfc - related deficits in psychopathy, such as deficits in reversal learning, and in gambling tasks. moreover, koenigs and colleagues reported that a subgroup of psychopathic offenders (ie, primary low - anxious psychopaths) performed similarly to vmpfc lesion patients in the ultimatum and dictator economic decision - making games. specifically, both primary psychopaths and vmpfc lesion patients accepted fewer unfair offers in the ultimatum game and offered lower amounts to others in the dictator game. moreover, they suggest that, as in narcissistic individuals, a deficit in integrating emotion with action may diminish the processes of self - insight and self - reflection in psychopathic individuals. further highlighting the potential utility of the somatic marker hypothesis for understanding the decision - making patterns of those with narcissism, individuals with high narcissism display poor performance on the iowa gambling task in a manner similar to those with vmpfc lesions and with psychopathy. specifically, individuals with high narcissism chose significantly more from the disadvantageous decks, which provided larger immediate reward but resulted in long - term net loss. lakey and colleagues suggested that narcissistic individuals are overly focused on reward, which biased the appraisals of reward and punishment, thus impeding adaptive decision - making. however, these results may also reflect impairment in the processing of negative affect (eg, fear), which often guide a shift in decision - making that would avoid repeated punishment. though the distinction between these perspectives may seem subtle they suggest differential sources of affective decision - making impairment : one related to hypersensitivity to reward and the other related to insensitivity to negative affects. overall, specific deficits in affect processing may contribute to this decision - making impairment in people with pathological narcissism and npd, and future research will need to parse and specify their decision - making and affective capability. regarding the brain mechanisms of pathological narcissism, there are multiple neural structures that overlap with decision - making processes, notably the amygdala and vmpfc. although at this time, it is impossible to make definitive statements about the neural root of pathological narcissism, the development of theoretical models that integrate emotion and decision - making would serve to more precisely understand the self - centered, self - serving, and self - enhancing actions of narcissistic individuals. in recent years, there has been a surge of research on decision making from a neuroscience perspective. though there are a number of decision - making models, in this review we focus on a particular neurobiological theory of decision making that highlights the interaction between experience and emotion : the somatic marker hypothesis. antonio damasio 's somatic marker hypothesis posits that physiological processes, such as emotion, act as signals to influence behavior. more specifically, for each experience an association between that situation and the corresponding somatic states (ie, emotions) is made. the recurrence of a particular situation triggers the reactivation of emotion - influenced neural patterns, which biases decision - making toward choices that maximize reward and minimize punishment. damasio and others propose that the orbitofrontal cortex, specifically the ventromedial prefrontal cortex (vmpfc), is central to decision - making. patients with lesions to the vmpfc display deficits in learning from previous experiences, poor decision - making, flat affect, and impairments in their ability to react to emotional situations. this pattern of impairment led damasio to hypothesize that the primary dysfunction of patients with vmpfc damage was an inability to use emotions to aid in decision - making (eg, in personal, monetary, and moral domains). to test this hypothesis in an experimental context this task consists of four decks of cards, each associated with varying levels of reward and punishment (two decks are low reward / low punishment [advantageous ] ; two decks are high reward / high punishment [disadvantageous ]). in general, participants sample both the advantageous and disadvantageous decks equally, but after experiencing a number of high punishments, they shift predominantly to advantageous decks. in contrast, subjects with vmpfc damage tend to continue choosing from the disadvantageous decks. moreover, vmpfc lesion patients did not display anticipatory emotional responses (eg, skin conductance), indicating a deficit in anticipating the emotional impact of future rewards and punishments (see ref 84 for review). finally, individuals with lesions to the amygdala also display impairments, similar to vmpfc patients, in performance on the iowa gambling task. however, unlike the vmpfc patients, those with amygdala lesions display impairments in registering the emotional impact of rewards and punishments, rather than the anticipation of this feedback. ultimately, emotional states are elicited during decision - making and are represented in the brain through both cortical (eg, insular cortex ; vmpfc) and subcortical pathways (eg, mesolimbic dopamine system ; amygdala). taken together, this model provides a basis for understanding how basic motivational and emotional processes are related to complex decision - making processes in a variety of contexts. increasingly, the principles gleaned from observing decision - making deficits in patients with lesions are being applied to understanding a diverse range of pathologies in which deficits in decision - making are evident and where emotions can play a critical role. individuals with npd are characterized by a sense of entitlement (ie, self - serving bias), taking advantage of others for personal gain, and hypersensitivity to criticism / punishment. however, it is possible that decision - making in such context is a consequence of weak somatic markers due to the underlying defect in emotional reactivity (see above section on fear). in a study by krusemark undergraduates who scored high on a questionnaire measure of narcissism displayed reduced brain activity for self - serving attributions following success (positive) feedback. more specifically, these individuals had decreased activity in bilateral occipital cortex, bilateral temporal cortex, left posterior parietal cortex, right dorsomedial prefrontal cortex, and bilateral vmpfc (see also ref 46 for similar neural patterns related to narcissism). as noted above, deficits in vmpfc are associated with poor decisionmaking, possibly because of the inability to integrate affective information from external stimuli. based on the evidence that individuals with high narcissism displayed reduced physiological responses to arousing cues, the results reported by krusemark and colleagues may suggest that they make more self - serving attributions following success because of weak stimulus registration, integration, and affective reactivity. the association between impaired decision - making and vmpfc activity has also been made with psychopathy. a number of laboratory paradigms demonstrate vmpfc - related deficits in psychopathy, such as deficits in reversal learning, and in gambling tasks. moreover, koenigs and colleagues reported that a subgroup of psychopathic offenders (ie, primary low - anxious psychopaths) performed similarly to vmpfc lesion patients in the ultimatum and dictator economic decision - making games. specifically, both primary psychopaths and vmpfc lesion patients accepted fewer unfair offers in the ultimatum game and offered lower amounts to others in the dictator game. moreover, they suggest that, as in narcissistic individuals, a deficit in integrating emotion with action may diminish the processes of self - insight and self - reflection in psychopathic individuals. further highlighting the potential utility of the somatic marker hypothesis for understanding the decision - making patterns of those with narcissism, individuals with high narcissism display poor performance on the iowa gambling task in a manner similar to those with vmpfc lesions and with psychopathy. specifically, individuals with high narcissism chose significantly more from the disadvantageous decks, which provided larger immediate reward but resulted in long - term net loss. lakey and colleagues suggested that narcissistic individuals are overly focused on reward, which biased the appraisals of reward and punishment, thus impeding adaptive decision - making. however, these results may also reflect impairment in the processing of negative affect (eg, fear), which often guide a shift in decision - making that would avoid repeated punishment. though the distinction between these perspectives may seem subtle they suggest differential sources of affective decision - making impairment : one related to hypersensitivity to reward and the other related to insensitivity to negative affects. overall, specific deficits in affect processing may contribute to this decision - making impairment in people with pathological narcissism and npd, and future research will need to parse and specify their decision - making and affective capability. regarding the brain mechanisms of pathological narcissism, there are multiple neural structures that overlap with decision - making processes, notably the amygdala and vmpfc. although at this time, it is impossible to make definitive statements about the neural root of pathological narcissism, the development of theoretical models that integrate emotion and decision - making would serve to more precisely understand the self - centered, self - serving, and self - enhancing actions of narcissistic individuals. the possible interactional patterns, both self - regulatory and neural, between fear and decision - making as outlined above, implicate the advantage of integrating clinical studies and neuroscience to improve our understanding of pathological narcissism and the diagnosis of npd. the psychoanalytic perspective of fear and decision - making provide more in - depth conceptualizations of narcissism to which neuroscience can add important information and perspectives to further identity the intervening processes in narcissistic personality functioning. this has the potential of significantly improving the diagnosis of npd and consequently also the treatment approach and strategies for patients with narcissistic personality functioning. accordingly, narcissistically based decision - making may be influenced by affect dysregulation, such as hypersensitivity to fear. in addition, fear in some individuals may be accompanied by other intense feelings (ie, secondary feelings) such as shame, rage, self - hatred, etc, or by early self - esteem related traumatic experiences, making feelings of fear intolerable and therefore especially challenging to appropriately integrate in the decision process. treatment focusing on increasing self -reflection, insight, and ability for emotion awareness and regulation would in such case potentially help to redirect or alter the narcissistic patient 's decision - making. alternatively, patients with npd may also be overly goal - focused in the service of self - enhancement, and hence, like people with psychopathy, be unable to redirect their attention. treatment efforts focusing on understanding and integrating vulnerability and feelings of fear in self - functioning and self - directedness would be most meaningful for these patients. importantly, these two treatment alternatives assume that narcissistic patients possess the ability to both recognize and process feelings of fear, even though they may for various reasons be insensitive, ignorant, hypersensitive or over - reactive to fear. however, it is also possible that neurocognitive limitations in recognizing and integrating feelings of fear greatly limit decision - making capabilities. in such case treatment focusing on learning alternative strategies may be more useful, but also require the patient 's motivation and realization of the necessity for change. strategies to address the role of fear and pattern of decision - making may potentially diminish the common risks for ruptures and premature termination, and ultimately promote collaborative alliance building with patients with pathological narcissism and npd. | linking psychoanalytic studies with neuroscience has proven increasingly productive for identifying and understanding personality functioning. this article focuses on pathological narcissism and narcissistic personality disorder (npd), with the aim of exploring two clinically relevant aspects of narcissistic functioning also recognized in psychoanalysis : fear and decision - making. evidence from neuroscientific studies of related conditions, such as psychopathy, suggests links between affective and cognitive functioning that can influence the sense of self - agency and narcissistic self - regulation. attention can play a crucial role in moderating fear and self - regulatory deficits, and the interaction between experience and emotion can be central for decision - making. in this review we will explore fear as a motivating factor in narcissistic personality functioning, and the impact fear may have on decision - making in people with pathological narcissism and npd. understanding the processes and neurological underpinnings of fear and decision - making can potentially influence both the diagnosis and treatment of npd. |
the temporomandibular joint (tmj) is a joint of the sinovial, double and bicondilar type. it is situated between the condylar process and the mandibular fossa of the temporal bone. it has an articular capsule, synovial membrane, ligaments and associated muscles. in comparison with other synovial joints, the tmj is unique because of the presence of the articular disc that separates it into two independent cavities, known as infra and supradiscal cavities, with distinct morphological features. at the end of the 12 gestational week morphologically, the articular disc is a small elliptic fibrocartilaginous plate shaped like an s in sagittal cut and having its bigger axis in the posteromedial direction. the articular disc is divided into four bands : anterior, intermediate, posterior and bilaminar zone. the disc also plays an important functional role in translation and rotation movements, in a broad and passive manner. the articular disc can be described as a concave lens with two faces (superior and inferior), two margins (medial and lateral) and two extremities (anterior e posterior). during the embryogenesis period, the articular disc of the tmj consists of a dense layer of mesenchymal cells that derived from the condylar blastema in the anterior region and from the glenoidal blastema in the posterior region. for sab, the architecture of the articular disc is complex, with collagen fibres predominating in the longitudinal direction of the superior and inferior faces and cross - sectional and interposed oblique fibres predominating in the intermediate portion. for minarelli and liberti, the disc has compact collagen bands in the intermediate band, with fibres running in the anteroposterior direction. takahashi and sato examined the distribution of types i and iii collagen, fibronectin and elastic fibres in the articular disc of 54 adult cadavers and 24 foetuses aged 12, 16, 20, 24, 28 and 32 weeks with light microscopy and scanning electron microscopy. according to the authors, at 12 weeks, the collagen fibres formed a thin layer on the entire disc ; at 16 weeks, the disc already had anteroposteriorly - oriented collagen fibres in the intermediate band and cross - sectional fibres were found in the most superficial layers of the disc ; finally, at 20 weeks, the authors found a disc consisting of a complex arrangement of collagen fibres, oriented in various directions. in terms of structural biology, the collagen fibres inside the articular disc of the tmj are composed mainly of type i collagen and small amounts of type iii collagen. according to takahashi and sato, the concentration of type - i collagens in the articular discs of foetuses aged 20 to 24 weeks increased in all bands, especially in the anterior and intermediate bands. meanwhile, the expression of type - three collagen in foetuses of the same gestational age increased only in the intermediate band. confirmed that a large number of fibroblast - like cells in the human tmj disc was co - localized with lumican expression in their specimens. in contrast, little lumican expression was observed in the vicinity of fibrochondrocytes and chondrocyte - like cells. matsumoto. demonstrated the presence of has3 in the chondrocyte - like cells of both normal and deformed tmj discs in adults. a study done at the institute of anatomy, university of catania, italy, used four articular discs from adult cadavers as controls. a multi - directional preservation of the collagen bands was found in all samples, in addition to a predominance of fibrocyte - like cells, and in a smaller aggregated amount, chondrocyte - like cells. the apparent presence of fibrocartilaginous areas also suggests the presence of type ii collagen fibers. fibrous proteins (collagen and elastin), responsible for the shape and structure of the lamina propria, are associated with resistance and elasticity, while interstitial proteins (glycoproteins and proteoglycans) control viscosity, hydration, and the tissue volume extracellular matrix. according to gray., the characteristics of the layers of the lamina propria are intimately associated with the arrangement and distribution of the fibrous proteins in the extracellular matrix, especially collagen fibres, which support and absorb the physical stress caused on the articular disc by joint movement. collagen belongs to a family of structural proteins with distinct chemical compositions, morphologies and functions. collagen is a component of many organs and tissues and is the main extracellular matrix element of multicellular animals, representing from 2530% of the protein mass of the organism. the different types of collagens have been organized into the following classes : fibrillar, associated with fibrils, non - fibrillar, filamentous, short- and long - chain. it has also been demonstrated that some types of collagen are capable of aggregating molecules to form heterotypical fibrils. the presence of heterotypical fibrils with different combinations in many tissues has already been reported. evidence of these combinations obtained with immunohistochemical analysis has been observed between types i and v collagen in human skin, tendon and cornea and between types ii and xi, and between i and ii in cartilage. given the scarcity of studies that focus morphologically on the types of collagen in the tmj disc of human foetuses, we believe a better structural knowledge of the relationship of these proteins with the articular disc of the tmj is necessary. thus, the objective of this study is to analyse the immunohistochemical expression of types i and iii collagen markers in the articular disc of the temporomandibular joint of human foetuses. the protocol of this study was submitted to the unifesp research ethics committee and approved under number 0225/05. our sample consisted of 10 human foetuses supplied by the universidade federal de uberaba, resulting in 20 tmj with gestational ages from 17 to 24 weeks. the gestational age of the foetuses was determined by measuring the crown - rump (cr) length. all foetuses were preserved in a 10% formalin solution and dissected by removing the skin, subcutaneous tissue and exposing the deep structures. two sections of 3 m were taken from each paraffin block containing the collected material and were then spread on glass slides. the streptavidin - biotin technique was used to analyse the immunohistochemistry reactions of types i and iii collagens. sections were deparaffinised in three changes of xylene (10 min each) and rehydrated in a graded series (100, 80 and 70%) of ethanol finishing in distilled water. antigen sites were retrieved by pretreatment with 0.5 m acetic acid for 90 min at 37c and multiple rinses with distilled water. endogenous peroxidases were quenched by incubating in 3% h2o2 (20 min, room temperature). the slides were then washed 32 min in pbs and incubated in milk powder solution for 20 min to block nonspecific binding of antibody. sections were incubated with 100 l of the primary antibody overnight at 4c.primary antibody was purified rabbit anti - human type i collagen, 1:800 (novatec inc., baltimore, md, usa) and rabbit monoclonal anti - human type iii collagen (novatec inc.). subsequently, slides were washed in pbs (32 min), and detection of the primary antibody was performed using the lsab kit (dako cytomation, carpinteria, ca, usa). sections were incubated with biotinylated secondary antibody for 30 min, washed (32 min in pbs), and incubated with streptavidin - peroxidase (hrp) conjugate for 30 min. the sections were again washed (32 min in pbs) before the antibody - hrp complex was visualized by incubation with diaminobenzidine (dab lsab + system - hpr dako cytomation) for 5 min. slides were briefly counter - stained in harris hematoxylin and dehydrated, and cover slips added. st louis, md, usa) resin, analysed, and photographed with a light microscope (olympus bx50). the tissues recommended by the manufacturer were used as positive antibody controls.the negative controls were the same cases used as positive controls, which were submitted to the immunohistochemical reaction described above, except for the incubation with the primary antibody, done with a buffer solution. figure 1a shows numerous type i collagen fibres throughout the extension of the articular disc of the tmj. however, these fibres are more abundant in the posterior band of articular disc (1b). additionally, figure 1c shows a large concentration of type iii collagen fibres on the inferior face of the articular disc. figure 1d shows type iii collagen fibres in the intermediate band of the articular disc. figure 1immunohistochemical micrograph of type i collagen. a), foetus aged 19 weeks of intrauterine life ; there are collagen fibres in the entire disc (a), mandibular head (b) and temporal bone (c) ; b), immunohistochemical photomicrograph of type i collagen in foetus at 22 weeks of intrauterine life ; the posterior band of the articular disc contains numerous complexly - arranged collagen fibres ; c), immunohistochemical micrograph of type iii collagen in foetus at 21 weeks of intrauterine life ; the inferior face (a) of the articular disc is delimited by numerous collagen fibres ; d), immunohistochemical micrograph of type iii collagen in foetus at 24 weeks of intrauterine life ; collagen fibres are arranged in many directions on the inferior face (intermediate). the heart tissues used as positive controls for collagen i and iii are represented in panels e) and f), respectively. a), foetus aged 19 weeks of intrauterine life ; there are collagen fibres in the entire disc (a), mandibular head (b) and temporal bone (c) ; b), immunohistochemical photomicrograph of type i collagen in foetus at 22 weeks of intrauterine life ; the posterior band of the articular disc contains numerous complexly - arranged collagen fibres ; c), immunohistochemical micrograph of type iii collagen in foetus at 21 weeks of intrauterine life ; the inferior face (a) of the articular disc is delimited by numerous collagen fibres ; d), immunohistochemical micrograph of type iii collagen in foetus at 24 weeks of intrauterine life ; collagen fibres are arranged in many directions on the inferior face (intermediate). the heart tissues used as positive controls for collagen i and iii are represented in panels e) and f), respectively. minarelli and liberti have a stated that the anterior band contains 85% of type i collagen fibres and 15% of type iii collagen fibres, while the posterior contain 94% of type i collagen fibres and 6% of type iii collagen fibres. consider that the fibres inside the disc are composed mainly of type i collagen and are responsible for roughly 80% of the dry mass of the disc. figures 1a and 1b show numerous type i collagen fibres throughout the tmj disc, especially in the posterior band. figure 1c shows a great concentration of type iii collagen fibres located on the inferior face of the disc, while figure 1d shows complexly arranged type iii collagen fibres running in many different directions. this does not mean that the anterior, intermediate and posterior bands of the disc were not marked by the antibody, but when type i collagen fibres are compared with those of type iii, there is a prevalence of type i in the posterior band of the disc and a prevalence of type iii on the entire inferior face of the disc. in foetuses and children, type i collagen fibres have a stratified arrangement, organized in fibre bundles with an anteroposterior, laterolateral and oblique orientation forming mesh. in the posterior band of the disc, in agreement with our findings, the laterolateral fibre bundles were marked and, in agreement with minarelli and liberti, they formed a ring that surrounded the disc. in contrast, in the intermediate band of the disc, there was a prevalence of longitudinally oriented fibres in the anteroposterior direction. descriptions of the arrangement that constitute the architecture of the articular disc have been made in humans of different ages and also in primates. again regarding the composition of collagen fibres, it is important to point out that the aggregation of fibrillar collagen molecules to form heterotypical fibrils has already been described in many structures such as skin, tendon, meniscus, cartilage, human cornea and heart. in our study we observed that these heterotypical fibrils had probably formed, in agreement with takahashi and sato who demonstrated that the disc of foetuses of various gestational ages contains types i and iii collagens, and probably type ii, as judged berkovitz. by the presence of fibrocartilaginous areas in the articular disc. the distribution pattern of the fibres throughout the foetuses articular discs is different from that observed in adults articular discs. the following hypothesis may be considered : for the distribution patterns of types i and iii collagens to change as the foetus develops and in adults, some events, such as sucking, small mandibular movements and load, begin during the maturation stage, after the twelfth week of development, and are lifelong. thus, findings from this study may lead us to conclude that immunohistochemical expression of types i and iii antibodies is positive for the presence for these collagen fibres and heterotypical fibril meshes form between those two types of collagen. | the objective was to study the morphology of the articular disc and analyse the immunohistochemical expression of types i and iii collagen markers in the temporomandibular joint (tmj) disc of human foetuses of different gestational ages. twenty tmj from human foetuses supplied by universidade federal de uberaba with gestational ages from 17 to 24 weeks were studied. the gestational age of the foetuses was determined by measuring the crown - rump (cr) length. macroscopically, the foetuses were fixed in 10% formalin solution and dissected by removing the skin and subcutaneous tissue and exposing the deep structures. immunohistochemical markers of type i and iii were used to characterize the existence of collagen fibres. analysis of the immunohistochemical markers of types i and iii collagen revealed the presence of heterotypical fibril networks. |
arterial ischemic stroke (ais) occurs more commonly in adults than children. in children the clinical presentation is often interpreted as other neurologic conditions or intoxications resulting in a delay in diagnosis due to the lack of awareness of pediatrician or that the symptoms do not systematically evoke a stroke. adult ais occurs in the setting of the principal risk factors of hypertension and atherosclerosis, while pediatric risk factors are heterogeneous and include focal intracranial arteriopathies, congenital cardiovascular diseases, and hemoglobinopathies [1, 2 ]. recent advances in the management of ais have emphasized the importance of timely diagnosis and restoration of arterial flow in the affected vascular territories. adult victims have access to time - sensitive or hyperacute therapies due to an awareness of the severity of the disease, organization of delivery of medical care from the emergency room that includes designation of stroke centers, and protocol - driven preparedness for such emergencies [36 ]. unfortunately, this is not the case for pediatric victims, because it is not known whether thrombolytic therapies are appropriate. we describe the case of a 12-year - old male with ais who presented to medical attention with a potentially devastating neurological injury. a resolute strategy utilizing hyperacute therapies aimed at restoring blood flow followed by neurointensive care support was provided. the patient had a very good outcome that may be related to this management but a complete causation can not be ascribed to it as 1 out of 3 patients with lenticulostriate stroke may have a spontaneous favorable evolution. herein we review the literature to date describing the challenges in analyzing and preparing for the application of time - sensitive therapies in the treatment of ais. the institutional irb was consulted and waved the need for approval of the publication of this case. the patient is a 12-year - old, right - handed, 52 kg african - american male without any significant medical history other than nocturnal enuresis. his father woke him up at 04:00 on the morning of admission as was customary in their home to prevent bedwetting. while initially he responded appropriately, the child collapsed to the ground after standing and became unresponsive. he was transported to an outlying emergency department where he was noted to have aphasia and left dense hemiplegia. computed tomography demonstrated signal attenuation and edema in the vascular distribution of the right middle cerebral artery. the patient was transferred to our institution by helicopter and was briefly examined in the emergency room prior to transfer to the magnetic resonance (mr) imaging suite. he was awake, with stable airway, anxious and frustrated by inability to speak, but was cooperative and able to follow some commands. heart rate was 5060 beats per minute and blood pressure was 130140/8090 mm hg. he demonstrated lack of language expression, flaccid paralysis of the left face, upper extremity, and lower extremity. mri and mra studies revealed occlusion of the right middle cerebral artery with restricted diffusion in the right basal ganglia and the posterior lateral aspect of the caudate head (figure 1). there was mass effect on the right lateral ventricle and midline shift. our information was that 6 h had elapsed since the onset of neurologic symptoms. taking into account the possibility of a devastating clinical outcome and in conjunction with adult stroke and interventional radiologist specialist, the decision was made to attempt to restore arterial patency. the patient was anesthetized, intubated, and cooled to 3335c (mild therapeutic hypothermia). a thrombus was located (figures 2 and 3) ; endovascular mechanical embolectomy with a merci retrieval device was attempted without success. recombinant tissue plasminogen activator (rt - pa) was infused into the mca in doses of 0.7, 2, and 4 mg with minimal improvement in blood flow. abciximab and nitroglycerin (25 g) were then infused with considerable improvement in blood flow (figures 4 and 5). neurosurgical consultation was obtained for an intracranial pressure (icp) monitoring, but this was not placed due to recent thrombolytic administration and the initiation of systemic anticoagulation. heparin was administered for 7 days, abciximab (10 g / min for the first 12 h), aspirin (325 mg / day), magnesium sulfate (2000 mg/24 hrs), 25% albumin (1 g / kg iv every 6 h for the first 5 days), and memantine hcl (10 mg nasogastric each day for 3 days). mild therapeutic hypothermia was maintained for 4 days to decrease acute postischemic cerebral edema followed by passive rewarming over a 24-hour period. a ct scan obtained at 24 hrs showed a decrease in the midline shift. the central venous pressure (cvp) was maintained at 810 cm h2o and mean arterial pressure (map) at 7580 mm hg. a dual channel near infrared spectroscopy (nirs) probe was applied to the scalp overlying both frontal lobes. initially, there was a discrepancy between the two sides with the ischemic right side 10%15% lower than the left side (40%50% versus 65%70%). subsequently, the partial pressure of arterial carbon dioxide was allowed to increase from 3540 to 4655 mm hg with resolution of the discrepancy (both sides equal to 60%65%). mr studies on the 7th day revealed appropriate mca blood flow and partial resolution of the ischemic changes in the basal ganglia. he was extubated on day 10 and was able to communicate verbally, written, and receptive but had a small degree of residual left hemiparesis. he was transferred to a rehabilitation facility on day 15 where he exhibited an almost complete recovery within the following two weeks and was discharged home. subsequent followup by our pediatric neurology service over the next year has demonstrated complete recovery to baseline without deficits. one year later he is taking advanced placement classes in school, playing junior high school basketball and baseball, and having no demonstrable neurological deficits. cerebral angiography and serologic studies did not find the presence of dissection, vasculitis, or autoimmune disease ; transthoracic echocardiography ruled out structural heart disease, intracardiac thrombus, and right to left intracardiac shunt, and hemoglobin electrophoresis, hypercoagulability studies, and lipid panels were all unremarkable. doppler studies of the veins of the lower extremities and pelvis were all negative for deep venous thrombosis. an mri performed 3 months after the events showed resolution of the edema and restricted diffusion with a residual hyperdense signal abnormally in the periventricular region (figure 6). our case report differs from others in the literature in that intra - arterial thrombolysis was undertaken 6 h after the onset of symptoms than most other reports of anterior circulation occlusion and few other patients have made as complete a recovery. several provocative issues relevant to ais in children are raised by his management : (a) the use of hyperacute therapies, (b) the role of antiplatelet agents and anticoagulants in the management of ais, and (c) the role of adjunctive pharmacological neuroprotective agents. the growing recognition that ais in adults is reversible if treated in a prompt manner has fostered a sense of urgency in clinicians who regard this condition as a brain attack and hospitals preparedness with designation of stroke centers for rapid triage and treatment. the resolve of the physician has a decisive effect on the timely implementation of therapies for stroke in adults. by comparison, little sense of urgency to aggressively restore vascular patency exists for pediatric patients, and with the exception of ais in the setting of sickle cell disease and moyamoya disease, therapeutic approaches that embrace supportive care and rehabilitation are more common in pediatric hospitals. first, pediatric emergency room practitioners encounter ais infrequently, and it is therefore not uncommon for considerable delay to occur in the diagnosis excluding the patient from the accepted time windows for hyperacute therapies. second, the outcome of children with stroke is incorrectly thought to be better than that of adults. the outcome of ais is mainly determined by size, location, and etiology of stroke. while the pediatric mortality is described to be between 5%10% and is favorable when compared to adult patients, pediatric survivors of ais are left with persistent neurologic and psychosocial deficit or seizure disorder for the rest of their lives [7, 8 ]. third, the pathophysiology underlying the heterogeneous disorders of pediatric ais makes it difficult to extrapolate the adult ais literature to children, and many children 's hospitals do not have preplanned algorithms for rapid diagnosis and treatment of ais. finally, many practitioners are uncomfortable administering thrombolytic medications in the absence of pediatric - specific data or clinical guidelines to support their use. indeed, most recommendations for the treatment of ais in children are derived from consensus and expert opinion, and neither the royal college of physicians (rcp) in the united kingdom nor the american college of chest physicians (accp) in the united states recommends the administration of rt - pa to children with ais [9, 10 ]. despite this reluctance, several case reports to date have described their use in children [1116 ], and a review of a national inpatient database registry indicates that a small minority of pediatric patients with ais are receiving thrombolytic therapy [17, 18 ]. the term hyperacute therapies has been coined to refer to therapies which must be initiated within an acceptable time interval in order to be effective, and they are designed to restore blood flow through obstructed vascular beds. hyperacute therapies that are offered to adults include intravenous thrombolysis with rt - pa, intra - arterial thrombolysis with rt - pa, and mechanical embolectomy. the use of intravenous thrombolysis is now well established in the literature and is recommended for the treatment of ais in adults without contraindications who present to medical attention within 3 h of symptoms onset [4, 5 ]. patients treated with 0.9 mg / kg rt - pa (10% as a bolus and the remainder infused over 1 hour) had better functional outcomes at 3 months when compared to patients who received placebo, and mortality rates were similar between the two groups. symptomatic intracranial hemorrhage occurred in 6.4% of patients treated with rt - pa and 0.6% of controls. the time period from onset of stroke symptoms to beneficial treatment with thrombolytic medications remains a subject of continuous debate. the debate addresses the observation that as time from symptom onset increases the likelihood of outcome benefit from thrombolysis decreases while thrombolytic therapy is associated with a substantial increase in likelihood of intracranial hemorrhage. however, a recent publication demonstrated benefit while extending the administration up to 4.5 h after the onset of symptoms. furthermore, whether strict adherence to pre - defined time interval cutoffs should be used to determine whether thrombolytic therapy should be offered is unclear. comparison of mri diffusion - weighted imaging (dwi) and perfusion - weighted imaging (pwi) may allow the clinician to determine for each patient how much brain tissue is receiving inadequate blood flow (abnormal pwi) to still viable brain tissue (intact dwi). such an examination might allow clinicians to determine for patients presenting after more prolonged time intervals from onset of neurologic symptoms whether thrombolytic therapy could still be beneficial to a given patient [10, 11, 18, 19 ]. intra - arterial administration of thrombolytics (urokinase and rt - pa) is sometimes offered as a potential, viable alternative for the delivery of thrombolytic medications to obstructed cerebral vascular beds for patients who are not candidates for systemic intravenous thrombolysis or those who present to medical attention between 3 and 6 h after symptom onset. intra - arterial administration of recombinant prourokinase and intravenous heparin within 6 h of onset of stroke symptoms resulted in improved functional outcomes when compared to intravenous heparin alone, but intra - arterial and intravenous thrombolyses have not been compared to each other. other options for recanalization of obstructed cerebral vasculature include the use of endovascular mechanical retrieval devices for clot removal. intra - arterial and endovascular mechanical embolectomies have extended the time window for treatment up to 8 h for ais in the anterior circulation, and even longer in cases involving the posterior circulation. while mechanical intra - arterial thrombectomy has demonstrated a high recanalization rate, randomized trials that describe functional outcome are lacking. there is evidence that thrombolytic medications are being administered to 1.6% of the children with ais, and this data suggests that children may have inferior clinical outcomes with respect to mortality. however, it can not be determined from this data whether inferior outcomes are related to ais severity or the treatment itself. optimal rt - pa dosing is likely to differ as there are age - dependent differences in hemostasis [22, 23 ], younger individuals demonstrating diminished specific indices of fibrinolysis and global, increased fibrinolytic capacity. case reports over the past eight years have described the administration of thrombolytic medications to pediatric patients with good result at widely different interval from onset of symptoms [13, 16, 2429 ]. indeed, due to the dire outcome of vertebrobasilar thrombosis (vbt), case reports in the pediatric literature describe successful administration of thrombolytic therapy 17 h after the onset of symptoms [24, 25 ]. the rcp and accp guidelines provide recommendations for acute treatment and secondary prevention of ais. their recommendations differ with respect to the role of anticoagulation in the acute management of ais in children. rcp guidelines for the management of acute ais in children (not associated with intracranial hemorrhage or sickle cell disease) recommend the administration of aspirin (5 mg / kg / day). the accp guidelines for management of acute ais in children have a class ii recommendation in regard to the initiation of unfractionated heparin or low - molecular weight heparin (lmwh) for 57 days or until cardioembolic stroke and vascular dissection have been excluded [2, 9, 10 ]. heparin may be safe for the treatment of strokes secondary to sinovenous thrombosis even though studies in regard to efficacy are lacking. the accp points out that while lmwh may offer reproducible pharmacokinetics and fewer monitoring tests in comparison with unfractionated heparin the effects of lmwh can not be rapidly reversed after its discontinuation. adult guidelines from the aha recommend the administration of aspirin (325 mg / day) within 2448 h of stroke onset [4, 5 ]. for secondary prevention of ais recurrence the accp recommends initiation of aspirin therapy (25 mg / kg / day) after discontinuation of anticoagulation. for children with vascular dissection or cardioembolic stroke the accp recommends continuation of anticoagulation with low - molecular weight heparin or vitamin k antagonists for 36 months. the rcp recommends continuation of aspirin (13 mg / kg / day) for all children except those with moyamoya disease, sickle cell disease, or arterial dissection. the rcp recommends treatment with anticoagulation for prevention of secondary ais in patients with arterial dissection or cardioembolic stroke. while aspirin is the major antiplatelet agent in clinical use, the roles for other antiplatelet and anticoagulant medications are under investigation. adjuvant medications such as thrombin inhibitors and inhibitors of platelet glycoprotein iib / iiia (giib / iiia) are being evaluated as monotherapy and in combination with rt - pa for the treatment of acute ischemic stroke. current studies also evaluate the use of argatroban (a thrombin inhibitor), ancrod (a viper - derived enzyme that cleaves fibrinogen and promotes endogenous release of plasminogen activator) [31, 32 ], and desmoteplase (a bat saliva - derived protease similar in function to human plasminogen activator) [33, 34 ]. a recently completed phase iii trial with an intravenous administration of a glycoprotein iib / iiia antagonists, abciximab, did not demonstrate safety or efficacy with an increased incidence of intracranial hemorrhage. once blood flow through the mca was restored, and measures were taken to protect the ongoing patency of the vessel, we next considered measures that attempted to minimize secondary neuronal injury during reperfusion of the ischemic regions of the brain. included among these were (a) the maintenance of adequate cerebral blood flow, (b) the prevention of hyperglycemia, (c) the continuation of mild therapeutic hypothermia, and (d) administration of 25% albumin, magnesium salts, and n - methyl - d - aspartate receptor (nmda) antagonists. maintenance of adequate cerebral perfusion pressure may be of importance in optimizing neurologic outcome after ais if regional autoregulation is lost or in cases of severe increase in intracranial pressure. while verification of adequacy of cerebral perfusion pressure might have been optimized with the assistance of an icp monitor, the risks of icp monitor placement in this anticoagulated child were felt to be prohibitive. in the absence of icp monitoring we assumed that our patient probably had at least moderately increased icp. accordingly, we allowed moderate permissive hypertension, maintained a high osmolarity with hypertonic saline and mild hypothermia. fever should be aggressively treated in ais, and mild hypothermia has shown to decreases acute postischemic cerebral edema, a transient phenomenon that is maximal at 24 days postinfarct [3739 ]. however it has been difficult to ascertain a positive impact in outcome with its use, and a large randomized study is needed. a rebound in intracranial pressure can occur when rewarming a patient that was hypothermic. as an indirect monitor of the adequacy of cerebral perfusion, near infrared spectroscopy (nirs) monitoring was applied to both cerebral hemispheres. in the initial h, regional cerebral oxygen saturation (rso2) the stepwise preload augmentation and addition of norepinephrine to augment mean arterial pressure only increased the differences as a the subsequent introduction of mild permissive hypercapnia produced an increase and equalization of rso2 in both hemispheres. while these trends in rso2 could be interpreted to indicate the restoration of adequate oxygen supply / demand in the affected hemisphere, prior studies employing nirs in the noninvasive monitoring of patients with middle cerebral artery infarction and cerebral edema suggest that disappearance of the rso2 difference between hemispheres may indicate worsening cerebral edema and herniation in the infarcted hemisphere. computed tomography of the brain on day 3 and reassuring neurologic examinations during improving rso2 values in the affected hemisphere assured us that the disappearance of the rso2 gradient between hemispheres was not due to an ominous process. the role of nirs as a noninvasive monitor of cerebral circulation in pediatric stroke remains to be delineated. our patient was treated with scheduled infusions of hypertonic albumin after returning from the angiography suite. the administration of hypertonic solutions of albumin is recommended for the treatment of adult ischemic stroke patients in our institution and is based on studies that demonstrate improved functional neurologic outcome in adult patients suffering from ischemic stroke [42, 43 ]. preclinical and phase i and ii trials of the administration of albumin in acute ischemic stroke suggest that the time to administration of albumin after ischemic stroke is critically important (within 4 - 5 h) and that a synergistic effect may exist when rt - pa is also administered. the physiologic benefit of albumin administration is believed to be related to its effect on the endothelium [4549 ], blood viscosity and aggregation, interactions with nitric oxide, and inhibition of platelet aggregation [52, 53 ]. studies of albumin administration in experimental models of central nervous system ischemia demonstrate that albumin administration rapidly improves blood flow in cerebral vessels with critically reduced flow and clears thrombotic material adherent to the endothelium in ischemic venules [53, 54 ]. while preliminary studies of high dose albumin administration to ais patients have been encouraging, the results of the multicenter, randomized, placebo - controlled efficacy trial of albumin in acute ischemic stroke alias phase iii trial is underway and may provide definitive answers on the role of albumin therapy in adult ais. hypertonic albumin administration may induce pulmonary edema in adults ; however it is frequently utilized in pediatric intensive care in patients with other conditions without deleterious effects. decompressive craniotomy is an effective treatment to treat increased intracranial pressures in patients with extensive stroke. this is the routine practice of our adult stroke team, and their administration is predicated on several theoretical principles. the magnesium ion regulates cellular energy metabolism, vascular tone, and cell membrane ion transport. magnesium may regulate atp concentrations and is a prerequisite for atp regeneration after ischemia and reperfusion [57, 58 ]. the magnesium ion also blocks the ion channel of the n - methyl - d - aspartate (nmda) receptor in a voltage - dependent fashion, and increasing extracellular magnesium concentrations in vitro cause noncompetitive nmda blockade [59, 60 ]. in vitro and in vivo models of focal and global ischemia have demonstrated neuronal protection that in some instances is as great as that seen with noncompetitive nmda antagonists. magnesium protects both hippocampal neurons from glutamate - mediated necrosis and white matter tracts from prolonged ischemia. systemic mgcl2 decreased cerebral infarct volume by 20% after permanent middle cerebral artery occlusion in the rat. memantine hydrochloride, another nmda receptor antagonist, was also administered to attenuate reperfusion injury. while a neuroprotective role has been ascribed to magnesium sulfate and nmda receptor antagonists, no data from human trials have shown conclusive benefit to adult patients with ais, and data is therefore certainly lacking to make recommendations regarding use in pediatric ais. the case described is a paradigmatic observation of the possibility of using hyperacute therapies in children with strokes : early recognition of the event, probable thromboembolism mechanism, mca distribution, and rapid consultation with multiple specialists were crucial factors influencing the decision process. thrombolysis is probably less favorable when other types of vasculopathy, that most often affect young children (e.g., basal arterial stenosis), are present. there is a need to increase awareness of childhood stroke by pediatricians and emergency room physicians caring for children with the aim of reducing the time lag from symptoms to the correct diagnosis. it is imperative to have in place a preplanned diagnostic algorithm as well as therapeutic algorithm. most of emergency departments do not receive regularly children with stroke. except in very specific cases, it is thus not possible to build autonomic pediatric stroke centers. nevertheless, as it is well documented in the present case report, collaboration between emergency room physicians, pediatric neurologists, and physicians working in adult stroke centers should be highly considered. | objective. the optimal management of pediatric patients with arterial ischemic stroke (ais) is not known. despite this, goal - oriented, time - sensitive therapies geared to rapid reestablishment of arterial blood flow are occasionally applied with beneficial effects. the inconsistent approach to ais is in part due to a lack of knowledge and preparedness. methods. case report of a 12-year - old male with right middle cerebral artery (mca) occlusion resulting in dense left hemiplegia and mutism and review of the literature. intervention(s). mechanical thrombectomy, intra - arterial administration of rt - pa, vasodilators, and platelet inhibitors, and systemic anticoagulation and subsequent critical care support. results. restoration of right mca blood flow and complete resolution of neurologic deficits. conclusion. we report the gratifying outcome of treatment of a case of ais in a pediatric patient treated with hyperacute therapies geared to arterial recanalization and subsequent neurologic critical care and review the pertinent literature. guidelines for the emergency room management of pediatric ais from prospective, randomized trials are needed. |
post - operative vaginal cuff brachytherapy is used to reduce the risk of vault recurrence for patients with endometrial or cervical carcinomas [1, 2 ]. although the choice of applicator for high - dose - rate (hdr) brachytherapy of vagina is both institutional and patient dependent, the most commonly used applicator is a vaginal cylinder. recently, the american brachytherapy society (abs) reported the recommendations for adjuvant vaginal hdr brachytherapy after hysterectomy, establishing dose prescription and optimization guidelines. as the prevalent location of the vaginal lymphatic channels is within 1 mm of tissue surrounding the vaginal cylinder, the abs report recognizes that it is imperative for the vaginal mucosa to be in contact with the cylindrical applicator surface to achieve the optimal dose distribution, and recommends computed tomography (ct) planning to confirm the presence of significant air gaps [3, 4 ]. their presence may result in the failure to eradicate all microscopic malignant cells, therefore increasing the risk of recurrence. to date, there is not enough literature addressing this subject [5, 6 ]. the purpose of this study is to retrospectively assess the incidence and magnitude of air pockets around vaginal cylinders, and its impact on dose distribution in ct - image based hdr brachytherapy. we retrospectively reviewed the data of 50 patients with endometrial carcinoma, treated between july 2010 and september 2013 by post - operative hdr vaginal cuff brachytherapy in the radiotherapy unit of king abdulaziz university hospital jeddah, saudi arabia. the patients had either post - operative external beam radiotherapy (ebrt ; 45 gy in 25 fractions, one fraction per day, five times per week) to the whole pelvis, using four - fields ct - based planning, followed by hdr brachytherapy (12 gy in 3 fractions, two fractions per week), or hdr brachytherapy alone (21 gy in 3 fractions, one fraction per week). clinical examination was performed before brachytherapy, to assess the patient and estimate the applicator size to be used. clinical judgement (inserting the cylinder according to the vaginal stump length and monitoring patient discomfort during insertion) was used to insure that the cylinder reaches the vaginal apex. application was completed without anesthesia, and the applicator was fixed in place by a perineal belt ensuring its immobilization. brachytherapy was carried out with cylindrical vaginal applicators of various diameters (2, 2.6, 3, and 3.5 cm), and the dose prescribed to 0.5 cm from the applicator 's surface, over a length of 5 cm from the applicator 's tip. computed tomography images were acquired for each brachytherapy fraction using a siemens somatom emotion ct scanner (siemens ag, erlangen, germany), with 2 mm slice intervals from the iliac crest to the ischial tuberosities, without intravenous contrast. dose distribution was calculated by varian brachyvision planning system, version 8.10 (varian medical systems, inc., palo alto, ca), for a brachytherapy remote afterloader varian hdr varisource ix (varian medical systems, inc.). the dose calculation algorithm is based on the tg-43 formalism, as recommended by the american association of physicists in medicine (aapm). all treatment plans were retrospectively analyzed for the presence of air pockets in the proximal 5 cm of the vaginal vault, that were contoured, and the volumes calculated by the treatment planning system, as shown in figure 1. computed tomography images of air pockets present around a vaginal cylinder for a representative patient, in axial and sagittal views the incidence, vaginal mucosa displacement, volume, and dosimetric effect of the air pockets around the vaginal cylinder were evaluated. for 50 patients, a total number of 135 ct - based brachytherapy plans were retrospectively reviewed. the average age of patients was 58.3 11.8 years (range : 31 - 87 years). we could not find any correlation between the incidence of air pockets and patient 's age. a total of 78 air pockets were found in 29/50 patients (58%) and 45/135 (33%) brachytherapy plans. they were located at the apex : 16/78 (20%) and lateral to the applicator : 62/78 (80%). the apex air pockets occurred during first brachytherapy fraction for 4 patients and in subsequent fractions for 6 patients. the incidence of air pockets per patient during the course of 3 brachytherapy fractions is presented in figure 2. among the 29 patients presenting air pockets, 21 (72%) had them occurring during first fraction of treatment, while 8 (28%) patients developed air pockets later in their brachytherapy course : 6 (21%) of them during second fraction and 2 (7%) during third fraction. three patients presented air pockets during all treatment. the maximum number of air pockets for a single patient during the whole brachytherapy course was 8. number of air pockets per patient over the course of 3 brachytherapy fractions the correlation between the incidence of air pockets and vaginal applicator size was analysed and is shown in figure 3. the highest incidence of air pockets occurred for 3 cm cylinder diameter, in 25/48 (52%) fractions, and the lowest for 2 and 2.6 cm diameter, in 7/9 (21%) and 7/11 (20%) fractions, respectively. the distribution of air pockets according to vaginal cylinder diameter the volume of air pockets ranged between 0.01 and 2.1 cm (mean : 0.15 cm 0.36 cm) and the maximum displacement of vaginal mucosa from cylinder surface was between 0.1 and 1.09 cm (mean : 0.34 cm 0.2 cm). the dosimetric impact of air pockets was the reduction of dose to the vaginal mucosa from 0.5% to 66% (mean : 26.4% 13.9%), as presented in figure 4. even for small displacements of vaginal wall (0.1 - 0.2 cm), the dose reduction exceeds 10%. the dose reduction is increasing with the air pockets displacement, this effect being more significant for cylinders with small diameters due to the inverse square factor. the percentage dose differences between cylinder lateral surface and vaginal mucosa surface at the point of maximum displacement created by the air pockets, for various diameters of vaginal cylinders. adapting a hdr vaginal brachytherapy plan to patient specific anatomy is challenging, as recognized by the recent abs guidelines for adjuvant vaginal cuff brachytherapy after hysterectomy. the applicator selection may be influenced by the post - operative shape of vagina, but is mainly physician or institutional depending, and the most commonly used applicator is a properly sized vaginal cylinder. regardless the choice of applicator, and though the total dose required to eradicate the disease is unknown, it is essential for the vaginal mucosa to be in contact with the applicator surface to achieve the optimal dose distribution [3, 9 ]. the vaginal cylinder should be fitted to the vagina in size and introduced to reach the vaginal vault. richardson. used gold seeds to mark the vaginal vault apex and ct - imaging to verify that the vaginal cylinder reached these seeds. in our study, we have used the clinical sense and patient 's feeling to ensure that the cylinder reached the vaginal vault apex. we reported 58% incidence of air gaps in our patients and 16/78 (20%) being at the apex, while richardson. reported 80% incidence of air gaps for all patients and 21/90 (23%) located at the apex. it seems that clinical judgement works well and gold seeds are not mandatory to verify the cylinder - vaginal vault relationship. in some patients, the vagina may not be cylindrical after surgery and have an enlargement in the lateral apices, due to surgical residues of the vaginal fornices. parity, hormonal status, or normal anatomic irregularities (e.g., larger vaginal apex in relation to introitus) can make difficult the adequate placement of a vaginal cylinder. also, if the chosen cylinder is too small, air gaps or folds may be present, leading to under - dosage of the vaginal target tissue. alternative approaches for a treatment individualized to the patient 's anatomy are the use of ovoids for vaginas with dog - ear configuration, or a vaginal custom mold [10, 11 ]. the use of anatomically conformal applicators, such as intra - vaginal balloon applicator or multi - channel applicator has been also suggested [12, 13 ]. vaginal relapse rates in post - operative vaginal cuff brachytherapy for endometrial carcinoma patients are reported as ranging from less than 5% to 18% [1, 1416 ]. since the predominant location of the vaginal lymphatic channels is within 1 mm of tissue surrounding the vaginal cylinder, a possible cause of relapse is that the target volume does not receive the prescribed dose, due to the presence of air pockets. besides, many institutions do not perform fractional imaging of brachytherapy treatments ; therefore the size and significance of these air pockets are widely undetermined. we found 78 air pockets in 29/50 patients (58%) and 45/135 (33%) brachytherapy plans. out of 50 patients, 29 (58%) did not reveal air pockets during the first fraction of treatment, but had air pockets present in following fractions, and 43 (86%) patients had no air pockets during last fraction of treatment. this indicates that ct - imaging only the first fraction of brachytherapy is insufficient for assessing the conformity of vaginal mucosa to the cylinder over the whole course of treatment, as suggested by other investigators [5, 17 ] as well. yaparpalvi. analysed the inter - fraction variations of cylindrical applicator insertion, as well as the fluctuations in bladder and rectal volumes, which have led to variations of bladder and rectal doses ; they concluded that each fraction of vaginal cuff brachytherapy should be image - based, in order to achieve an accurate and complete dosimetric assessment of the treatment. analysing the correlation between the incidence of air pockets and vaginal applicator size, we found that the highest incidence of air pockets occurred for 3 cm cylinder diameter, and the lowest for 2 and 2.6 cm diameter. although selecting the largest tolerated applicator was suggested to ensure an optimal contact with the vaginal mucosa, it is not necessary to be associated with fewer incidences of air pockets. a prospective study is necessary to explore the role of using larger applicator in the remaining fractions in patients having air pockets in the first brachytherapy application. our data show that, even for small displacements of vaginal wall (1 - 2 mm), the dose reduction exceeds 10%. the dose reduction is increasing with the displacement generated by air pockets, this effect being more significant for cylinders with small diameters due to the inverse square factor. in a study analysing brachytherapy plans of 25 patients with post - operative endometrial carcinoma, they found an average pocket volume of 0.34 cm (range : 0.01 - 1.32 cm), and an average mucosa displacement of 0.37 mm (range : 0.13 - 0.8 mm), the dosimetric impact being a reduction of the dose to the vaginal mucosa of about 27% (range : 9 - 58%). cameron. compared the dose to 0.5 cm from the surface of the vaginal mucosa at air gaps location to the dose that mucosa would have received if there was no air gap, and found it ranging between 54.7% and 97.3% (average : 86.7%). although barely reported, the presence of air pockets around vaginal cylinders seems to be a frequent event in vaginal cuff brachytherapy. however, brachytherapy appears to reduce the risk of vaginal relapse despite the vaginal mucosa potentially being displaced from the applicator by air pockets [1, 1416, 18 ]. even if air pockets have a considerable dosimetric effect, it seems that it may be of minor clinical consequence. the highest risk of microscopic malignant cells prevails for vaginal mucosa located at the apex of applicator. our data show that only 20% of air pockets were located at the apex, this finding decreasing the risk of not treating the microscopic disease. moreover, except for one patient, the position of air gaps was different for each brachytherapy fraction, additionally attenuating their overall effect, as also noticed by cameron.. furthermore, for the most treatment planning systems currently available, the brachytherapy dose calculation algorithms do not take in account the tissue inhomogeneity, and the dose is calculated as if the sources are surrounded by water. yet, if low density air pockets are present, the delivered doses are fairly increased by scattering effects from adjacent tissues. the literature indicates that post - hysterectomy vaginal hdr brachytherapy is an efficient treatment, resulting in good clinical outcome for many different dose and fractionation schedules. however, the dose required to eradicate microscopic disease is still unknown. randomizing 290 patients with low - risk endometrial carcinoma to 15 vs. 30 gy in 6 fractions, prescribed to 0.5 cm vaginal tissue, sorbe. reported no difference in local recurrence. it is not yet established if this is valid for high - intermediate - risk disease too, but it is possible that a lower dose would be similarly efficient. therefore, even if air pockets are present, the dose to vaginal mucosa may still be sufficient to treat microscopic malignant cells. the magnitude of air pockets around vaginal cylinders and their effect on dose distribution was scarcely reported in the literature [5, 6 ], and our report will add more evidence that this issue substantiate attention and more research. further clinical trials are needed to estimate the uncertainties in delivering vaginal cuff hdr brachytherapy [18, 19 ]. | purposeto retrospectively assess the incidence and magnitude of air pockets around vaginal cylinders and its impact on dose distribution in vaginal cuff image - guided high - dose - rate (hdr) brachytherapy.material and methodsfifty endometrial carcinoma patients treated by postoperative hdr vaginal cuff brachytherapy were included in the study. the average age of patients was 58.3 11.8 years (range : 31 - 87 years). brachytherapy was performed using cylindrical applicators, and the dose prescribed to 0.5 cm from the applicator 's surface, over a length of 5 cm from the applicator 's tip. computed tomography (ct) simulation was used for each brachytherapy fraction. the incidence, vaginal mucosa displacement, volume, and dosimetric effect of air pockets around the vaginal cylinder were evaluated.resultsa total of 78 air pockets were found in 29/50 patients (58%) and 45/135 (33%) brachytherapy plans. they were located at the apex : 16/78 (20%) and lateral to the applicator : 62/78 (80%). the volume of air pockets ranged between 0.01 and 2.1 cm3 (mean : 0.15 cm3 0.36 cm3), and the maximum displacement of vaginal mucosa from cylinder surface was between 0.1 and 1.09 cm (mean : 0.34 cm 0.2 cm). the dose reduction to the vaginal mucosa generated by the air pockets ranged from 0.5 to 66% (mean : 26.4% 13.9%).conclusionsthe presence of air pockets around vaginal cylinder applicators is frequently noticed in post - operative vaginal cuff brachytherapy. the dose to the vaginal mucosa is reduced, as a result of displacement generated by air pockets. the effect on the clinical outcome of this dose reduction is yet to be determined. |
the fundamental notion that perception and action are based on discontinuous processing of information in discrete units is characterized by the idea of co - temporality, i.e., events within such a time unit have no before after relation (ruhnau, 1995). this is demonstrated by the analysis of psychophysical experiments assessing the perception of successiveness of two events. the sensory systems have different temporal resolutions for the detection of successiveness or non - simultaneity. the highest temporal resolution (the lowest threshold of detection) is observed in the auditory system, where two short acoustic stimuli which are only 23 ms apart are detected as non - simultaneous. the visual and the tactile system have a lower temporal resolution with respect to non - simultaneity with thresholds of some tens of milliseconds ; inter - modal stimulation leads to the highest thresholds (exner, 1875 ; lackner and teuber, 1973 ; kirman, 1974 ; lotze., 1999). the detection of non - simultaneity of two short events, however, is not perceptually sufficient to indicate their temporal order. although we may be aware that two events did not occur simultaneously, we can still be unable to tell which one of the two stimuli occurred first. the temporal order threshold, which defines the inter - stimulus interval between two events at which an observer can reliably indicate the temporal order is more comparable across senses and lies roughly at 2060 ms, to some extent depending on physical stimulus properties (hirsh and sherrick, 1961 ; fink.. a similar minimal threshold of at least 20 ms is necessary for the identification of temporal order of onset between two longer complex acoustic events, adding to the notion that the temporal - order threshold marks a fundamental limit of temporal perception (pastore and farrington, 1996). temporal order is a primary experiential temporal datum, connecting subjective experience with the objective order of events (wackermann, 2007, 2008). ultimately, the notion of time is based on the elementary temporal relation of two events, a and b, which can be judged in their temporal order, a occurs before b or a occurs after b. for example, music and spoken language are only meaningful if the correct temporal order of individual components is detected. the inversion of temporal order would lead to a different and new experience. for this reason, experienced temporal order as retrieved from memory appears in the same temporal order as when it was perceived (mach, 1911). below the experimentally assessed threshold of some tens of milliseconds the temporal order of events elements that are perceived as non - simultaneous can be inter - changed without a noticeable effect for an observer. this has, for example, lead to the idea that temporal information within a segment of the speech signal not exceeding the functional moment might not be relevant for decoding spoken language (kiss., the relation between the perception of speech and the perception of temporal order has repeatedly been demonstrated in studies with neurological patients suffering from aphasia and with adolescents who have language - learning impairments (wittmann and fink, 2004). these individuals have difficulties in discriminating consonants, which requires the ability to detect temporal order of speech signal components, because they have increased auditory temporal order thresholds (wittmann., 2004 ; fink., 2006b). based on the empirical findings of discrete processing in perception and action it has therefore been suggested that the brain creates a - temporal system states during which incoming information is treated as co - temporal, and which are on the one hand responsible for binding intra- and inter - modal information and on the other hand create the experience of temporal order (pppel., 1990 ; ruhnau, 1995 ; pppel, 1997). the idea that perceptual information as well as motor commands might be processed in discrete packets, at regular moments in time (dehaene, 1993 ; vanrullen and koch, 2003 ; van wassenhove, 2009), is in accordance with the conception of a functional moment, a snapshot of perception. findings of several independent empirical approaches in neuroscience have lead to the suggestion that temporal building blocks in sensory and cognitive processing exist responsible for creating discrete functional units in time as well as binding spatial features into perceptual wholes. these temporal units have been related to rhythmic brain activity of thalamo - cortical loops, the gamma, 1994 ; basar - eroglu., 1996 ; fries., 2007 however, periodicities in the alpha band (around 10 hz) as well as the theta band (48 hz), and potentially related to functional integration on a time scale of 100 ms and above (see below), may additionally contribute to temporal integration phenomena (vanrullen and koch, 2003 ; van wassenhove, 2009). it is important to note that basic temporal integration mechanisms uniting disparate events into perceptual segments have been identified on different time scales and also in more complex inter - sensory perceptual tasks. for example, a time frame of about 200 ms determines the integration of auditory visual input in speech processing when probing for the mcgurk effect an illusory fusion percept created when lip movements are incongruent to heard syllables (van wassenhove., 2007). the fusion percept was reported if the onset of lip movement and syllable did not exceed this time lag. temporal integration in a time frame of around 250 ms was reported in sensory motor processing distinguishing maximum tapping speed from a personal, controlled motor speed (peters, 1989 ; wittmann. when inter - tap intervals are around 150 ms, movements are too fast to be represented as individual button presses within an ordered sequence. only when the movement slows down and inter - tap intervals exceed at least 250 ms individuated button presses are experienced as following each other. stimulus durations of 200300 ms (and minimum inter - onset intervals) are a necessary prerequisite for the establishment of temporal order representation for the detection of the correct sequence of four acoustic or visual events (warren and obusek, 1972 ; ulbrich., 2009). when stimuli are shorter, or the inter - onset between stimuli is smaller, subjects can not reliably report the temporal order of the presented sequence. an interpretation of these finding is that if two or more stimulus onsets fall within one window of temporal integration then temporal order can not be experienced as the onsets are treated as co - temporal. regarding this approximate time range, it has been proposed that anterior insular cortex function may provide the continuity of subjective awareness by temporally integrating a series of elementary building blocks successive moments of self - realization informed by the interoceptive system (craig, 2009). the continuous processing from moment to moment would advance with a frame rate of about 8 hz, these temporal building blocks of perception lying in the range of 125 ms (picard and craig, 2009). neural microstates with average duration of 125 ms as derived from electrophysiological recordings have been discussed as potential atoms of thought, constituting critical time windows within which neural events are functionally integrated (lehmann., 1998). in combining the two kinds of functional moments presented here, endogenous cortical rhythms in the gamma and theta range involved in speech perception and production speech would be processed by the left auditory cortex, integrating the signal into 2060 ms segments which would correspond to phoneme length ; at the same time speech would be processed in the right auditory cortex, integrating the signal into segments of 100300 ms corresponding to syllabic analyzes (poeppel, 2003 ; giraud., 2007). in the context of findings of various temporal integration phenomena it has moreover been proposed that mental processing is organized in multiple ranges of discrete periods which are all multiples of an absolutely smallest quantal period estimated to lie at approximately 4.5 ms (geissler and kompass, 2003). it has to be noted that research has identified further integration phenomena, all being on comparable time scales (fraisse, 1984). for example, a temporal integration window of 100150 ms duration has been suggested to operate for perceptual grouping mechanisms of target and distractor tones in sensori - motor processing (repp, 2004). using the paradigm of mismatch negativity of magnetic brain responses a window of integration of 160170 ms was estimated to bind successive auditory input into an auditory percept (yabe., 1998). further integration levels below 1 s as related to the processing of static stimuli as well as associated with motion perception are not presented, but see fraisse (1984) and dainton (2010). what these divergent findings have in common is that on a temporally fine grained level in the range of tens of milliseconds as well as of hundreds of milliseconds temporal integration phenomena occur that are the basis for the experience of temporal unity of events (below the threshold) and of succession and temporal order (above the threshold). below the reported thresholds of around 30 ms (when two short events are presented) or of thresholds ranging between 100 and 300 ms (when a stream of events is presented) temporal integration provides functional moments of experienced co - temporality. one could argue that these functional moments, within which events are fused together, are not experienced as having duration. although the sensation of non - simultaneity implies two temporally separated events, one could nevertheless say that the experience of duration necessitates a clearly demarcating onset a and offset b defining an interval (with the inherent temporal order a before b). in this sense one can state that below the temporal order threshold, when two events have no clear temporal relation, subjective duration between the two stimuli is not experienced ; the functional moment has no perceivable duration. despite the possibly discrete nature of underlying processes in perception and cognition, our phenomenal experience is nevertheless characterized as evolving continuously (vanrullen and koch, 2003). only in rare neurological disorders or under the influence of pharmacological agents such as lsd individuals occasionally report of perceiving a series of discrete stationary images (dubois and vanrullen, 2011). however, we typically do not perceive static snapshots of the world but perceived events are embedded in an ongoing stream of experience. music and language are only conceivable as consisting of extended moments, melodies, and phrases, which inter - connect individual musical and linguistic elements (wittmann and pppel, 2000). even when we focus on an individual note in a musical piece or a word in a spoken sentence, these acoustic events can only be understood in its temporal relation to the preceding and the following musical or language structure. lloyd (2004, 2011) has an intuitive example for temporality in music : when we hear paul mccartney land on jude, the hey is still somehow present although no longer sensed (husserl s retention). a listener familiar with the beatles, when she hears the hey can not help but already hear the jude. the jude is somehow present but it is actually only anticipated (husserl s protention). in phenomenological terms, what we perceive at present is strongly intertwined with what has just happened and what is about to happen (lloyd, 2004, 2011 ; kiverstein, 2010). when listening to a metronome at moderate speed, we do not hear a train of individual beats, but automatically form perceptual gestalts as an accent is perceived on every nth beat (12, 12, or 123, 123). these temporal units are mental constructs physically speaking, they do not exist (pppel, 2009). if the metronome is too fast, the inter - beat intervals are very short, a fast train of beats is perceived that can not be experienced as containing temporally separated events with an ordered temporal structure. if on the other hand the metronome is too slow, inter - beat intervals are too long, only individual beats which are not related to each other are perceived (111 etc.). this lower and upper range of the metronome speed at which accentuated temporal structures can be heard defines the temporal limits of perceptual grouping on this time scale. empirical evidence suggests that these mental units comprising several individual beats have a lower limit of around 250 ms and an upper limit of approximately 2 s (szelag., 1996 ; further empirical observations revealed through a systematic variation of duration indicate that empty intervals marked by two acoustic events larger than 150250 ms and shorter than 2 s are perceived as qualitatively different than intervals beyond these temporal boundaries (benussi, 1913 ; nakajima., 1980). for example, two sound bursts separated by an interval below 150 ms were perceived as one double - peaked sound ; between 150 ms and 2 s the two sound bursts were clearly separated from each other but subjects still felt a relation between them and they tried to automatically synchronize their body movements to the stimulus pair ; with intervals larger than 2 s subjects reported that the two sounds were difficult to relate to each other and synchronization of body movements was not attempted (nakajima., 1980). whereas findings of qualitative as well as quantitative differences between intervals below and above 2 s are predominantly found by presenting empty intervals that are marked by two sounds (for a psychophysical study see getty, 1975), also regarding filled intervals it has been shown that duration up to 23 s is differently processed than duration exceeding 3 s (ulbrich., 2007). however, results are not as clear cut as with empty intervals and break points are not always found (noulhiane., 2008 ; lewis and miall, 2009). subjects can synchronize their motor actions to a sequence of presented tones with inter - stimulus intervals of above 250 ms (peters, 1989). however, this synchronization ability can only be maintained when inter - tone intervals do not exceed durations of about 23 s. with longer intervals precise anticipation of tones effortless timing of behavior breaks down (mates., 1994). in a further analysis of this type of timing behavior, time ranges between 0.45 and 1.5 s seemed to be processed automatically, i.e., not strongly affected by secondary task fulfillment, whereas concomitant processing of a secondary task affected intervals in the range between 1.8 and 3.6 s (miyake., 2004). also the phenomenon of perceptual bi - stability suggests itself for studying temporal constraints of conscious experience as one can easily tap into the subjective percepts (leopold., 2002 ; the temporal analysis of bi - stable perception has been suggested as primary experimental approach for the understanding of the dynamics of mental states (atmanspacher and filk, 2010). in essence, an ongoing competition between the neural representations of the two aspects of an ambiguous figure, such as the necker cube, has to be resolved leading to the experience of one of the two perspectives at a given point in time. during continuous presentation, one aspect lasts on average for around 3 s before a switch in perspectives occurs, with some inter - individual variability and variance attributable to stimulus characteristics of the particular ambiguous figure (gmez., 1995 ; given its temporal dynamics, the spontaneous switching rate has been discussed as stemming from the discussed temporal segmentation mechanism related to the subjective present (varela, 1999 ; franck and atmanspacher, 2009 ; pppel, 2009). for example, it has been proposed that the temporal integration mechanism of around 3 s, evoking our feeling of nowness, integrates successive processing units of around 30 ms, functional moments (pppel, 1997, 2009 ; szelag., 2004). in another line of research that treats bi - stable perception as evolving from unstable two - state systems it was proposed that different mental processing stages have temporal properties matching the found temporal integration levels of 30, 300, and 3 s (atmanspacher., 2004 ; atmanspacher and filk, 2010). in combining the empirical findings, temporal integration of a few seconds has been suggested to provide the logistical basis for the subjective present (pppel, 1978, 2009 ; fraisse, 1984 ; szelag., 2004). whereas the duration of the functional moment is not perceived, an experienced moment relates to the experience of an extended now. according to this conception, the experienced moment has duration. a temporal interval with duration exceeding about 3 s is experienced as being qualitatively different than shorter duration. when two events are separated by an interval of, say, 6 s, the experience of emptiness evolves, events are not bound together and the length of the interval separating the two becomes the focus of attention (wackermann, 2007). a pause in a conversation, if it reaches 6 s, might be felt as disturbingly long. in that sense, duration longer than 3 s leads to the predominant experience of an extended temporal interval. on the other side of the spectrum, it is well possible to judge the duration of 1 h, i.e., pressing a button every time one thinks that an hour has passed (aschoff, 1998). however, it is impossible to maintain a 1-h time interval continuously in the focus of awareness (wackermann, 2007). during such a period of 1 h a multitude of experiences accumulates that later can be retrieved from memory forming temporal cues that can be used to judge duration retrospectively (zakay and block, 1997). but then the question remains what the upper limit of integration in prospective time perception might be, that is, of the perception of duration as presently and continuously experienced ? more generally formulated and more importantly, what are the temporal boundaries of perception that allow us to hold events in present experience, in mental presence ? whereas the experienced moment forms an elementary unit, a temporally unified percept, mental presence involves the experience of a perceiving and feeling agent (my self) within a window of extended presence, a phenomenon that is based on working memory function. working memory provides a temporal bridge between events both those that are internally generated and environmentally presented thereby conferring a sense of unity and continuity to conscious experience mental presence encloses a sequence of such moments for the representation of a unified experience of presence. reports from neurological case studies with individuals who suffer from anterograde amnesia after bilateral damage to the hippocampus indicate that these patients live within a moving temporal window of presence that does not reach beyond their short - term or working memory span, incapable of storing incidents into episodic long - term memory (scoville and milner, 1957). these patients can hold information for a limited time in memory, they perform short tasks accurately and seemingly behave normal ; but already after a few minutes they can not recall what has just happened (for a striking description of a patient with anterograde amnesia, see sacks, 1986). due to their neurological impairment, patients with anterograde amnesia can act adequately within the temporal constraints of their functioning working memory, which accordingly must be a temporal constraint for mental presence, the continuous awareness of oneself as presently perceiving and acting within an environment. these clinical cases emphasize the functioning of short - term memory in healthy humans and how it can be interpreted as forming temporal boundaries of present awareness. experimental investigations of short - term memory show how the number of correct recalls of presented syllables decreases with increasing interval length between stimulus presentation and recall in the range of multiple seconds if the rehearsal of syllables is prevented (peterson and peterson, 1959 ; baddeley, 1990). the capacity of short - term retention is defined as gradual loss of memorized elements as time passes. the typical retention functions described by logarithmic and exponential fits decrease rapidly at first and then levels out on a plateau (rubin and wenzel, 1996). one could state that the time frame provided by short - term memory (related, working memory) creates a temporal horizon of experience which in humans contains descriptive narrative elements created by our capacity for language (varela, 1999). within this temporal horizon mental presence unfolds integrating mental processes and enabling conscious experience of a narrative self that has personal identity and continuity over time (gallagher, 2000). mental presence is bound to the ability of maintaining mental representations in an active state for a certain period of time. it depends on the integration of multiple mental operations that lead to intentional behavior created by ongoing activity of a global workspace, integrating activity from multiple distributed and specialized brain areas (baars, 1988 ; dehaene and naccache, 2001). essentially, it is working memory, a system of limited attentional capacity, supplemented by visuospatial, episodic, and phonologic storage systems, which holds information for temporal storage and manipulation (baddeley, 2003). in duration reproduction tasks, individuals have to reproduce temporal intervals by pressing a key to indicate that a second comparison stimulus has reached the duration of a previously presented stimulus. the mean of reproduced intervals is accurate for shorter intervals of up to 3 s but with increasing interval lengths are progressively under - reproduced relative to physical time (eisler and eisler, 1992 ; wackermann, 2005 ; ulbrich. the negative curvature of the duration reproduction function results in an asymptotic upper limit of duration accessible to experience, i.e., a temporal horizon of experienced time in the range of roughly 10 s (wackermann, 2007). note, that the negative curvature in duration reproduction performance is found in those studies where subjects are instructed or discouraged from counting (rattat and droit - volet, 2011). without chronometric counting, the immediate experience of duration is limited by an ultimate temporal horizon of reproducibility due to memory - loss of duration representation over time ; temporal resolution of duration blurs with increasing interval length (wackermann, 2007, 2008). it is tempting to suggest that a healthy individual s short - term memory span is related to the upper limit of prospective time perception ; the limits of temporal experience, of perceiving duration continuously, would thereafter rely on the basic temporal properties of working memory. in fact, decay of memory traces has been discussed to underlie the experience of duration. since memory strength decreases with time, memory trace decay could actually function as a clock (staddon, 2005). this memory - loss component is an intrinsic feature of the dual klepsydra model (wackermann and ehm, 2006 ; wackermann, 2008), where subjective duration is represented by the state of a lossy accumulator. this accumulator receives inflow for the build - up of duration presentation of a stimulus that has to be judged. a simultaneous outflow reflects the loss of representation leading to typical responses in psychophysical tasks, indicative of subjective shortening of stored duration over time. the reports of a limiting value in the order of magnitude of 10 s in the ability to reproduce duration indicate that the representation of increasing duration becomes more and more compressed. the duration reproduction curve becomes increasingly flatter, i.e., with increasing temporal intervals differences in physical duration are represented with decreasing resolution (wackermann, 2007). that is, the reported limits in duration reproduction and short - term memory do not point to absolute and static boundaries correspondingly, mental presence has no fixed duration but to a gradual dissolving of representation with increasing duration. related to this temporal characteristic, mental presence is related to the fact that once attended objects slowly phase out of experience over time ; that is, the phenomenally experienced sliding window of mental presence co - occurs with the constant loss of memory contents. the moving window of presence is related to the constant sequential input of a sequence of perceived events, which each fade out of working memory one after the other after some time. mental presence is a temporal platform of multiple seconds within which an individual is aware of herself and the environment, where sensory motor perception, cognition, and emotion are interconnected features of representation leading to phenomenal experience. facing the puzzle of how we can perceive duration of events if our perception is bound to the present moment, more than 1600 years ago st. although the present (praesens tempus) has no extension as it passes away in a moment (in puncto praeterit), an observation nevertheless has extension (distentio) through temporally lasting attention (attentio) which encompasses the anticipation of events (expectatio) that eventually fade into memory (memoria) ; here, anticipation and memory are part of an extended present where attention lasts for some time (confessions, book xi ; section xxviii.37 in flasch, 1993). moreover, conscious experience involving a sense of self may only be understood as an entity that is extended over time (zahavi, 2005 ; wittmann, 2009 ; stolzenberg, 2010). augustine s conception of time has strong similarities to husserl (1928) tripartite structure of present experience. although a variety of philosophical models concerning the present moments exists, important in the present context is the common insight that the present moment is experienced as extended. also our everyday language use implies that what is happening at the present moment typically has duration. present experience on the level of content is a continuously evolving phenomenon and individual events are embedded in an extended temporal field (stern, 1897 ; lloyd, 2004, 2011). a suddenly occurring short stimulus of a few milliseconds moreover, psychophysical investigations in which stimulus properties are systematically varied reveal thresholds of experience below which no temporal relationship is perceived. in these limiting cases disclosed by psychophysics one can actually speak of functional moments without perceivable duration because temporal order, a primary experiential datum, is not detected. for example, if a short tone with a lower pitch is followed by another short tone with a slightly higher pitch, the impression of a single tone rising in pitch can be elicited (fink., 2006a). within different modalities, the rapid serial appearance of two or more stimuli at different positions can give the impression of apparent motion (exner, 1875 ; kirman, 1974). that is, the common cases of perception, in accordance with the phenomenological analysis of our lebenswelt, suggest that subjective time provides a frame of reference within which moments are stretched out over time. the integration level of the experienced moment is the basic operational platform within which temporality can evolve. form, as short as the overall duration may seem, involves anticipation and memory which are activated while the some people might even anticipate the do nt make it bad. then, however, the two verses may already fall into two different experienced moments. the break between the two subunits hey jude and do nt make it bad points to a general principle in poems and in music where caesurae form boundaries between which individual verses are recited ; across different languages and cultures the duration of these lyrical and musical units seems not to exceed 3 s (pppel, 1988 ; turner and pppel, 1988). beyond the experienced moment is a stronger bounded unit than hey jude, do nt make it bad. and with increasingly longer intervals temporally separate components within these units will become less strongly connected ; retention and protention are temporally limited in the way that they cover only what has just happened and is about to happen in the range of a few seconds, working memory span forming a boundary of present experience as mental presence. the discussed examples in literature of experienced temporality refer to situations concerning temporal integration in the several seconds range. husserl (1928, p. 383) discusses the situation of hearing a melody which is only possible because individual tones are integrated to form a perceptual whole. kelly (2005) presents the example of hearing a steady high c produced by an opera singer who holds that note for some time for the listener subjectively going on for a long time that is, for several seconds. different from this phenomenological notion of retention and protention is the conception of time perspective as a fundamental cognitive dimension partitioning human experience into past, present, and future (zimbardo and boyd, 1999). past and future in this context can span decades, as far as long - term memory reaches back in time and as far as we plan our future. through this partitioning of time, the notion of a presence becomes meaningful since the explicit representation of the present perspective is only possible through its distinction from past and future (droege, 2009). within the realm of the mental presence an individual can be considered fully operational as she tracks current conditions, compares them with memories of past incidences, and makes plans for the future. the past and the future as presently experienced can also be explicitly judged in relation to subjective time. when judging the past, we typically perceive the decades of our lives to speed up as we get older (wittmann and lehnhoff, 2005). related to the future, we constantly generate predictions about how long it will take for some events to occur, these temporal estimates eventually leading to decisions regarding options with different delays (wittmann and paulus, 2009). for example, a person who chooses to save money opts for a momentary loss of money that otherwise would be available now in order to gain a future greater benefit. a fundamental question remains of how lower - level temporal units are bound together forming higher - level units ; a variety of concepts has been rigorously discussed by dainton (2010). independent of how the sequential units of lower - level units might be related to each other, potentially overlapping each other or following in direct succession, continuity of experience has been considered to stem from an ongoing semantic connection across individual segments, which masks the discontinuity of the sequential units (pppel, 1997 ; droege, 2009). regarding specifically the continuity of experience across experienced moments, working memory related to semantic and episodic content might bind together the sequence of temporal segments of nowness that leads to the experience of mental presence. it is also a prerequisite for interpersonal communication between two individuals made possible by synchronizing the moments of individuals, thereby creating shared moments of presence for effortless interaction an essential feature in music, conversation, and dance (wittmann and pppel, 2000). however, the experience of a self acting in its environment, remembering the past and planning the future necessitates an integration interval as has been related to mental presence exceeding the postulated 3-s time window of the experienced moment. continuity of experience only unfolds as mental presence, which is a floating window of feeling present and acting at present. the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. | it has been suggested that perception and action can be understood as evolving in temporal epochs or sequential processing units. successive events are fused into units forming a unitary experience or psychological present. studies have identified several temporal integration levels on different time scales which are fundamental for our understanding of behavior and subjective experience. in recent literature concerning the philosophy and neuroscience of consciousness these separate temporal processing levels are not always precisely distinguished. therefore, empirical evidence from psychophysics and neuropsychology on these distinct temporal processing levels is presented and discussed within philosophical conceptualizations of time experience. on an elementary level, one can identify a functional moment, a basic temporal building block of perception in the range of milliseconds that defines simultaneity and succession. below a certain threshold temporal order is not perceived, individual events are processed as co - temporal. on a second level, an experienced moment, which is based on temporal integration of up to a few seconds, has been reported in many qualitatively different experiments in perception and action. it has been suggested that this segmental processing mechanism creates temporal windows that provide a logistical basis for conscious representation and the experience of nowness. on a third level of integration, continuity of experience is enabled by working memory in the range of multiple seconds allowing the maintenance of cognitive operations and emotional feelings, leading to mental presence, a temporal window of an individual s experienced presence. |
three broad categories of journals are available for faculty scholarly publications, excluding individual or standalone publications not connected to a publisher platform. traditional journals are available through hardcopy and/or electronic journal subscription. as a gold standard, such journals are affiliated with reputable publishers with archiving and indexing capabilities. the journals are : a) free of charge to readers, b) free of permission barriers (copyright law, licenses, hardware / software digital rights management), c) indexed for easy location, d) deposited in supported oa archives or repositories, and e) contained in a readily identifiable oa label (suber, 2003). a number of definitions of scholarly oa exist and are available at the www.mlanet.org/resources/publish_/sc_open-access.html website. to maintain the more traditional, scholarly standards for manuscript publication, three declarations, known as the bbb declarations, were among the first established as the basis for designating oa journal credibility : budapest open access initiative defined the term open access in 2002 and recommended institutions of higher education develop a policy for institutional repositories in 2012 (www.budapestopenaccessinitiative.org/read). bethesda statement on open access publishing drafted statements of principle for oa publishing (2003) (http://legacy.earlham.edu/~peters/fos/bethesda.htm). berlin declaration on open access to knowledge in the sciences and humanities specified the measures for consideration of oa dissemination (2003) (http://openaccess.mpg.de/berlin-declaration). predatory publishing is a term conceived by university of colorado denver librarian and researcher jeffrey beall. defining a predatory journal is complex and requires examination of the publisher s content, practices, and websites. predatory oa publishers do not follow established academic practices for publication and primarily exist as fee - collecting operations (berger & cirasella, 2015). traditional journals are available through hardcopy and/or electronic journal subscription. as a gold standard, such journals are affiliated with reputable publishers with archiving and indexing capabilities. the journals are : a) free of charge to readers, b) free of permission barriers (copyright law, licenses, hardware / software digital rights management), c) indexed for easy location, d) deposited in supported oa archives or repositories, and e) contained in a readily identifiable oa label (suber, 2003). a number of definitions of scholarly oa exist and are available at the www.mlanet.org/resources/publish_/sc_open-access.html website. to maintain the more traditional, scholarly standards for manuscript publication, three declarations, known as the bbb declarations, were among the first established as the basis for designating oa journal credibility : budapest open access initiative defined the term open access in 2002 and recommended institutions of higher education develop a policy for institutional repositories in 2012 (www.budapestopenaccessinitiative.org/read). bethesda statement on open access publishing drafted statements of principle for oa publishing (2003) (http://legacy.earlham.edu/~peters/fos/bethesda.htm). berlin declaration on open access to knowledge in the sciences and humanities specified the measures for consideration of oa dissemination (2003) (http://openaccess.mpg.de/berlin-declaration). predatory publishing is a term conceived by university of colorado denver librarian and researcher jeffrey beall. defining a predatory journal is complex and requires examination of the publisher s content, practices, and websites. predatory oa publishers do not follow established academic practices for publication and primarily exist as fee - collecting operations (berger & cirasella, 2015). there has been ongoing popularity growth due to the timely publication for scholarly work (suber, 2013). key, identifiable characteristics of each major category of journals discussed are presented in table 1, which is synthesized from beall s (2012, 2015) work and supported by others (nicoll, 2014). traditional, scholarly open access, and predatory open access journal credibility characteristics the medical library association supports the bbb declarations, and strong support exists for timely access to useful content via faster publication in oa journals. ethics concerns tend to focus on whether charging authors to publish in an oa journal represents a conflict of interest. alternatively, charging both readers and authors to subscribe to or purchase traditional hardcopy and/or electronic journal access has been accepted as the way publications can occur and could also be considered a conflict of interest. the primary ethical issue surrounding publication in predatory oa journals is based on the purpose for establishing the journal, specifically, the for profit focus of the fraudulent journal owner(s) (coyle, 2013). the alert author is aware (or needs to be aware) of warning signs to distinguish predatory oa publication opportunities from scholarly oa journal opportunities. there are a number of important questions authors need to consider before submitting a manuscript. a brief list follows (beall, 2012) : does the publishing opportunity sound too good to be true ?) are articles from the journal available only on google scholar ? (google scholar provides access to all publications without screening for quality.) is the publisher listed on beall s list of either questionable journals or questionable publishers (updated at least annually) ? is the journal name interesting or a variation of a scholarly journal name, for example, the american journal of _ _ _ _, national journal of _ _ _, or international journal of _ _ _ _ ? is the home office located in an emerging or third - world country (e.g., india, kenya, malaysia, nigeria, nigeria, pakistan, romania) ? is the home office located in a small, old storefront or listed as a post office number ? the medical library association supports the bbb declarations, and strong support exists for timely access to useful content via faster publication in oa journals. ethics concerns tend to focus on whether charging authors to publish in an oa journal represents a conflict of interest. alternatively, charging both readers and authors to subscribe to or purchase traditional hardcopy and/or electronic journal access has been accepted as the way publications can occur and could also be considered a conflict of interest. the primary ethical issue surrounding publication in predatory oa journals is based on the purpose for establishing the journal, specifically, the for profit focus of the fraudulent journal owner(s) (coyle, 2013). the alert author is aware (or needs to be aware) of warning signs to distinguish predatory oa publication opportunities from scholarly oa journal opportunities. there are a number of important questions authors need to consider before submitting a manuscript. a brief list follows (beall, 2012) : does the publishing opportunity sound too good to be true ?) are articles from the journal available only on google scholar ? (google scholar provides access to all publications without screening for quality.) do the journal board members list any articles actually published in the journal ? is the publisher listed on beall s list of either questionable journals or questionable publishers (updated at least annually) ? is the journal name interesting or a variation of a scholarly journal name, for example, the american journal of _ _ _ _, national journal of _ _ _, or international journal of _ _ _ _ ? is the home office located in an emerging or third - world country (e.g., india, kenya, malaysia, nigeria, nigeria, pakistan, romania) ? is the home office located in a small, old storefront or listed as a post office number ? one strategy to use is the directory of open access journals (doaj), where journals accepted after march 2014 have met revised, stringent criteria for inclusion. journals without the symbol were approved prior to march 2014, and the publishers have been asked to reapply for acceptance under the more stringent criteria. another option is to evaluate relative importance of a published article via impact factor, a calculation used as a measure of article quality, based on frequency of article citation by others in the field (springer, 2013). the assumption is, if other scientists / authors are willing to build on content published in the original article, then the original article must be valid (garfield, 1996). reputable examples of databases with impact factor include scopus, journal of citation reports, web of science, or the new scielo citation index. unfortunately, impact factors may be used by questionable publishers and databases, as well, and not all reputable publications have an actual calculated impact factor. if navigation goes in circles, the topic being searched can not be located in a database about the topic, and/or the editors do not reply to requests for additional information, then the website / database may be questionable. finally, a useful strategy for scholarly publication is to use the public library of science and biomed central websites. each site started as a location where authors could publish faster with credibility. both currently charge for publication. public library of science charges approximately $ 2,500 per article, and biomed central charges from $ 1,450 to $ 3,000 (suber, 2003). the essential peer review process is critical for ensuring quality, ethical publication and tends to be a source of publication and production fees. thus, requiring authors to pay for peer review may not be all bad as some individuals believe, but costs need to be reasonable. characteristics of scholarly oa journals are compatible with many characteristics of traditional journals, including the four key criteria of archiving / preservation, reputable board members, indexing, and peer review. thus, authors need to complete due diligence in reviewing both publisher and journal with the help of the aforementioned resources and then decide where and whether to submit a manuscript for publication. do not prematurely limit publication possibilities because of professional blindness toward author - pays requirements, while evaluating opportunities for publication in scholarly oa journals. in addition, promotion and tenure committees need to recognize the value of oa publications. finally, check beall s list of predatory publishers (https://scholarlyoa.com/publishers/) to identify predatory publishers and the doaj after march 2014 (https://doaj.org) to identify safe journals before submitting a manuscript. | abstractaimthe purpose of the article is to alert faculty about predatory online journals, review characteristics of three broad categories of journals, and provide suggestions for faculty evaluation of journals before submission of scholarship for publication.backgroundthe availability of online journals in recent years has rapidly increased the number of journals available for publication of faculty scholarship. however, not all online journals meet the same standards as traditional journals.methodthe article is not a report for a research study.resultscurrently, there are three broad categories of journals for faculty scholarship publication : traditional, open access scholarly, and predatory open access journals.conclusionfaculty authors need to carefully evaluate the journal characteristics and publisher business practices before submitting a manuscript for publication to prevent inadvertent submission to a predatory open access journal. |
lipopolysaccharide (lps) is a biomedically highly relevant glycolipid located in the outer leaflet of the cell membrane of gram - negative bacteria. lps is essential for many functions of the bacterial membrane, providing stability, a protective shield, and permeation control, and is thus indispensable for the viability of bacteria. lps is also a major virulence factor which is involved in a multitude of interactions with the adaptive and innate immune system of respective host organisms. in structural terms, lps of enterobacteriaceae comprises a highly variable o - antigenic polysaccharide chain followed by a core region which provides the link to the endotoxic lipid a domain. within the core region, the inner part of enterobacterial lps is composed of the higher carbon sugars 3-deoxy - d - manno - oct-2-ulosonic acid (kdo) and l - glycero - d - manno - heptose (l, d - hep) residues. specifically, phosphorylated heptosyl units are important antigens suitable for the development of vaccines against pathogenic bacteria such as haemophilus influenzae and neisseria meningitidis. a prototype phosphorylated heptose core structure as found in escherichia coli and salmonella enterica lps is shown in figure 1. common inner core oligosaccharide domains in enterobacterial lps. the branched 4-o - phosphorylated heptosyl trisaccharide core domain has recently been reported as essential part mediating the binding to the cross - reactive antibacterial monoclonal antibody wn1 222 - 5. of note, the paratope of this antibody mimics the receptor binding site of toll - like receptor 4 (tlr-4), wherein the 4-o - phosphoryl heptosyl domains are involved in multiple ionic and hydrogen - bonded interactions. in addition, l, d - heptosyl residues have recently been reported to interact with c - type lectins such as lung surfactant protein d (sp - d), concanavalin a, as well as bacterial lectins from burkholderia cenocepacia. thus, the chemical synthesis of defined heptosyl oligosaccharides is a challenging task in order to further elucidate the molecular basis of these biomedically relevant protein lps interactions and to provide ligands for immunochemical applications to be translated into future vaccine development. the chemical synthesis of suitably protected heptosyl building blocks has been accomplished by de novo approaches as well as by exquisite orthogonal protecting group manipulations followed by selective glycosylation strategies to produce spacer - equipped oligosaccharides corresponding to lps - inner core part structures related to yersinia pestis, pseudomonas aeruginosa, n. meningitidis, and h. influenzae antigens. the latter target structures comprised 4-o--d - glucopyranosylheptosyl units with 6-o - phosphorylethanolamine substitution introduced at the neighboring l, d - hep residue. the disaccharide fragment hep4p-(13)-hep4p has previously been synthesized from an orthogonally protected disaccharide precursor which was phosphorylated following cleavage of the respective protective group at either of the 4-positions. herein we disclose a highly convergent approach allowing access to -glucosylated 4-o - phosphorylated heptosides based on an early introduction of a 4-o - phosphotriester moiety, thereby minimizing the number of protection and deblocking steps. in addition, in combination with a regioselective opening of a 1,1,3,3-tetraisopropyldisiloxane-1,3-diyl (tipds) protecting group to give o-7 acceptor heptoside derivatives, a straightforward assembly of several (17)- and (13)-linked 4-o - phosphorylated heptosides, has been elaborated. the common enterobacterial heptose region as shown in figure 1 reveals a repetitive substitution pattern comprising two heptosyl units harboring a 4-o - phosphoryl substituent which are extended at position 3 by another glycose residue (note : a reversed pattern is seen in n. meniningitidis and h. influenzae lps, wherein a -d - glucopyranosyl residue is present at o-4). the 3-o--d - glucosyl substituted heptose residue is additionally substituted at o-7 by a second heptose unit. thus, retrosynthetic analysis would suggest assembling these ligands from a side - chain protected heptoside precursor to be converted into the corresponding 4-o - phosphotriester intermediate (figure 2). regioselective 2,3-o - orthoester opening should then allow for extension at o-3, while regioselective opening of the protecting group at the side chain would generate a 7-oh glycosyl acceptor. thus, several phosphorylated heptosyl lps ligands would be accessible from a common 4-o - phosphorylated building block, thereby minimizing the number of protecting group manipulations a highly important issue in current oligosaccharide synthesis. the presence of a protected phosphate moiety to be kept throughout the synthesis, however, clearly implicates additional synthetic challenges to be met during the assembly of the oligosaccharide. methyl l - glycero - d - manno - heptopyranoside 1, obtained previously by the brimacombe approach, was used for the regioselective protection of the side - chain diol at c6 and c7. reaction of crystalline 1 with tipdscl2 in the presence of imidazole gave directly the 6,7-o - tipds - protected derivative 2 in 87% yield (scheme 1). starting from the tipds - protected heptoside 2, two approaches for the differentiation of the remaining hydroxyl groups were developed via initial selective syn-2,3-o - orthoester formation. the first approach utilized the 2,3-o - orthobenzoate as a temporary protecting group that is sufficiently stable to allow phosphorylation using phosphoramidite - based coupling methodology. indeed, reaction of the intermediate orthoester with dibenzyl n, n - diisopropylaminophosphoramidite/1h - tetrazole followed by oxidation with m - cpba gave a separable mixture of the two diastereomers 3a/3b (3:1) in 89% yield. the phosphoester substitution at position 4 was confirmed by the respective heteronuclear h / p and c / p spin - coupling interactions. selective acid - induced orthoester opening then furnished 2-o - benzoate 4 in 72% yield together with minor amounts of the corresponding 3-o - benzoate 5. the orthoester mixture was also directly converted into 2,3-di - o - benzoate 6 in a two - step yield of 89%. alternatively, the intermediate orthobenzoate was directly opened by the action of camphorsulfonic acid to give 2-o - benzoate 7 (82%) which was further transformed into the 3-o - lev - protected compound 8 via a steglich - type protocol in 96% yield. almost complete regioselectivity in the latter step was achieved by gradually treating the reaction mixture with a solution of dcc (note : the 3,4-di - o - levulinated byproduct, however, was readily formed when the reaction was performed under conventional reaction conditions). formation of the corresponding 4-o - lev - protected isomer was not observed. subsequent phosphotriester formation at position 4 of the 3-o - lev derivative 8 was uneventful and delivered the orthogonally protected compound 9 in 81% yield. next, the 7-oh acceptor derivatives 10 and 11 were approached by a selective partial cleavage of the 6,7-o - tipds group of fully protected intermediates 6 and 9, respectively. this methodology was originally developed by the group of ziegler for hexopyranosides and has so far been exclusively used in this context. initially, this protodesilylation was attempted with hf - pyridine as reagent, according to the published protocol, but turned out to be difficult to elaborate into a reliable procedure for the regioselective opening of the exocyclic tipds group in heptoside derivatives 6 and 9, respectively. even when the reagent was added in moderate excess and at low temperatures, the undesirable cleavage of both still, by close monitoring of the reaction and careful handling during the workup, a good selectivity and high isolated yield could be achieved for compound 10. eventually, exchange of the reagent to triethylamine trihydrofluoride (treat) allowed for a better control of the reaction progress and resulted in a reliable and scalable preparation of 10 and 11, respectively, since complete desilylation by treat would require substantially more reagent amounts, higher temperatures, and extended reaction times. the 6-o - ftipds - protected acceptor 10 was stable upon storage for several months in a refrigerator. also, a trial experiment of acceptor 11 under glycosylation conditions (at 40 c in the presence of boron trifluoride etherate) indicated that the 6-o - ftipds group was not affected. the 4-o - phosphorylated heptoside acceptor derivatives 4, 10, and 11 then served as versatile precursors for ready attachment of sugars at o-3 (4) as well as o-7 (10, 11), allowing also for subsequent coupling steps to give o-7/o-3 disubstituted products. for the synthesis of heptosyl oligosaccharides, trihaloacetimidate leaving groups developed by schmidt initially, the readily available per - o - acetylated n - phenyltrifluoroacetimidate donor 13 was tested, since it usually provides a good stereocontrol in the glycosylation step via 2-o - acyl group participation leading to 1,2-trans glycosides. several glycosylation attempts utilizing 13 and the glycosyl acceptor molecules 4 or 10, however, produced mainly orthoester species accompanied by other byproducts which were not further analyzed. as the imidate 13 has recently been reported to be a suitable donor for the glycosylation of a 2,3,4,6-tetra - o - acetylheptoside acceptor, the poor outcome of the glycosylation reactions at the primary alcohol position of 10 may thus have been due to the steric bulk imposed by the adjacent ftipds group. per - o - benzylated heptosyl donor was envisaged to be more effective and was prepared in a straightforward sequence from the known hexa - o - acetyl heptose derivative 12 (scheme 2). first, treatment of 12 with thiophenol in the presence of excess boron trifluoride etherate gave a separable mixture of the anomeric phenyl 1-thioglycoside in 88% yield. the anomeric mixture as well as the isolated -glycoside 14 was then processed into the phenyl penta - o - benzyl-1-thioglycoside 16 in a combined yield of 80% via sodium methoxide catalyzed transesterification to give 15 followed by benzylation. subsequent hydrolysis of the thioglycoside with nbs gave the lactol 17 in 85% yield, which was eventually converted into the n - phenyltrifluoroacetimidate (nptfa) derivative 18. the corresponding tetra - o - benzyl d - glucopyranosyl nptfa donor 19 was prepared according to published procedures. depending on the mode of activation of the leaving group of donor 19 and the solvent used, highly selective glucosylation reactions have been reported leading to either - or -selective glycoside formation. glycosylation of alcohol 10 using a slight excess of heptosyl donor 18 was performed in dichloromethane in the presence of 0.05 equiv of tmsotf. the coupling step proceeded smoothly and gave a separable 4.9:1 anomeric mixture of disaccharides 20a and 20b in 83% yield (scheme 3). no relevant side reaction was observed when working at a temperature of 78 c. the -anomeric configuration of the distal heptose unit in 20a was inferred from the value of the heteronuclear jc-1,h-1 coupling constant (172 hz)being in the expected range for an -d - manno - configuration in contrast to 20b, which had a jc-1,h-1 coupling constant of 153.5 hz. in addition, the assigned substitution at position 7 was supported by hmbc correlation signals from h-1 of the distal heptose (5.02) to c-7 (66.9) of the methyl heptoside unit in 20a as well as, conversely, from h-7b (3.74) to the anomeric carbon of the distal heptose unit in 20b, respectively. similarly, the glycosylation reaction of the 3-o - levulinoyl derivative 11 was performed at 39 c and provided a 4.1:1 anomeric mixture (ratio based on the integration values of the downfield - shifted h-2 signals) of 21a and 21b in 72% yield. disaccharide 21a was isolated by hplc and was then subjected to treatment with hydrazine acetate to furnish the glycosyl acceptor derivative 22 in 84% yield. in contrast to the smooth glycosylation of the primary alcohol 10, glycosylation at position 3 of acceptor 4 using heptosyl imidate 18 met with difficulties (scheme 4). specifically, the reaction required a higher temperature, and the donor 18 was consumed by debenzylation at o-6/intramolecular cyclization with formation of the known 1,6-anhydro sugar 25. in order to suppress this side reaction, heptosyl donor 18 (2 equiv) was slowly added to a mixture of acceptor and promotor, but the isolated yield of pure -anomer 24 did not exceed 36% under various reaction conditions tested. in contrast, the synthesis of the related 3-o - glucosyl derivative 23 using donor 19 was robust (75% glycosylation yield) and gave good isolated yields of the pure -anomer (> 60%) with a temperature - dependent to ratio between 3:1 to 5:1 (with slightly increased -selectivity observed for glycosylations at higher temperatures). the -anomeric configuration of the d - glucosyl residue in compound 23 was inferred from the jh-1,h-2 coupling constant (3.4 hz). based on the results obtained for the synthesis of the disaccharides, trisaccharide 27 was targeted via a short route by converting -glucosyl-(13)-heptoside 23 into the corresponding 7-o - acceptor 26 (scheme 5). again, regioselective tipds cleavage of 23 by the action of treat was accomplished in high yield thereby providing evidence that this methodology is also applicable at the disaccharide stage. gratifyingly, the 6-o - ftipds group was compatible with the ensuing coupling step using 1.5 equiv of donor 18 and 0.05 equiv of tmso - triflate as promotor. additional promotor, however, had to be added, and the temperature was gradually raised from 40 to 0 c in order to drive the reaction to completion. thus, a 3.3:1 / anomeric mixture of trisaccharides was formed in 69% yield, from which the -glycoside 27 was eventually isolated in 51% yield by hplc separation. as minor byproducts, the 1,6-anhydroheptose derivative 25 and a 7-o - trimethylsilyl acceptor derivative alternatively, the glycosylation sequence was reversed and the (17)-linked heptobioside 22 was subjected to glycosylation with the trifluoroacetimidate glucosyl donor 19. the coupling reaction of 19 with 22 produced a separable mixture of the anomeric trisaccharides in 82% yield and in high -selectivity (/ = 9:1). both pathways delivered the protected trisaccharide 27 in comparable overall yield (17% versus 19%) but in a different number of steps (five versus seven steps when starting from 2). in summary, the side - chain tipds protecting group pattern turned out as a versatile means for chain elongation at the primary alcohol group of l, d - heptose. global deprotection of 6, 20a, 23, 24, and 27 was performed by initial desilylation, followed by hydrogenation and final alkaline saponification of the benzoate ester groups, and was optimized using monosaccharide 6 (scheme 6). the choice to first cleave the silyl - protecting group allowed for an additional chromatographic purification step prior to final deprotection. however, careful optimization of the desilylation conditions and close monitoring was necessary to prevent partial debenzylation of the phosphate moiety forming water - soluble compounds, in particular, when the more reactive hf pyridine reagent was used. hydrogenolysis of 28 followed by alkaline transesterification / ester hydrolysis of the dibenzoate 29 yielded the known 4-o - phosphoryl heptoside 30. the di- and trisaccharide derivatives were treated similarly to the deprotection of the monosaccharide 6. the desilylation of 23 and 24 was accomplished under mild treat conditions affording 34 and 37 without significant phosphate by contrast, the 7-o - heptosides 20a and 27, respectively, required the more reactive hf pyridine reagent under carefully monitored reaction conditions to afford 31 and 40 in good isolated yields and without hydrolysis of the benzyl phosphate group. hydrogenolysis and alkaline ester cleavage was uneventful in all cases, complicated only by the fact that benzoylated heptosides 32, 38, and 41 were insoluble in meoh at basic ph and needed addition of water to achieve quantitative cleavage. the formed methylbenzoate and benzoic acid were subsequently extracted with et2o or chcl3, respectively. the structures of 33, 39, and 42 were fully assigned on the basis of one- and two - dimensional nmr measurements, and this data will be used in ongoing std - nmr experiments with heptose - binding lectins. a straightforward synthetic route toward lps - oligosaccharide fragments containing a central 4-o - phosphorylated heptosyl residue has been established. this strategy capitalizes on the introduction of the required phosphorylation pattern already at the early stage of monosaccharide building blocks followed by a regioselective partial cleavage of a side - chain tipds protecting group as a robust and high - yielding method to generate fully protected 7-o - heptosyl acceptor derivatives. in addition, the presence of the resulting 6-o - fluorosilyl protecting group after regioselective tipds cleavage may also be exploited for subsequent selective substitution at position 6 of heptoses. the selective ring - opening of a side - chain locked tipds group should also work for the synthesis of other higher - carbon sugars such as kdo and provide rapid access to 8-o - substituted kdo derivatives. in conclusion, starting from a methyl heptoside, less than 10 steps were needed to complete the synthesis of a series of -(13)- and -(17)-connected lps heptose fragments (figure 3). summary of di- and trisaccharide ligands derived from the tipds - protected 4-o - phosphotriester precursor. all starting materials and reagents were purchased from commercial sources and used without further purification. residual moisture was confirmed by karl fischer tritration to be at least below 5 ppm. reactions were monitored by tlc on silica gel 60 f254 plates ; spots were detected by uv light examination or visualized by spraying with anisaldehyde sulfuric acid and heating. normal - phase column chromatography was performed on silica gel 60 (230400 mesh) or on prepacked spe columns (2 g/6 ml). preparative normal - phase hplc was performed on column a (250 20 mm, s-5 m, 6 nm) or column b (250 10 mm, s-5 m, 6 nm). nmr spectra were recorded at 297 k in the solvent indicated, with 300 and 600 mhz instruments, respectively, employing standard software provided by the manufacturer. h nmr spectra were referenced to tetramethylsilane (tms, = 0) or by calibration with the residual solvent peaks for solutions in organic solvents and to dss for solutions in d2o. c nmr spectra were referenced to tms (= 0), residual solvent peaks (cdcl3, = 77.0, cd3od, = 49.0) in organic solvents and to 1,4-dioxane (= 67.4) for solutions in d2o. p nmr spectra in d2o were referenced to external h3po4 (= 0). ms monitoring was done by injection of 0.010.1% solutions (520 l) on a system with two gradient pumps, degasser, and a lcms-2200 ev detector with mobile phase a = h2o (0.1% hcooh) and mobile phase b = ch3cn (0.1% hcooh) on a column (3.5 m, 100, 4.6 150 mm). method a : flow rate : 0.75 ml / min (022 min) ; gradient : 02 min : 85% b, 217 min : 8540% b, 1722 min : 40% b. method b : flow rate : 0.75 ml / min (022 min) ; gradient : 02 min : 95% b, 217 min : 9540% b, 1722 min : 40% b. accurate mass analysis (2 ppm mass accuracy) was carried out from 10100 mg / l solutions via lc tofms measurements using an autosampler, an hplc system with binary pumps, degasser, and column thermostat and esi - tof mass spectrometer. the fully protected heptoside was coevaporated with toluene, dissolved in dry dcm (16 ml), and transferred into a teflon flask. treat (0.3 ml, 150 equiv of f) was added dropwise to the vigorously stirred solution, which was kept at rt for 1521 h. the solution was poured into a stirred mixture of cold 50% aq nahco3 and etoac. the combined organic layers were washed with satd aq nahco3 and brine, dried (na2so4), concentrated, and coevaporated with toluene. a solution of the heptoside in dry meoh (58 ml) was purged with argon, and 10% pd / c was added. the atmosphere was exchanged for h2, and the suspension was stirred at rt for the time indicated. the atmosphere was exchanged to argon, and the suspension was filtered through celite and washed repeatedly with meoh. the filtrate was concentrated to yield the acidic form or was treated with et3n and concentrated to afford the target compound as et3n species. compound 1 (300 mg, 1.338 mmol, crystallized from hot 2-propanol) was coevaporated with toluene (3) and then dissolved in dry dmf (4.7 ml). 1h - imidazole (228 mg, 3.35 mmol, 2.5 equiv) was added, and the solution was cooled to 55 c. tipdscl2 (0.444 ml, 1.40 mmol, 1.05 equiv) was added via a syringe during 10 min, and the solution was slowly warmed to room temperature during 2.5 h. the reaction mixture was diluted with etoac (40 ml) and washed with satd aq nahco3. the crude material was purified by flash column chromatography on a silica gel cartridge (100 g, toluene / etoac 2:31:4) to give compound 2 (546.5 mg, 87%) as a syrup : rf 0.29 (toluene / etoac 1:1) ; []d + 48.4 (c 1.3, meoh) ; h nmr (600 mhz, cdcl3) 4.71 (d, j = 1.3 hz, 1h, h-1), 4.30 (dt, j = 3.0, 5.0 hz, 1h, h-6), 4.03 (d, j = 5.0 hz, 2h, h-7a, h-7b), 3.85 (dd, j = 1.8, 3.5 hz, 1h, h-2), 3.84 (t, j = 9.5 hz, 1h, h-4), 3.75 (dd, j = 3.4, 9.2 hz, 1h, h-3), 3.58 (dd, j = 2.9, 9.6 hz, 1h, h-5), 3.35 (s, 3h, och3), 1.120.94 (m, 28h, tipds - ch3, tipds - ch) ; c nmr (151 mhz, cdcl3) 100.8 (c-1), 75.3 (c-6), 72.1 (c-3), 70.95 (c-5), 70.2 (c-2), 68.2 (c-4), 67.3 (c-7), 55.0 (och3), 17.4, 17.4, 17.29, 17.27, 17.24, 17.21 (8 tipds - ch3), 12.9, 12.7, 12.4, 12.3 (tipds - ch) ; hrms (esi - tof) m / z [m + na ] calcd for c20h42nao8si2 489.2310, found 489.2312. triethyl orthobenzoate (0.59 ml, 2.61 mmol) and camphorsulfonic acid (25 mg, 0.11 mmol) were added to a solution of triol 2 (1.00 g, 2.14 mmol) in dry dcm (18 ml). the reaction mixture was stirred at rt for 30 min, when tlc (toluene / etoac 5:1 + et3n) indicated almost complete conversion to the intermediate orthoester diastereoisomers (2:1 ratio). et3n (0.3 ml, 2.14 mmol) was added, and the solution was concentrated and coevaporated with dry toluene. dibenzyl n, n - diisopropylphosphoramidite (1.08 ml, 3.21 mmol) was added followed by dropwise addition of a 0.45 m solution of 1h - tetrazole in acetonitrile (6.2 ml, 2.785 mmol). the solution was stirred for 45 min at rt and then cooled to 78 c. a solution of m - cpba (0.96 g ; approximately 77%, 4.29 mmol) in dcm (6 ml) was added, and the reaction mixture was stirred for 75 min at this temperature. triethylamine (0.75 ml, 5.36 mmol) was added, and the solution was warmed to rt. the reaction mixture was then added to a stirred mixture of satd aq nahco3 (40 ml) and etoac (40 ml). phases were separated, and the aqueous layer was extracted with etoac (30 ml). the combined organic layers were washed with satd aq nahco3 (30 ml) and brine (30 ml), dried (na2so4), and concentrated. the crude was purified by chromatography (sio2 : 150 g, toluene / etoac 15:112:1 ; the column was preconditioned with toluene / etoac 15:1 containing 0.3% et3n) to give 3a (0.532 g, 29%) and 3b (1.103 g, 60%) as a syrup. data for isomer 3a : rf 0.73 (toluene / etoac 3:1) ; []d + 25 (c 0.6, chcl3) ; h nmr (600 mhz, cdcl3) 7.477.44 (m, 2h, 2 phh), 7.357.28 (m, 13h, 13 phh), 5.14 (dd, j = 11.9, jh, p = 8.2 hz, 1h, poch2), 5.10 (dd, j = 11.6, jh, p = 6.9 hz, 1h, poch2), 5.085.03 (m, 2h, poch2), 5.01 (app dt, j = 9.6, jh, p = 6.8 hz, 1h, h-4), 4.95 (bs, 1h, h-1), 4.60 (app t, j = 6.9 hz, h-3), 4.35 (app d, j = 9.1 hz, 1h, h-6), 4.12 (dd, j = 12.2, 8.8 hz, 1h, h-7a), 3.94 (dd, j = 6.9, 1.1 hz, 1h, h-2), 3.88 (m, 3h, och2, h-7b), 3.68 (dd, j = 10.0 hz, 1.4 hz, 1h, h-5), 3.34 (s, 3h, och3), 1.19 (t, j = 7.1 hz, 2h, och2ch3), 1.121.03 (m, 26h, tipds), 1.000.92 (m, 2h, tipds) ; c nmr (151 mhz, cdcl3) 139.07, 136.11, 136.0, 128.9, 128.5, 128.4, 128.3, 128.1, 127.82, 127.79, 126.0, 121.7 (cq, orthoester), 98.4 (c-1), 76.3 (c-3), 75.2 (c-2), 74.8 (d, jc, p = 6.5 hz, c-4), 73.55 (c-6), 69.45 (d, jc, p = 5.5 hz, poch2), 69.3 (d, jc, p = 5.5 hz, poch2), 69.0 (d, jc, p = 9.5 hz, c-5), 68.3 (c-7), 59.0 (och2ch3), 55.4 (och3), 17.7, 17.6, 17.43, 17.36, 17.35, 17.3, 17.2 (tipds - ch3), 15.0 (och2ch3), 13.23, 13.20, 12.7, 12.6 (4 tipds - ch). hrms (esi - tof) m / z [m + na ] calcd for c43h63nao12psi2 881.3488, found 881.3491. data for 3b : rf 0.66 (toluene / etoac 3:1) ; []d + 9 (c 0.5, chcl3) ; h nmr (600 mhz, cdcl3) 7.717.65 (m, 2h, phh), 7.377.23 (m, 13h, phh), 5.074.89 (m, 4h, 1.5 poch2, h-1), 4.90 (dd, j = 11.8, jh, p = 8.5 hz, 1h, poch2), 4.86 (app t, j = 6.7 hz, 1h, h-3), 4.55 (app dt, j = 10.4, jh, p = 6.9 hz, 1h, h-4), 4.51 (dd, j = 6.5, 1.0 hz, 1h, h-2), 4.27 (app d, j = 8.8 hz, 1h, h-6), 4.06 (dd, j = 12.1, 8.8 hz, 1h, h-7a), 3.79 (dd, j = 12.2, j = 1.5 hz, 1h, h-7b), 3.67 (dd, j = 9.9, 1.6 hz, 1h, h-5), 3.35 (q, j = 7.1 hz, 2h, och2ch3), 3.36 (s, 3h, och3), 1.13 (t, j = 7.1 hz, 3h, och2ch3), 1.070.85 (m, 28h, tipds) ; c nmr (151 mhz, cdcl3) 137.0, 136.0, 135.9, 129.0, 128.5, 128.5, 128.4, 128.31, 128.27, 128.21, 128.18, 127.9, 127.83, 127.77, 126.5 (phc), 120.9 (cq, orthoester), 98.0 (c-1), 77.4 (c-3), 75.75 (c-2), 75.05 (d, jc, p = 6.5 hz, c-4), 73.3 (c6), 69.33 (d, jc, p = 5.5 hz, poch2), 69.28 (d, jc, p = 5.5 hz, poch2), 68.6 (d, jc, p = 8.9 hz, c-5), 68.0 (c-7), 59.3 (och2ch3), 55.4 (och3), 17.6, 17.5, 17.4, 17.3, 17.25, 17.22, 17.21, 17.1 (8 tipds - ch3), 15.1 (och2ch3), 13.2, 13.1, 12.7, 12.6 (4 tipds - ch). hrms (esi - tof) m / z [m + na ] calcd for c43h63nao12psi2 881.3488, found 881.3494. orthoester 3a/3b (1.61 g, 1.877 mmol) was dissolved in dcm (32 ml), and csa (44 mg, 0.188 mmol) was added at rt followed by dropwise addition of h2o (0.1 ml). the reaction mixture was stirred at rt for 1 h. the organic layer was washed with satd aq nahco3 (30 ml), and the aqueous layer was reextracted with dcm (30 ml). the combined organic layers were washed with satd aq nahco3 (30 ml) and brine (15 ml), dried (na2so4), and concentrated. the crude (1.593 g) was purified by column chromatography (sio2 : 150 g, toluene / etoac 10:1 7:1 5:1) to yield pure target compound 4 (1.129 g, 72%) as a syrup and a second fraction containing a mixture of 2-o- and 3-o - monobenzoate 4 and 5 (280 mg, 18%). data for 4 : rf 0.30 (toluene / etoac 3:1) ; []d + 26 (c 0.3, chcl3) ; h nmr (600 mhz, cdcl3) 8.068.04 (m, 2h, bzh2/h6), 7.597.55 (m, 1h, bzh4), 7.437.39 (m, 2h, bzh3/h5), 7.387.33 (m, 3h, phh), 7.237.32 (m, 7h, phh), 5.40 (dd, j = 3.6, 1.7 hz, 1h, h-2), 5.085.00 (m, 4h, 2 poch2), 4.80 (td, j = 9.4, jh, p = 7.8 hz, 1h, h-4), 4.79 (d, j = 1.4 hz, 1h, h-1), 4.76 (d, j = 4.6 hz, 1h, 3-oh), 4.35 (dt, j = 9.9, 4.6 hz, 1h, h-3), 4.32 (d, j = 9.3 hz, 1h, h-6), 4.08 (dd, j = 12.4, 8.7 hz, 1h, h-7a), 3.79 (dd, j = 12.3, 1.2 hz, 1h, h-7b), 3.60 (dd, j = 9.5, 1.0 hz, 1h, h-5), 3.34 (s, 3h, och3), 1.100.83 (m, 28h, tipds) ; c nmr (151 mhz, cdcl3) 165.8 (phc = o), 135.5 (d, jc, p = 6.8 hz, poch2phc1), 135.4 (d, jc, p = 6.9 hz, poch2phc1), 133.15, 130.0, 129.7, 128.60, 128.58, 128.5, 128.3, 128.2, 127.9 (phc), 98.439 (c-1), 76.8 (c-4), 73.4 (c-6), 72.2 (c-2), 71.0 (d, jc, p = 1.6 hz, c-5), 70.2 (d, jc, p = 5.5 hz, poch2), 69.8 (d, jc, p = 5.4 hz, poch2), 68.5 (c-3), 68.15 (c-7), 55.2 (och3), 17.62, 17.56, 17.4, 17.3, 17.25, 17.2, 17.1 (8 tipds - ch3), 13.3, 13.2, 12.6 (4 tipds - ch) ; hrms (esi - tof) m / z [m + h ] calcd for c41h60o12 psi2 831.3355, found 831.3355. data for 5 : rf 0.29 (hexane / etoac 3:1) ; []d + 21.4 (c 0.9, chcl3) ; h nmr (600 mhz, cdcl3) 8.168.13 (m, 2h, bzh2/h6), 7.537.50 (m, 1h, bzh4), 7.407.36 (m, 2h, bzh3/h5), 7.287.25 (m, 3h, phh), 7.227.18 (m, 1h, phh), 7.187.14 (m, 4h, phh), 6.93 6.90 (m, 2h, phh), 5.52 (dd, j = 9.7, 3.2 hz, 1h, h-3), 5.17 (app q, j = 9.3 hz, 1h, h-4), 4.91 (dd, j = 11.8, jh, p = 7.0 hz, 1h, poch2), 4.84 (dd, j = 11.8, jh, p = 8.5 hz, 1h, poch2), 4.73 (d, j = 2.1 hz, 1h, h-1), 4.71 (dd, j = 11.7, jh, p = 6.8 hz, 1h, poch2), 4.54 (dd, j = 11.8, jh, p = 8.0 hz, 1h, poch2), 4.38 (app dt, j = 8.7, 1.3 hz, 1h, h-6), 4.194.14 (m, 2h, h-2, h-7a), 3.85 (dd, j = 12.4, 1.4 hz, 1h, h-7b), 3.75 (dd, j = 9.5, 1.2 hz, 1h, h-5), 3.40 (s, 3h, och3), 2.10 (d d, j = 6.8 hz, 1h, 2-oh), 1.120.87 (m, 28h, tipds) ; c nmr (151 mhz, cdcl3) 165.7 (phc = o), 135.6 and 135.4 (d, jc, p= 7.7 hz, poch2phc1), 133.2, 130.2, 129.5, 128.4, 128.35, 128.3, 128.27, 128.1, 127.7, 127.4 (phc), 100.5 (c-1), 73.7 (c-6), 73.3 (c-3), 72.0 (d, jc, p = 8.8 hz, c-5), 71.8 (d, jc, p = 6.1 hz, c-4), 69.1 (c-2), 69.06 (d, jc, p = 5.5 hz, poch2), 68.95 (d, jc, p = 5.5 hz, poch2), 67.9 (c-7), 55.3 (och3), 17.6, 17.4, 17.4, 17.3, 17.2 (8 tipds - ch3), 13.55, 13.4, 12.72, 12.69 (4 tipds - ch) ; p nmr (243 mhz, cdcl3) 2.77 ; hrms (esi - tof) m / z [m + h ] calcd for c41h60o12psi2 : m / z = 831.3355, found 831.3363. a solution of orthoester 3a/3b (1.09 g, 1.268 mmol) in dcm (22 ml) was treated with csa (29 mg, 0.127 mmol) and water (75 l) as described for the preparation of 4. the crude material (1.163 g) was dissolved in pyridine (5.3 ml) and bzcl (0.3 ml, 2.5 mmol) was added dropwise at rt and the reaction mixture was stirred overnight. the reaction was quenched by addition of meoh (0.1 ml, 2.5 mmol) and stirring was continued for 30 min. the solution was diluted with etoac (50 ml), washed with cold 1 m hcl (with re - extraction of the aqueous phase). the combined organic layers were washed with water, satd aq nahco3, brine, dried (na2so4) and concentrated. the crude material (1.291 g) was purified by column chromatography (sio2 : 20 g, dcm dcm / etoac 30:1) to give compound 6 (1.051 g, 88.6% for 2 steps) as a colorless oil that crystallized upon storage, m.p. : 97100 c (etoac) ; rf 0.33 (toluene / etoac 9:1) ; []d 35 (c 0.5, chcl3) ; h nmr (600 mhz) 8.038.01 (m, 2h, bzh2/h6), 7.987.97 (m, 2h, bzh2/h6), 7.607.57 (m, 1h, bzh4), 7.457.40 (m, 3h, bzh3/h5, bzh4), 7.287.23 (m, 6h, bzh3/h5, 4 phh), 7.207.12 (m, 4h, phh), 6.916.88 (m, 2h, phh), 5.76 (dd, j = 9.8, 3.5 hz, 1h, h-3), 5.58 (dd, j = 3.5, 1.7 hz, 1h, h-2), 5.32 (app q, j = 9.4 hz, 1h, h-4), 4.91 (dd, j = 11.8, jh, p = 7.1 hz, 1h, poch2), 4.89 (d, j = 1.4 hz, 1h, h-1), 4.81 (dd, j = 11.8, jh, p = 9.0 hz, 1h, poch2), 4.68 (dd, j = 11.8, jh, p = 6.7 hz, 1h, poch2), 4.50 (dd, j = 11.8, jh, p = 7.9 hz, 1h, poch2), 4.45 (app d, j = 8.6 hz, 1h, h-6), 4.17 (dd, j = 12.4, 8.7 hz, 1h, h-7a), 3.87 (dd, j = 12.4, 1.1 hz, 1h, h-7b), 3.86 (app d, j = 9.5 hz, 1h, h-5), 3.43 (s, 3h, och3), 1.130.94 (m, 28 h, tipds) ; c nmr (151 mhz, cdcl3) 165.5 (phc = o), 165.4 (phc = o), 135.6 (d, jc, p = 7.3 hz, poch2phc1), 135.4 (d, jc, p = 7.7 hz, poch2phc1), 133.3, 133.0, 130.0, 129.9, 129.4, 129.35, 128.41, 128.36, 128.3, 128.25, 128.2, 128.1, 127.7, 127.3 (phc), 98.35 (c-1), 73.5 (c-6), 71.9 (d, jc, p = 6.5 hz, c-5), 71.85 (d, j = 4.4 hz, c-4), 70.9 (c-3), 70.4 (c-2), 69.1 (d, jc, p = 6.2 hz, poch2), 68.9 (d, jc, p = 5.2 hz, poch2), 68.1 (c-7), 55.3 (och3), 17.7, 17.6, 17.39, 17.36, 17.3, 17.25, 17.21, 17.17 (8 tipds - ch3), 13.6, 13.5, 12.7, 12.7 (4 tipds - ch) ; hrms (esi - tof) m / z [m + h ] calcd for c48h64o13psi2 935.3618, found 935.3625. a solution of triol 2 (2.00 g, 4.28 mmol) in dry dcm (40 ml) and triethyl orthobenzoate (1.17 ml, 5.16 mmol) was treated with csa (49 mg, 0.22 mmol) at rt. tlc monitoring (hexane / etoac 2:1) revealed almost complete conversion into the intermediate orthoester after 10 min. after 30 min, h2o (160 l, 8.5 mmol) was added, and stirring was continued for 1 h at rt. the solution was diluted with dcm (60 ml) and washed with satd aq nahco3 (90 ml). the aqueous layer was reextracted with dcm (70 ml), and the combined organic layers were washed with brine (60 ml), dried (na2so4), and concentrated. the crude material (2.5 g) was purified by vacuum flash chromatography (sio2 : 50 g, toluene / etoac 5:1) to give 7 (2.00 g, 81.7%) as a white solid foam : []d + 12.8 (c 2.1, cdcl3) ; h nmr (600 mhz, cdcl3) 8.068.03 (m, 2h, bzh2/h6), 7.607.56 (m, 1h, bzh4), 7.457.41 (m, 2h, bzh3/h5), 5.32 (dd, j = 1.7, 2.9 hz, 1h, h-2), 4.81 (d, j = 1.6 hz, 1h, h-1), 4.38 (ddd, j = 1.4, 2.3, 8.5 hz, 1h, h-6), 4.134.06 (m, 3h, h-4, h-7a, h-3), 4.00 (dd, j = 1.4, 12.2 hz, 1h, h-7b), 3.66 (dd, j = 2.4, 9.2 hz, 1h, h-5), 3.37 (s, 3h, och3), 2.77 (d, j = 1.8 hz, 1h, 4-oh), 2.29 (d, j = 5.3 hz, 1h, 3-oh), 1.130.94 (m, 28h, tipds) ; c nmr (151 mhz, cdcl3) 166.2 (phc = o), 133.4, 129.9, 129.5, 128.4 (phc), 98.8 (c-1), 74.4 (c-6), 72.1 (c-2), 71.9 (c-5), 70.8 (c-3), 68.15 (c-4), 67.8 (c-7), 55.1 (och3), 17.5, 17.42, 17.39, 17.30, 17.26, 17.2 (tipds - ch3), 13.0, 12.7, 12.6, 12.4 (tipds - ch) ; hrms (esi - tof) m / z [m + na ] calcd for c27h46nao9si2 593.2573, found 593.2575. a solution of dcc (304 mg, 1.472 mmol) in dry dcm (2.4 ml) was slowly added in several portions for a period of 45 min to a solution of diol 7 (700 mg, 1.23 mmol), levulinic acid (157 mg, 1.249 mmol), and dmap (7.5 mg, 0.061 mmol) in dry dcm (14 ml) at rt. the reaction mixture was then diluted with dcm (40 ml) and filtered over a plug of cotton. the organic layer was washed with satd aq nahco3 (30 ml), and the aqueous layer was re - extracted with dcm (40 ml). the combined organic phases were washed with brine (30 ml), dried (na2so4), and concentrated. the residue was dissolved in a small volume of dcm, filtered, and concentrated. the crude material (870 mg) was purified by mplc - column chromatography (sio2 : 60 g, flow - rate : 40 ml / min toluene / etoac 7:1) to afford compound 8 (785 mg, 96%) as a glasslike solid : []d + 8.4 (c 1.1, chcl3) ; h nmr (600 mhz, cdcl3) 8.078.05 (m, 2h, bzh2/h6), 7.617.58 (m, 1h, bzh4), 7.477.42 (m, 2h, bzh3/h5), 5.42 (dd, j = 1.8, 3.5 hz, 1h, h-2), 5.31 (dd, j = 3.4, 9.8 hz, 1h, h-3), 4.78 (d, j = 1.7 hz, 1h, h-1), 4.454.42 (m, 1h, h-6), 4.26 (dt, j = 3.4, 9.7 hz, 1h, h-4), 4.11 (dd, j = 8.6, 12.1 hz, 1h, h-7a), 3.98 (dd, j = 1.3, 12.1 hz, 1h, h-7b), 3.73 (dd, j = 2.2, 9.6 hz, 1h, h-5), 3.38 (s, 3h, och3), 2.83 (d, j = 3.4 hz, 1h, 4-oh), 2.77 (ddd, j = 6.1, 7.7, 18.4 hz, 1h, lev - h3a), 2.66 (dt, j = 6.2, 18.4 hz, 1h, lev - h3b), 2.57 (ddd, j = 5.7, 7.8, 17.0 hz, 1h, lev - h2a), 2.48 (dt, j = 6.4, 17.0 hz, 1h, lev - h2b), 2.08 (s, 3h, lev - ch3), 1.150.94 (m, 28h, tipds) ; c nmr (151 mhz, cdcl3) 207.15 (lev - c4), 172.3 (lev - c1), 165.4 (phc = o), 133.4, 129.8, 129.5, 128.4 (phc), 98.7 (c-1), 73.8 (c-6), 72.8 (c-3), 72.5 (c-5), 70.0 (c-2), 68.1 (c-7), 65.5 (c-4), 55.0 (och3), 38.1 (lev - c3), 29.6 (lev - c5), 28.1 (lev - c2), 17.44, 17.42, 17.41, 17.36, 17.3, 17.2 (8 tipds - ch3) 13.0, 12.6, 12.35 (4 tipds - ch) ; hrms (esi - tof) m / z [m + na ] calcd for c32h52nao11si2 691.2940, found 691.2940. a solution of 1h - tetrazole in ch3cn (0.45 m, dried over molecular sieves) was added dropwise at rt to a solution of alcohol 8 (86 mg, 0.129 mmol) and dibenzyl n, n - diisopropylphosphoramidite (60 l, 0.18 mmol) in dry dcm (1.7 ml). tlc (toluene / etoac 5:1) indicated complete conversion to the intermediate phosphite species after 10 min. the reaction mixture was cooled to 72 c, and m - cpba (71 mg, 0.315 mmol) was added in one portion. after the mixture was stirred for 40 min, et3n (54 l, 0.386 mmol) the solution was warmed to rt and was separated between etoac and satd aq nahco3. the aqueous layer was again extracted with etoac, and the combined organic layers were washed with satd aq nahco3 and brine, dried (na2so4), and concentrated. the crude material was purified by repeated chromatography on silica gel (sio2 : 2 g cartridge, toluene toluene / etoac 3:1), then by preparative hplc (column a, toluene / etoac 10:1 to 5:1), to give 9 (97 mg, 81%) : []d 4.5 (c 0.9, chcl3) ; h nmr (600 mhz, cdcl3) 8.078.04 (m, 2h, bzh2/h6), 7.627.58 (m, 1h, bzh4), 7.457.41 (m, 2h, bzh3/h5), 7.297.23 (m, 10h, phh), 5.54 (dd, j = 3.5, 9.8 hz, 1h, h-3), 5.46 (dd, j = 1.8, 3.6 hz, 1h, h-2), 5.03 (app q, j = 9.4 hz, 1h, h-4), 5.014.91 (m, 4h, 2 poch2), 4.79 (d, j = 1.6 hz, 1h, h-1), 4.454.42 (m, 1h, h-6), 4.13 (dd, j = 8.7, 12.4 hz, 1h, h-7a), 3.84 (dd, j = 1.2, 12.3 hz, 1h, h-7b), 3.823.79 (m, 1h, h-5), 3.38 (s, 3h, och3), 2.562.47 (m, 2h, lev - h3a / h3b), 2.462.32 (m, 2h, lev - h2a / h2b), 1.97 (s, 3h, lev - ch3), 1.120.91 (m, 28h, tipds) ; c nmr (151 mhz, cdcl3) 206.1 (lev - c4), 171.8 (lev - c1), 165.5 (phc = o), 135.7 (d, jc, p = 7.1 hz, poch2phc), 135.6 (d, jc, p = 6.6 hz, poch2phc), 133.4, 130.0, 129.3, 128.49, 128.47, 128.44, 128.38, 127.95, 127.8 (phc), 98.4 (c-1), 73.4 (c-6), 72.0 (d, jc, p = 7.1 hz, c-4), 71.65 (d, jc, p = 8.4 hz, c-5), 70.3 (c-3), 70.0 (c-2), 69.3 (d, jc, p = 5.3 hz, poch2), 69.2 (d, jc, p = 5.6 hz, poch2), 68.1 (c7), 55.2 (och3), 37.7 (lev - c3), 29.55 (lev - ch3), 27.9 (lev - c2), 17.6, 17.5, 17.4, 17.3, 17.2, 17.1 (8 tipds - ch3), 13.36, 13.34, 12.64, 12.62 (4 tipds - ch) ; p nmr (243 mhz, cdcl3) 3.47 ; hrms (esi - tof) m / z [m + h ] calcd for c46h66o14psi2 929.3723, found 929.3731. compound 6 (290 mg, 0.310 mmol) was dissolved in dry dcm (15 ml), transferred into a teflon flask, and cooled in an ice bath. pyridine reagent (88 l, 10 equiv of f) was added in four portions every 2 min to prevent local overheating. the reaction was quenched by dropping the solution into stirred ice - cold satd aq nahco3 (60 ml). the aqueous layer was extracted with dcm (3 30 ml), and the combined organic layers were washed with satd aq nahco3, and brine, dried (na2so4), and concentrated. the crude material (19.8 mg) was purified by column chromatography (sio2 : 17 g, toluene / etoac 2:3) to furnish 10 (245 mg, 83%). a solution of compound 6 (20 mg, 21.4 mol) in dry dcm (1.0 ml) was treated with treat (174 l, 150 equiv of f) at 0 c for 1 h according to general procedure 1. the residue (19.8 mg) was purified by column chromatography (sio2 : 2 g cartridge, hexane / etoac 3:1) to give 10 (18.3 mg, 90%) which solidified upon storage in a refrigerator. analytical data for 10 : rf 0.20 (toluene / etoac 5:1) ; []d 51.8 (c 0.5, chcl3) ; h nmr (600 mhz, cdcl3) 8.048.01 (m, 2h, bzh2/h6), 7.957.93 (m, 2h, bzh2/h6), 7.607.57 (m, 1h, bzh4), 7.457.41 (m, 3h, bzh3/h5, bzh4), 7.277.22 (m, 5h, bzh3/h5, 3 phh), 7.197.17 (m, 1h, phh), 7.147.10 (m, 4h, phh), 6.916.89 (m, 2h, phh), 5.80 (dd, j = 3.5, 9.7 hz, 1h, h-3), 5.59 (dd, j = 1.7, 3.5 hz, 1h, h-2), 5.33 (app q, j = 9.7 hz, 1h, h-4), 4.90 (d, j = 1.9 hz, 1h, h-1), 4.88 (dd, j = 7.1, 11.6 hz, 1h, poch2), 4.78 (dd, j = 8.8, 11.8 hz, 1h, poch2), 4.70 (dd, j = 6.9, 11.8 hz, 1h, poch2), 4.55 (dd, j = 8.5, 11.8 hz, 1h, poch2), 4.374.34 (m, h-6), 4.10 (dd, j = 1.9, 9.8 hz, 1h, h-5), 3.93 (dd, j = 4.7, 11.1 hz, 1h, h-7a), 3.86 (dd, j = 6.1, 11.1 hz, 1h, h-7b), 3.47 (s, 3h, och3), 1.160.95 (m, 28h, tipds) ; c nmr (151 mhz, cdcl3) 165.6 (phc = o), 165.4 (phc = o), 135.6 (d, jc, p = 7.0 hz, poch2phc), 135.4 (d, jc, p = 7.5 hz, poch2phc), 133.4, 133.0, 123.0, 129.9, 129.42, 129.38, 128.41, 128.39, 128.31, 128.26, 128.24, 128.1, 127.8, 127.5 (phc), 98.35 (c-1), 72.4 (d, jc, p = 6.3 hz, c-4), 71.4 (c-6), 71.0 (c-3), 70.6 (c-2), 70.4 (d, jc, p = 7.6 hz, c-5), 69.2 (d, jc, p = 5.9 hz, poch2), 69.1 (d, jc, p = 5.4 hz, poch2), 63.6 (c-7), 55.4 (och3), 17.5, 17.4, 17.3, 17.2, 16.7, 16.6 (8 ftipds - ch3), 13.6, 13.1 (2 ftipds - ch), 12.61 and 12.59 (2 d, j = 16.5 hz, ftipds - ch) ; hrms (esi - tof) m / z [m + h ] calcd for c48h65fo13psi2 955.3680, found 955.3693. a solution of compound 9 (45 mg, 48.4 mol) in dry dcm (1.2 ml) was treated with treat (158 l, 60 equiv of f) at 0 c for 3.5 h according to general procedure 1. the residue was purified by column chromatography (sio2 : 2 g cartridge, toluene / etoac 10:15:12:1) to give 11 as colorless solid (37.8 mg, 82%). alternatively, compound 11 was prepared in a two - step sequence from 8 : alcohol 8 (350 mg, 0.522 mmol) was treated as described for 11 with phosphoramidite (245 l, 0.731 mmol) and a solution of 1h - tetrazole in ch3cn (0.45 m, 0.68 mmol, dried over molecular sieves) followed by oxidation with m - cpba (287 mg, 1.28 mmol). the crude material was directly submitted to selective tipds cleavage as described above with treat (1.7 ml, 60 equiv of f) and purified by column chromatography (sio2 : 50 g, toluene / etoac 5:1) to afford 11 (397 mg, 80% for two steps) as a colorless foam : []d 20 (c 1.1, chcl3) ; h nmr (600 mhz, cdcl3) 8.078.04 (m, 2h, bzh2/h6), 7.617.58 (m, 1h, bzh4), 7.45- 7.42 (m, 2h, bzh3/h5), 7.307.20 (m, 10h, phh), 5.55 (dd, j = 3.5, 9.8 hz, 1h, h-3), 5.46 (dd, j = 1.7, 3.6 hz, 1h, h-2), 5.08 (app q, j = 9.8 hz, 1h, h-4), 5.014.94 (m, 3h, poch2), 4.92 (dd, j = 6.8, 11.7 hz, 1h, poch2), 4.81 (d, j = 1.6 hz, 1h, h-1), 4.344.32 (m, 1h, h-6), 4.04 (dd, j = 2.0, 9.8 hz, 1h, h-5), 3.90 (dd, j = 4.8, 11.1 hz, 1h, h-7a), 3.83 (dd, j = 6.2, 11.1 hz, 1h, h-7b), 3.42 (s, 3h, och3), 2.49 (t, j = 7.1 hz, 2h, lev - h3a, h3b), 2.412.28 (m, 2h, lev - h2a, h2b), 1.97 (s, 3h, lev - ch3), 1.070.94 (m, 28h, ftipds) ; c nmr (151 mhz, cdcl3) 206.1 (lev - c4), 171.8 (lev - c1), 165.5 (phc = o), 135.7 (d, jc, p = 7.5 hz, 2 poch2phc1), 133.4, 129.9, 129.35, 128.50, 128.47, 128.46, 128.42, 128.39, 128.0, 127.8 (phc), 98.4 (c-1), 72.4 (d, jc, p = 6.7 hz, c-4), 71.2 (c-6), 70.5 (c-3), 70.3 (d, jc, p = 7.8 hz, c-5), 70.25 (c-2), 69.44 (d, jc, p = 5.5 hz, poch2), 69.36 (d, jc, p = 5.5 hz, poch2), 63.6 (c-7), 55.3 (och3), 37.65 (lev - c3), 29.6 (lev - ch3), 27.9 (lev - c2), 17.5, 17.4, 17.3, 17.2 (4 ftipds - ch3) 16.7 (2 ftipds - ch3), 16.6 (2 ftipds - ch3), 13.6, 13.1 (2 ftipds - ch), 12.57 and 12.56 (2 d, j = 16.4 hz, ftipds - ch) ; hrms (esi - tof) m / z [m + h ] calcd for c46h67fo14psi2 949.3786, found 949.3792. peracetate 12(31) (1.00 g, 2.16 mmol) was dissolved in dry dcm (10 ml), and the solution was stripped with argon for several minutes. thiophenol (0.44 ml, 4.3 mmol) and then bf3oet2 (1.33 ml, 10.8 mmol) were added dropwise, each within 15 min, and the solution was stirred at rt for 23 h. the reaction mixture was poured into 1:1 dcm / satd aq nahco3 (100 ml). the phases were separated, and the aqueous layer was extracted with dcm (3). the combined organic layer was washed twice with satd aq nahco3, 0.5% aq i2-solution, 5% aq na2s2o3, and brine. the crude material was purified by vacuum flash chromatography (sio2 : 22 g, hexane / etoac 2:1) to give 14 as an anomeric mixture (980 mg, 88.4%, / 93:7) that was used for the further steps. to obtain pure -anomer, the material was crystallized from hot dry etoh (10 ml) to give 14 as colorless crystals (657 mg, 59%), mp 109111 c (etoh). the anomers can alternatively be separated by chromatography using et2o / hexane 3:2 as eluent. analytical data for -anomer 14 : rf 0.21 (hexane / et2o 2:3) ; []d + 93.9 (c 1.9, chcl3) ; h nmr (600 mhz, cdcl3) 7.437.40 (m, 2h, phh), 7.347.26 (m, 3h, phh), 5.62 (d, j = 1.6 hz, 1h, h-1), 5.52 (dd, j = 1.5, 3.4 hz, 1h, h-2), 5.37 (app t, j = 10.1, 1h, h-4), 5.31 (dd, j = 3.4, 10.1 hz, 1h, h-3), 5.29 (ddd, j = 2.0, 5.6, 7.5 hz, 1h, h-6), 4.55 (dd, j = 2.1, 10.0 hz, 1h, h-5), 4.03 (dd, j = 5.8, 11.4 hz, 1h, h-7a), 3.99 (dd, j = 7.5, 11.4 hz, 1h, h-7b), 2.18, 2.12, 2.04, 2.01, and 1.90 (5s, 5 3h, ch3c = o) ; c nmr (151 mhz, cdcl3) 170.3, 170.2, 169.9, 169.8, 169.55 (5 c = o), 132.5, 130.9, 129.4, 127.9 (phc), 85.6 (c-1), 70.9 (c-2), 69.7 (c-5), 69.6 (c-3), 67.0 (c-6), 64.8 (c-4), 61.8 (c-7), 20.9, 20.64, 20.59, 20.58, 20.5 (5 ch3c = o) ; hrms (esi - tof) m / z [m + nh4 ] peracetate 14 (48 mg, 1.26 mmol) was dissolved in dry meoh (15 ml), 1 m naome (0.38 ml, 0.25 equiv) was added, and the reaction mixture was stirred at rt for 4 h. the ph was adjusted 7.0 by addition of dowex 50 cation - exchange resin (h - form). the resin was removed by filtration and washed with meoh, and the filtrate was concentrated to give 15 (373 mg, 98%) as a colorless syrup : rf 0.24 (etoac / meoh 4:1) ; []d + 251.8 (c 1.2, meoh) ; h nmr (600 mhz, meod) 7.527.48 (m, 2h, phh), 7.357.31 (m, 2h, phh), 7.307.26 (m, 1h, phh), 5.48 (d, j = 1.6 hz, 1h, h-1), 4.07 (dd, j = 1.5, 3.3 hz, 1h, h-2), 3.993.96 (m, 2h, h-5, h-6), 3.93 (app t, j = 9.6 hz, 1h, h-4), 3.70 (dd, j = 3.3, 9.3 hz, 1h, h-3), 3.48 (dd, j = 7.4, 11.0 hz, 1h, h-7a), 3.39 (dd, j = 5.2, 11.0 hz, 1h, h-7b) ; c nmr (151 mhz, meod) 135.5, 133.0, 130.1, 128.6 (phc), 90.3 (c-1), 74.25 (c-5), 73.6 (c-2), 73.4 (c-3), 71.0 (c-6), 68.0 (c-4), 65.0 (c-7) ; hrms (esi - tof) m / z [m + hcoo ] calcd for c14h19o8s 347.0806, found 347.0803. thioglycoside 15 (310 mg, 1.025 mmol) was coevaporated with toluene and dissolved in dry dmf (20 ml). the solution was cooled to 0 c, and nah (60%, 226 mg, 5.65 mmol, 5.5 equiv) was added in portions. the resulting slurry was stirred for 0.5 h, and benzyl bromide (0.74 ml, 6.15 mmol, 6.0 equiv) was added dropwise. the reaction mixture was warmed to rt and was stirred for 18 h. since mainly two polar spots were visible on tlc (toluene / etoac 20:1, etoac / meoh = 4:1), additional nah (75 mg, 1.87 mmol, 1.8 equiv) was added at 0 c. the ice bath was removed, and stirring was continued for 20 min. bnbr (0.06 ml, 0.25 mmol, 0.24 equiv) was added, and the mixture was stirred for an additional 45 min without significant additional formation of product. meoh (0.84 ml, 20 equiv) was added leading to formation of h2 (caution !). after 30 min, the reaction mixture was separated between et2o (50 ml) and satd aq nahco3 (100 ml). the aqueous layer was extracted with et2o (50 ml), and the combined organic layers were washed with satd aq nahco3 (50 ml), water (50 ml), and brine (50 ml). the organic layer was dried (na2so4), filtered, and concentrated, and the residue was coevaporated with toluene. the crude material was submitted to vacuum flash chromatography (sio2 : 30 g toluene toluene / etoac 120:180:1) to give 16 (625.5 mg, 81%) as a colorless oil : []d + 73.6 (c 0.8, chcl3) ; h nmr (600 mhz, cdcl3) 7.397.36 (m, 4h, phh), 7.347.19 (m, 26h, phh), 5.75 (d, j = 1.7 hz, 1h, h-1), 4.88 and 4.34 (2 d, j = 11.0 hz, 2h, ch2ph), 4.84 and 4.52 (2 d, j = 11.8 hz, 2h, ch2ph), 4.76 and 4.63 (2 d, j = 12.2 hz, 2h, ch2ph), 4.57 (bs, 2h, ch2ph), 4.38 and 4.35 (2 d, j = 11.9 hz, 2h, ch2ph), 4.25 (app t, j = 9.5 hz, 1h, h-4), 4.154.11 (m, 2h, h-5, h-6), 3.98 (dd, j = 2.0, 2.9 hz, 1h, h-2), 3.88 (dd, j = 3.0, 9.1 hz, 1h, h-3), 3.69 (dd, j = 6.7, 10.0 hz, 1h, h-7a), 3.46 (dd, j = 5.1, 10.0 hz, 1h, h-7b) ; c nmr (151 mhz, cdcl3) 138.7, 138.7, 138.2, 138.1, 137.95 (phc), 134.4 (sphc-1), 130.7, 128.9, 128.4, 128.33, 128.29, 128.2, 127.9, 127.73, 127.71, 127.68, 127.6, 127.50, 127.47, 127.35, and 127.0 (phc), 85.2 (c-1), 80.6 (c-3), 76.0 (c-2), 75.3 (c-6), 74.7 (ch2ph), 74.35 (c-4), 73.3 (ch2ph), 73.1 (c-5), 72.8 (ch2ph), 72.1 (ch2ph), 72.0 (ch2ph), 71.0 (c-7) ; hrms (esi - tof) m / z [m + nh4 ] calcd for c48h52no6s 770.3510, found 770.3544. thioglycoside 16 (833 mg, 1.106 mmol) was dissolved in 24:1 acetone / water (28 ml) and stirred with external cooling using an etoh ice bath. a solution of nbs (591 mg, 3.32 mmol) in 24:1 acetone / water (6 ml) was added dropwise within 5 min keeping the temperature below 5 c. after 15 min, the reaction was quenched by pouring the reaction mixture into an ice - cold stirred mixture of satd aq nahco3 (25 ml), 5% aq na2s2o3 (25 ml), and etoac (50 ml). the aqueous layer was extracted with etoac (20 ml) and the combined organic layers were washed with satd aq nahco3 (30 ml), water (30 ml), brine (30 ml), dried (na2so4) and concentrated. the crude material (785 mg) was submitted to preparative mplc (sio2 : 60 g ; flow rate : 35 ml / min ; hexane / etoac 3:1) to give 17 (621 mg, 85%) as a colorless oil : 0.32 (hexane / etoac 2:1) ; h nmr (600 mhz, cdcl3, / ratio appr. 2:1, denotes signals assigned to the -anomer) 7.387.17 (m, 25h, phh), 5.25 (d, j = 1.9 hz, 1h, h-1), 5.12 (d, j = 11.6 hz, 1h, phch2), 4.88 (d, j = 11.4 hz, 1h, phch2), 4.86 (d, j = 8.2 hz, 1h, phch2), 4.84 (d, j = 9.0 hz, 1h, phch2), 4.81 (d, j = 11.6 hz, 1h, phch2), 4.73 (d, j = 11.6 hz, 1h, phch2), 4.72 (bs, 2h, phch2), 4.70 (d, j = 11.7 hz, 1h, phch2), 4.66 (d, j = 11.6 hz, 1h, phch2), 4.594.58 (m, 3h, h-1, 2 phch2), 4.57 (d, j = 11.9 hz, 1h, phch2), 4.54 (d, j = 11.9 hz, 1h, phch2), 4.54 (d, j = 12.1 hz, 1h, phch2), 4.53 (d, j = 11.4 hz, 1h, phch2), 4.51 (d, j = 11.5 hz, 1h, phch2), 4.46 (d, j = 11.9 hz, 1h, phch2), 4.374.34 (m, 2h, phch2, phch2), 4.19 (t, j = 9.5 hz, 1h, h-4), 4.16 (t, j = 9.5 hz, 1h, h-4), 4.124.08 (m, 2h, h-6, h-6), 3.96 (dd, j = 3.0, 9.2 hz, 1h, h-3), 3.92 (dd, j = 1.7, 9.7 hz, 1h, h-5), 3.863.83 (m, 2h, h-2, h-7a), 3.803.75 (m, 3h, h-2, h-7a, h-7b), 3.72 (dd, j = 6.2, 9.8 hz, 1h, h-7b), 3.63 (dd, j = 2.7, 9.4 hz, 1h, h-3), 3.42 (dd, j = 1.9, 9.6 hz, 1h, h-5) ; c nmr (151 mhz, cdcl3) 138.9, 138.8, 138.7, 138.6, 138.45, 138.4, 138.33, 138.30, 138.2, 137.9, 128.5, 128.5, 128.4, 128.36, 128.34, 128.28, 128.25, 128.2, 128.05, 127.9, 127.86, 127.85, 127.80, 127.77, 127.74, 127.72, 127.69, 127.6, 127.54, 127.50, 127.46, 127.38 (phc), 94.0 (c-1), 92.8 (c-1), 83.9 (c-3), 80.1 (c-3), 76.3 (c-2), 75.3 (c-5), 75.0 (c-6, c-6), 74.71 (ch2ph), 74.68 (c-2), 74.6 (ch2ph), 74.5 (ch2ph), 74.4 (c-4), 74.0 (c-4), 73.4 (ch2ph), 73.25 (ch2ph), 72.75 (ch2ph) 72.7 (ch2ph), 72.55 (ch2ph), 72.4 (ch2ph), 72.1 (ch2ph), 71.7 (c-5), 70.5 (c-7), 70.0 (c-7) ; hrms (esi - tof) m / z [m + nh4 ] calcd for c42h48o7n 678.3435, found 678.3431. reducing sugar 17 (100 mg, 0.151 mmol) was dissolved in acetone (2.0 ml). k2co3 (42 mg, 0.303 mmol) and n - phenyltrifluoroacetimidoyl chloride (63 mg, 0.30 mmol) were added rapidly, and the reaction mixture was stirred at rt for 4.5 h. the suspension was filtered through a plug of celite and washed thoroughly with acetone. after addition of one drop of triethylamine, the solution was concentrated and the crude material was purified by column chromatography (sio2 : 15 g, toluene / etoac 20:1 + 0.2% et3n) to give 18 (119 mg, 94.5%) as a colorless oil : h nmr of main isomer (600 mhz, cdcl3) 7.377.18 (m, 28h, phh), 6.73 6.68 (m, 2h, phh), 6.30 (bs, 1h, h-1), 4.874.83 (m, 2h, ch2ph), 4.764.60 (bs, 1h, ch2ph), 4.63 (d, j = 11.7 hz, 1h, ch2ph), 4.584.49 (m, 4h, 2 ch2ph), 4.384.34 (m, 1h, ch2ph), 4.294.23 (m, 1h), 4.144.10 (m, 1h, h-6), 3.943.88 (m, 2h), 3.85 (dd, j = 6.8, 10.0 hz, 1h, h-7a), 3.833.78 (bs, 1h), 3.70 (dd, j = 5.2, 10.0 hz, 1h, h-7b) ; c nmr (151 mhz, cdcl3) 143.5, 142.8, 142.55, 138.6, 138.4, 138.2, 138.0, 137.7, 128.8, 128.43, 128.37, 128.36, 128.32, 128.27, 128.05, 128.0, 127.82, 127.77, 127.72, 127.69, 127.6, 127.4, 119.4 (phc), 79.3, 75.0 (c-6), 74.75 (ch2ph), 74.5, 73.7, 73.5 (ch2ph), 73.2, 72.7, 72.53, 72.48 (3 ch2ph), 70.66 (c-7). dry dcm (5 ml) and ground molecular sieves (4, 300 mg) were placed in a flame - dried, two - necked 10 ml flask. acceptor 10 (207 mg, 0.217 mmol) and donor 18 (216 mg, 260 mmol, 1.2 equiv) were combined and coevaporated with dry toluene, transferred into the suspension, stirred under ar for 30 min at rt, and then cooled to 78 c. a solution of tmsotf (2 l, 0.011 mmol, 0.05 equiv) in dry dcm (in 200 l) was added, and the reaction mixture was stirred for 1.5 h at 78 c. the reaction was quenched by dropwise addition of et3n (57 l, 0.412 mmol) in dcm (1 ml). the suspension was allowed to warm to rt, filtered through celite, and washed with dcm. the residue was first subjected to chromatography on an mplc column (sio2 : 21 g, flow rate 20 ml / min with a stepwise gradient hexane / etoac 5:1 3:1) to furnish a fraction containing the disaccharides. final purification was achieved by preparative hplc (in three portions on column a, flow rate 15 ml / min, hexane / etoac 7:1) to give first -anomer 20a (238 mg, 69%), followed by -anomer 20b (46.6 mg, 13.5%) as a colorless oils. analytical data for 20a : rf 0.4 (hexane / etoac) ; []d 15.6 (c 0.9, chcl3) ; h nmr (600 mhz, cdcl3) 8.048.01 (m, 2h, bzh2/h6), 7.997.96 (m, 2h, bzh2/h6), 7.607.56 (m, 1h, bzh4), 7.467.40 (m, 3h, bzh4, bzh3/h5), 7.387.36 (m, 2h, phh), 7.357.32 (m, 2h, phh), 7.317.14 (m, 27h, bzh3/h5, 25 phh), 7.117.07 (m, 4h, phh), 6.876.84 (m, 2h, phh), 5.79 (dd, j = 3.6, 9.8 hz, 1h, h-3), 5.53 (dd, j = 1.8, 3.7 hz, 1h, h-2), 5.30 (app q, j = 9.7 hz, 1h, h-4), 5.02 (d, j = 1.8 hz, 1h, h-1), 4.90 and 4.33 (2 d, 2 j = 11.2 hz, 2h, ch2ph), 4.884.84 (m, 2h, poch2, ch2ph), 4.784.64 (m, 4h, 2 poch2, 2 ch2ph), 4.68 (bs, 1h, h-1), 4.584.45 (m, 7h, 5 ch2ph, poch2, h-6), 4.32 (d, 1h, ch2ph), 4.21 (app t, j = 9.5 hz, 1h, h-4), 4.10 (ddd, j = 1.3, 5.4, 6.6 hz, 1h, h-6), 3.943.84 (m, 4h, h-7a, h-5, h-7a, h-3), 3.82 (dd, j = 2.0, 3.0 hz, 1h, h-2), 3.79 (dd, j = 5.4, 10.0 hz, 1h, h-7b), 3.74 (dd, j = 1.5, 9.7 hz, 1h, h-5), 3.71 (dd, j = 6.0, 10.0 hz, 1h, h-7b), 3.26 (s, 3h, och3), 1.160.96 (m, 28h, tipds) ; c nmr (151 mhz, cdcl3) 165.6 (c = o), 165.4 (c = o), 138.9, 138.8, 138.4, 138.21, 138.17, 135.6 (d, jc, p = 7.9 hz, poch2phc1), 135.4 (d, jc, p = 7.6 hz, poch2phc1), 133.3, 133.0, 130.0, 129.9, 129.45, 129.4, 128.45, 128.4, 128.36, 128.25, 128.22, 128.18, 128.1, 127.77, 127.76, 127.73, 127.68, 127.64, 127.59, 127.5, 127.42, 127.41, 127.34, 127.31, 127.27 (phc), 98.4 (c-1), 98.25 (c-1), 80.1 (c-3), 75.1 (c-6), 74.55 (ch2ph), 74.3 (c-2), 74.15 (c-4), 73.35 (ch2ph), 72.8 (ch2ph), 72.5 (ch2ph), 72.15 (c-5), 71.7 (c-4), 71.7 (ch2ph), 71.1 (c-3), 71.0 (c-7), 70.7 (c-2), 70.0 (d, jc, p = 8.7 hz, c-5), 69.2 (d, jc, p = 5.5 hz, poch2), 69.0 (c-6), 68.85 (d, jc, p = 5.4 hz, poch2), 66.9 (c-7), 55.6 (och3), 17.64, 17.58, 17.5, 17.4 (4 ftipds - ch3), 16.8, 16.7 (bs, 2 ftipds - ch3), 13.8, 13.4 (2 ftipds - ch), 12.6 (d, jc, f = 16.5 hz, 2 ftipds - ch) ; p nmr (243 mhz, cdcl3) 2.76. hrms (esi - tof) m / z [m + nh4 ] calcd for c90h110fo19npsi2 1614.692, found 1614.6907. analytical data for 20b : rf 0.4 (hexane / etoac 3:1) ; []d 37.8 (c 1.0, chcl3) ; h nmr (600 mhz, cdcl3) 8.038.00 (m, 4h, 2 bzh2/h6), 7.597.55 (m, 1h, bz h4), 7.467.39 (m, 5h, bz h4, bzh3/h5, 2 phh), 7.377.35 (m, 2h, phh), 7.337.15 (m, 26h, bzh3/h5, phh), 7.15 7.11 (m, 3h, phh), 7.107.06 (m, 2h, phh), 6.826.79 (m, 2h, phh), 5.82 (dd, j = 3.5, 9.8 hz, 1h, h-3), 5.56 (dd, j = 1.6, 3.6 hz, 1h, h-2), 5.39 (app q, j = 9.8 hz, 1h, h-4), 4.964.83 (m, 5h, 4 ch2ph, poch2), 4.85 (bs, 1h, h-1), 4.78 (dd, jh, p = 8.6, j = 11.9 hz, 1h, poch2), 4.664.58 (m, 3h, poch2, ch2ph, h-6), 4.564.45 (m, 5h, 4 ch2ph, poch2ph), 4.39 (d, j = 11.0 hz, 1h, ch2ph), 4.29 (bs, 1h, h-1), 4.26 (t, j = 9.5, 1h, h-4), 4.204.16 (m, 2h, h-7a, h-6), 4.11 (br d, 1h, h-5), 4.01 (dd, j = 7.3, 10.5 hz, 1h, h-7a), 3.83 (dd, j = 4.0, 10.5 hz, 1h, h-7b), 3.79 (d, j = 2.8 hz, 1h, h-2), 3.74 (dd, j = 8.4, 10.2 hz, 1h, h-7b), 3.48 (dd, j = 2.7, 9.6 hz, 1h, h-3), 3.39 (dd, j = 2.0, 9.6 hz, 1h, h-5), 3.16 (s, 3h, och3), 1.070.95 (m, 28h, ftipds) ; c nmr (151 mhz, cdcl3) 165.8 (c = o), 165.5 (c = o), 138.9, 138.8, 138.5, 138.45, 138.0, 135.4 (d, jc, p = 7.7 hz, poch2phc1), 135.38 (d, jc, p = 7.1 hz, poch2phc1), 133.3, 133.0, 130.1, 129.9, 129.41, 129.38, 128.5, 128.4, 128.33, 128.28, 128.25, 128.19, 128.15, 128.0, 127.85, 127.75, 127.7, 127.6, 127.53, 127.50, 127.4, 127.24, 127.23 (phc), 102.3 (c-1), 98.4 (c-1), 82.8 (c-3), 76.6 (c-5), 75.45 (c-6), 74.7 (ch2ph), 74.2 (c-4), 73.6 (ch2ph), 73.5 (c-2), 73.3 (ch2ph), 72.2 (ch2ph), 72.0 (c-7), 71.9 (ch2ph), 71.5 (d, jc, p = 5.5 hz, c-4), 71.2 (c-3), 70.8 (c-2), 69.3 (d, jc, p = 5.5 hz, poch2), 69.1 (d, jc, p = 7.7 hz, c-5), 68.8 (d, jc, p = 5.5 hz, poch2), 68.7 (c-6), 68.15 (c-7), 55.0 (och3), 17.67, 17.66, 17.44, 17.37, 16.75, 16.70, 16.67 (8 ftipds - ch3), 13.7, 13.4 (2 ftipds - ch), 12.6 (d, jc, f = 16.5 hz, ftipds - ch), 12.5 (jc, f = 16.5 hz, ftipds - ch) ; p nmr (243 mhz, cdcl3) 2.42. hrms (esi - tof) m / z [m + nh4 ] calcd for c90h110fo19npsi2 1614.6927, found 1614.6921. acceptor 11 (25.0 mg, 26 mol) and donor 18 (32.9 mg, 40 mol, 1.5 equiv) were combined and coevaporated with dry toluene. the residue was dissolved in dry dcm (1.2 ml) and transferred into a flame - dried flask containing ground molecular sieves 4 (30 mg). the suspension was stirred under ar for 30 min at rt and then cooled to 39 c. a solution of tmsotf (0.37 l, 2 mol) in dry dcm (0.15 ml) was added dropwise, and the suspension was stirred for 60 min. the reaction was quenched by dropwise addition of et3n (6.9 l, 50 mol) in dcm (0.5 ml) and was warmed to rt. the suspension was filtered over a plug of cotton and washed with dcm, and the filtrate was concentrated. the crude material (59.6 mg) was first purified on silica gel (sio2 : 2 g cartridge, hexane / etoac 3:1 1:2) to give a disaccharide fraction (41.2 mg) that was further purified by preparative hplc (column a, flow rate 10 ml / min, hexane / acetone 5:1) to provide pure -anomer 21a (24.2 mg, 58%) followed by elution of the -anomer 21b (5.9 mg, 14%) both as a colorless oils. analytical data for 21a : rf = 0.22 (hexane / etoac 2:1) ; []d 5.3 (c 0.7, toluene) ; h nmr (600 mhz, cdcl3) 8.078.05 (m, 2h, bzh2/h6), 7.627.58 (m, 1h, bzh4), 7.457.41 (m, 2h, bzh3/h5), 7.397.36 (m, 2h, phh), 7.347.32 (m, 2h, phh), 7.317.15 (m, 33h, phh), 5.55 (dd, j = 3.6, 9.9 hz, 1h, h-3), 5.41 (dd, j = 1.7, 3.6 hz, 1h, h-2), 5.03 (ddd, j = 8.6, 9.8, 9.8 hz, 1h, h-4), 5.01 (d, j = 1.7 hz, 1h, h-1), 4.96 (dd, j = 7.1, 11.8 hz, 1h, poch2), 4.954.90 (m, 3h, poch2), 4.89 and 4.31 (2 d, j = 11.3 hz, 1h, och2), 4.85 and 4.53 (2d, j = 11.85 hz, 2h, ch2ph), 4.73 and 4.69 (2 d, j = 12.2 hz, 2h, ch2ph), 4.56 and 4.49 (2 d, j = 11.75 hz, 2h, ch2ph), 4.56 (d, j = 1.7 hz, 1h, h-1), 4.51 and 4.46 (2 d, j = 11.85 hz, 1h, ch2ph), 4.474.44 (m, 1h, h-6), 4.20 (t, j = 9.5 hz, 1h, h-4), 4.09 (ddd, j = 1.4, 5.3, 6.8 hz, 1h, h-6), 3.89 (dd, j = 8.0, 9.8 hz, 1h, h-7a), 3.883.83 (m, 3h, h-5, h-7a, h-3), 3.82 (dd, j = 1.9, 2.9 hz, 1h, h-2), 3.77 (dd, j = 5.5, 10.1 hz, 1h, h-7b), 3.72 (dd, j = 1.6, 9.8 hz, 1h, h-5), 3.67 (dd, j = 5.9, 9.8 hz, 1h, h-7b), 3.21 (s, 3h, och3), 2.552.45 (m, 2h, lev - h2a / h2b), 2.432.31 (m, 2h, lev - h3a / h3b), 1.98 (s, 3h, lev - h5), 1.140.88 (m, 28h, ftipds) ; c nmr (151 mhz, cdcl3) 206.1 (lev - c4), 171.9 (lev - c1), 165.6 (phc = o), 138.9, 138.7, 138.4, 138.2, 138.1, 135.7 (d, j = 7.9 hz, poch2phc1), 135.6 (d, j = 6.7 hz, poch2phc1), 133.1, 130.0, 129.4, 128.54, 128.48, 128.39, 128.37, 128.36, 128.22, 128.18, 127.9, 127.8, 127.72, 127.67, 127.64, 127.58, 127.5, 127.43, 127.40, 127.34, 127.31 (phc), 98.4 (c-1), 98.2 (c-1), 80.1 (c-3), 75.1 (c-6), 74.5 (ch2ph), 74.14 (c-2), 74.09 (c-4), 73.3 (ch2ph), 72.8 (ch2ph), 72.4 (ch2ph), 72.1 (c-5), 71.8 (d, j = 6.7 hz, c-4), 71.6 (ch2ph), 71.0 (c-7), 70.6 (c-3), 70.2 (c-2), 69.8 (d, jc, p = 8.5 hz, c-5), 69.4 (d, jc, p = 5.5 hz, poch2), 69.2 (d, jc, p = 5.6 hz, poch2), 68.8 (c-6), 66.9 (c-7), 55.6 (och3), 37.7 (lev - c3), 29.6 (lev - c5), 27.9 (lev - c2), 17.6, 17.55, 17.4, 17.35, 16.8, 16.73, 16.71 (8 ftipds - ch3), 13.7, 13.25 (2 ftipds - ch), 12.58 and 12.56 (2 d, jc, f = 16.8 hz, ftipds - ch) ; p nmr (243 mhz, cdcl3) 3.21. hrms (esi - tof) m / z [m + na ] calcd for c88h108fnao20psi2 1613.6586, found 1613.6575. analytical data for 21b : []d 19.0 (c 1.4, chcl3) ; h nmr (600 mhz, cdcl3) 8.068.04 (m, 2h, bzh2/h6), 7.607.57 (m, 1h, bzh4), 7.447.40 (m, 4h, bzh3/h5, 2phh), 7.377.34 (m, 2h, phh), 7.337.16 (m, 31h, phh), 5.60 (dd, j = 3.6, 9.8 hz, 1h, h-3), 5.43 (dd, j = 1.7, 3.6 hz, 1h, h-2), 5.11 (ddd, j = 8.2, 9.8, 9.8 hz, 1h, h-4), 4.97 (dd, jh, p = 7.3, j = 11.8 hz, 1h, poch2), 4.95 and 4.82 (2 d, j = 12.4 hz, 2h, ch2ph), 4.964.86 (m, 5h, 3 poch2, 2 ch2ph), 4.76 (d, j = 1.5 hz, 1h, h-1), 4.604.57 (m, 1h, h-6), 4.60 (d, j = 11.7 hz, 1h, ch2ph), 4.544.47 (m, 4h, 2 ch2ph), 4.38 (d, j = 10.8 hz, 1h, ch2ph), 4.26 (bs, 1h, h-1), 4.25 (app t, j = 9.4, 1h, h-4), 4.184.14 (m, 2h, h-6, h-7a), 4.04 (bd, j = 10.0 hz, 1h, h-5), 4.01 (dd, j = 7.2, 10.3 hz, 1h, h-7a), 3.83 (dd, j = 4.0, 10.3 hz, 1h, h-7b), 3.76 (d, j = 2.8 hz, 1h, h-2), 3.70 (dd, j = 8.5, 10.1 hz, 1h, h-7b), 3.46 (dd, j = 2.9, 9.5 hz, 1h, h-3), 3.38 (dd, j = 2.0, 9.5 hz, 1h, h-5), 3.10 (s, 3h, och3), 2.562.47 (m, 2h, lev - h3a / h3b), 2.462.34 (m, 2h, lev - h2a / h2b), 1.97 (s, 3h, lev - h5), 1.050.87 (m, 28h, tipdsf) ; c nmr (151 mhz, cdcl3) 206.2 (lev - c4), 171.9 (lev - c1), 165.7 (phc = o), 138.9, 138.75, 138.5, 138.45, 138.0, 135.7 (d, jc, p = 8.0 hz, poch2phc1), 135.5 (d, j = 6.6 hz, poch2phc1), 133.4, 123.0, 129.4, 128.54, 128.46, 128.44, 128.39, 128.35, 128.26, 128.24, 128.15, 128.1, 128.0, 127.9, 127.7, 127.6, 127.52, 127.50, 127.4, 127.2 (phc), 102.3 (c-1), 98.4 (c-1), 82.7 (c-3), 76.6 (c-5), 75.4 (c-6), 74.7 (ch2ph), 74.2 (c-4), 73.5 (ch2ph), 73.4 (c-2), 73.3 (ch2ph), 72.2 (ch2ph), 71.9 (c-7), 71.8 (ch2ph), 71.65 (d, jc, p = 6.6 hz, c-4), 70.6 (c-3), 70.5 (c-2), 69.4 (d, jc, p = 5.5 hz, poch2), 69.2 (d, j = 5.5 hz, poch2), 69.0 (d, j = 8.1 hz, c-5), 68.5 (c-6), 68.2 (c-7), 54.9 (och3), 37.7 (lev - c3), 29.6 (lev - c5), 28.0 (lev - c2), 17.69, 17.66, 17.4, 17.35, 16.75, 16.70, 16.65 (ftipds - ch3), 13.7, 13.3 (2 ftipds - ch), 12.54 and 12.49 (2 d, jf, c = 16.5 hz, ftipds - ch) ; p nmr (243 mhz, cdcl3) 2.88. hrms (esi - tof) m / z [m + na ] calcd for c88h108fnao20psi2 1613.6586, found 1613.6590. disaccharide 21a (34 mg, 0.021 mmol) was dissolved in 6:4 pyridine / acoh (1.2 ml). a solution of n2h4h2o (3.2 l, 0.043 mmol, 2 equiv) in 6:4 pyridine / acoh (50 l) was added, and the solution was stirred at rt for 40 min. the reaction was quenched by addition of acetone (40 l) and diluted with etoac (20 ml). the organic layer washed with satd aq nahco3 (5 ml) and brine, dried (naso4), concentrated, and coevaporated with toluene. the crude material (31.2 mg) was purified by column chromatography (sio2 : 2 g cartridge, stepwise gradient hexane / etoac 4:1 2:1) to give 22 (26.7 mg, 84%) as a glasslike solid : rf 0.65 (hexane / etoac 1:1) ; []d + 18.5 (c 1.3, chcl3) ; h nmr (600 mhz, cdcl3) 8.078.04 (m, 2h, bzh2/h6), 7.587.55 (m, 1h, bzh4), 7.427.39 (m, 1h, bzh3/h5), 7.377.35 (m, 2h, phh), 7.337.31 (m, 2h, phh), 7.297.14 (m, 33h, phh), 5.37 (dd, j = 1.7, 3.6 hz, 1h, h-2), 5.054.98 (m, 4h, 2 poch2), 4.86 and 4.30 (2 d, j = 11.1 hz, 2h, ch2ph), 4.83 and 4.52 (2 d, j = 11.8 hz, 2h, ch2ph), 4.81 (ddd, j = 6.1, 9.5, 9.5 hz, 1h, h-4), 4.72 and 4.67 (2 d, j = 12.2 hz, 2h, ch2ph), 4.70 (d, j = 5.0 hz, 1h, 3-oh), 4.58 (d, j = 1.3 hz, 1h, h-1), 4.54 and 4.47 (2 d, j = 11.6 hz, 2h, ch2ph), 4.48 and 4.42 (2 d, j = 11.8 hz, 2h, ch2ph), 4.354.29 (m, 2h, h-3, h-6), 4.19 (app t, j = 9.5 hz, 1h, h-4), 4.08 (ddd, j = 1.2, 5.5, 6.7 hz, 1h, h-6), 3.883.82 (m, 3h, h-7a, h-3, h-7a), 3.813.80 (m, 1h, h-2), 3.75 (dd, j = 5.7, 9.9 hz, 1h, h-7b), 3.72 (dd, j = 1.4, 9.8 hz, 1h, h-5), 3.70 (bd, j = 10.0 hz, 1h, h-5), 3.61 (dd, j = 6.2, 9.9 hz, 1h, h-7b), 3.21 (s, 3h, och3), 1.110.82 (m, 28h, ftipds) ; c nmr (151 mhz, cdcl3) 165.9 (c = o), 138.9, 138.7, 138.4, 138.2, 138.1,135.5 (d, jc, p = 6.9 hz, poch2phc1),135.3 (d, jc, p = 6.6 hz, poch2phc1), 133.15, 130.0, 129.8, 128.65, 128.57, 128.56, 128.34, 128.30, 128.26, 128.22, 128.2, 127.74, 127.73, 127.68, 127.60, 127.57, 127.5, 127.4, 127.35 (phc), 98.5 (c-1), 98.3 (c-1), 80.3 (c-3), 76.55 (d, jc, p = 6.6 hz, c-4), 75.0 (c-6), 74.5 (ch2ph), 74.15 (c-2), 74.1 (c-4), 73.3 (ch2ph), 72.75 (ch2ph), 72.4 (ch2ph), 72.3 (c-2), 72.0 (c-5), 71.7 (ch2ph), 70.7 (c-7), 70.2 (d, jc, p = 5.3 hz, poch2ph), 69.7 (d, jc, p = 5.4 hz, poch2ph), 69.45 (d, jc, p = 11.0 hz, c-5), 68.9, 68.9 (c-3, c-6), 67.4 (c-7), 55.6 (och3), 17.6, 17.5, 17.3, 16.74, 16.69 (ftipds - ch3), 13.6, 13.2 (2 ftipds - ch), 12.55 and 12.53 (2 d, jf, c = 16.5 hz, ftipds - ch) ; p nmr (243 mhz, cdcl3) 0.64. hrms (esi - tof) m / z [m + na ] calcd for c83h102fnao18psi2 1515.6219, found 1515.6245. a suspension of acceptor 4 (100 mg, 0.120 mmol), donor 19 (130 mg, 0.180 mg, 1.50 equiv) and molecular sieves 4 (150 mg) in dry dcm (2.5 ml) was stirred for 20 min at rt and then cooled to 7 c. a solution of tmsotf (2.2 l, 0.012 mmol, 0.10 equiv) in dry dcm (0.5 ml) was added. tlc control (toluene / etoac 5:1) indicated complete consumption of acceptor and donor after 5 min. the reaction was quenched by slow addition of et3n (33.4 l, 0.241 mmol) in dcm (0.3 ml) and stirring for several min. the suspension was filtered through a bed of celite and washed with dcm, and the filtrate was concentrated. the crude material was passed over a short bed of silica gel (sio2 : 2 g cartridge, hexane / etoac 5:1) to furnish a disaccharide - containing fraction which was further purified by preparative hplc on column a (hexane / etoac 6:1) to give first a fraction containing mainly the -(13)-isomer (23 mg 14%) : h nmr (600 mhz, cdcl3) 8.007.97 (m, 2h, bzh2/h6), 7.487.45 (m, 1h, bzh4), 7.317.19 (m, 22h, bzh3/h5, phh), 7.147.08 (m, 4h, phh), 7.077.04 (m, 1h, phh), 6.996.93 (m, 4h, phh), 5.53 (dd, j = 2.2, 3.1 hz, 1h, h-2), 5.22 (app q, j = 10.0 hz, 1h, h-4), 5.16 (dd, jh, p = 6.2, j = 12.1 hz, 1h, poch2), 5.04 (dd, jh, p = 7.2, 13.1 hz, 1h, poch2), 5.03 (d, jh, p = 7.2 hz, 2h, poch2), 4.86 (d, j = 1.9 hz, 1h, h-1), 4.774.74 (m, 3h, h-3, 2 ch2ph), 4.64 and 4.35 (2d, j = 11.0 hz, 2h, ch2ph), 4.61 (d, j = 11.1 hz, 1h, ch2ph), 4.58 (d, j = 7.7 hz, 1h, h-1), 4.554.52 (m, 1h, h-6), 4.514 (d, j = 11.9 hz, 1h, ch2ph), 4.510 and 4.48 (2 d, j = 12.25 hz, 2h, ch2ph), 4.15 (dd, j = 8.6, 12.4 hz, 1h, h-7a), 3.86 (dd, j = 1.3, 12.4 hz, 1h, h-7b), 3.82 (dd, j = 0.9, 9.6 hz, 1h, h-5), 3.71 (dd, j = 1.7, 10.8 hz, 1h, h-6a), 3.533.49 (m, 2h, h-6b, h-3), 3.453.42 (m, 1h, h-5), 3.34 (s, 3h, och3), 3.34 (dd, j = 7.8, 9.1 hz, 1h, h-2), 3.15 (t, j = 9.4 hz, 1h, h-4), 1.130.82 (m, 28h, ftipds) ; c nmr (151 mhz, cdcl3) 165.8 (c = o), 138.8, 138.6, 138.4, 138.3 (phc), 136.4 (d, jc, p = 7.6 hz, poch2phc1), 136.1 (d, j = 7.7 hz, poch2phc1), 133.2, 130.0, 129.2, 128.4, 128.33, 128.30, 128.24, 128.16, 128.1, 127.9, 127.82, 127.79, 127.71, 127.68, 127.6, 127.5, 127.25, 126.8 (phc), 98.4 (bs, c-1), 98.3 (c-1), 84.4 (c-3), 81.6 (c-2), 77.7 (c-4), 75.2 (ch2ph), 75.0 (c-5), 74.8 (ch2ph), 74.4 (ch2ph), 73.7 (c-6), 73.2 (ch2ph), 73.0 (bs, c-4), 72.2 (d, jc, p = 6.3 hz, c-5), 71.4 (bs, c-3), 68.968.7 (bs, 2 poch2, c-6), 68.2 (c-7), 67.8 (bs, c-2), 55.2 (och3), 17.7, 17.5, 17.4, 17.31, 17.26, 17.24, 17.16 (8 tipds - ch3), 13.6, 13.4, 12.73, 12.71 (4 tipds - ch) ; hrms (esi - tof) m / z [m + na ] calcd for c75h93nao17psi2 1375.5581, found 1375.5601. continued elution of the column gave -isomer 23 (99.4 mg, 61%) as a colorless oil : []d + 20 (c 1.0, chcl3) ; h nmr (600 mhz, cdcl3) 8.038.00 (m, 2h, bz - h2/h6), 7.56 (t, j = 7.4 hz, 1h, bzh4), 7.37 (t, j = 7.8 hz, 2h, bz - h3/h5), 7.337.30 (m, 2h, phh), 7.287.16 (m, 24h, phh), 7.097.05 (m, 2h, phh), 6.966.91 (m, 2h, phh), 5.38 (dd, j = 2.3, 3.2 hz, 1h, h-2), 5.23 (d, j = 3.4 hz, 1h, h-1), 5.114.96 (m, 5h, 2 poch2, h-4), 4.90 (d, j = 1.8 hz, 1h, h-1), 4.79 and 4.53 (2d, j = 12.15 hz, 2h, ch2ph), 4.75 (dd, j = 3.2, 9.4 hz, 1h, h-3), 4.66 and 4.34 (2 d, j = 11.15 hz, 2h, ch2ph), 4.62 and 4.47 (2 d, j = 10.8 hz, 2h, ch2ph), 4.58 (d, j = 8.5 hz, 1h, h-6), 4.46 and 4.23 (2 d, j = 12.15 hz, 2h, ch2ph), 4.02 (dd, j = 8.6, 12.3 hz, 1h, h-7a), 3.92 (d, j = 9.5 hz, 1h, h-5), 3.92 (t, j = 9.4 hz, 1h, h-3), 3.77 (bd, j = 10.0 hz, 1h, h-5), 3.69 (d, j = 12.0 hz, 1h, h-7b), 3.61 (d, j = 9.5 hz, 1h, h-4), 3.48 (dd, j = 3.5, 9.9 hz, 1h, h-2), 3.34 (s, 3h, och3), 3.24 (dd, j = 2.6, 10.8 hz, 1h, h-6a), 3.17 (bd, j = 11.0 hz, 1h, h-6b), 1.070.99 and 0.900.86 (m, 20h and 8h, tipds) ; c nmr (151 mhz, cdcl3) 165.8 (c = o), 138.9, 138.6, 138.5, 138.0, 136.01, 135.96 (phc), 135.9 (d.i., 2 poch2phc), 133.2, 129.9, 129.6, 128.5, 128.45, 128.38, 128.3, 128.24, 128.19, 128.12, 128.09, 128.07, 127.93, 127.88, 127.8, 127.6, 127.5, 127.45, 127.37, 127.2 (phc), 98.2 (c-1), 97.7 (c-1), 81.2 (c-3), 79.75 (c-2), 77.2 (c-4), 75.8 (c-3), 75.2 (ch2ph), 74.6 (d, jc, p = 6.9 hz, c-4), 74.4 (ch2ph), 73.5 (c-6), 73.4 (ch2ph), 72.5 (c-2), 72.46 (ch2ph), 71.4 (bs, c-5), 71.3 (c-5), 69.5 (d, jc, p = 5.6 hz, poch2), 69.3 (d, jc, p = 4.9 hz, poch2), 68.03, 67.99 (c-6, c-7), 55.3 (och3), 17.6, 17.5, 17.4, 17.34, 17.32, 17.25 (8 tipds - ch3), 13.35, 13.2, 12.7, 12.6 (tipds - ch) ; hrms (esi - tof) m / z [m + na ] calcd for c75h93nao17psi2 1375.5581, found 1375.5562. acceptor 4 (57 mg, 0.069 mmol) was coevaporated with dry toluene and dissolved in dry dcm (0.8 ml) in a flame - dried flask. ground molecular sieves (4, 40 mg) were added, and the suspension was stirred for 30 min at rt under ar. a solution of tbdmsotf (1.57 l, 0.007 mmol, 0.10 equiv) in dry dcm (in 0.1 ml) was added dropwise. then a solution of donor 18 (103 mg, 0.123 mmol, 1.8 equiv) in dry dcm (1 ml) was added continuously within 1 h via a syringe pump. additional tbdmsotf solution (0.05 equiv) was added after 1.5 h, and the suspension was stirred for 1 h at rt. the reaction was quenched by dropwise addition of et3n (10 l) in dcm (0.5 ml). the suspension was filtered over celite and washed with dcm, and the filtrate was concentrated. the crude material was first purified by mplc (sio2 : 18 g, flow rate 15 ml / min, dcm / etoac 30:1) to give the anhydro byproduct 25 (17.3 mg, 25% of donor used) followed by a disaccharide - containing fraction (53.9 mg). analytical data for 25 : nmr data were in full agreement with published values : hrms (esi - tof) m / z [m + nh4 ] calcd for c35h40no6 570.2850, found 570.2846. the disaccharide fraction was further purified by preparative hplc (in three portions on column a, flow rate 15 ml / min, hexane / etoac 7:1) to furnish -anomer 24 (36.3 mg, 36%) as an oil ; rf 0.56 (hexane / etoac 3:1) : []d 11.3 (c 1.6, chcl3) ; h nmr (600 mhz, cdcl3) 8.078.03 (m, 2h, bzh2/h6), 7.587.54 (m, 1h, bzh4), 7.397.35 (m, 4h, bzh3/h5, 2 phh), 7.09 (m, 31h, phh), 7.077.03 (m, 2h, phh), 5.41 (d, j = 1.7 hz, 1h, h-1), 5.40 (dd, j = 2.0, 3.2 hz, 1h, h-2), 5.034.94 (m, 4h, h-4, 3 poch2), 4.92 (dd, jh, p = 7.0, j = 11.8 hz, 1h, poch2), 4.82 and 4.47 (2 d, j = 11.85 hz, 2h, ch2ph), 4.76 and 4.31 (2 d, j = 11.55 hz, 2h, ch2ph), 4.76 (d, j = 1.9 hz, 1h, h-1), 4.62 and 4.52 (2d, j = 12.1 hz, 2h, ch2ph), 4.61 and 4.60 (2 d, j = 12.15 hz, 2h, ch2ph), 4.554.45 (m, 1h, h-6), 4.43 (dd, j = 3.3, 9.5 hz, 1h, h-3), 4.20 and 4.16 (2 d, j = 11.6 hz, 2h, ch2ph), 4.19 (app t, j = 9.6 hz, 1h, h-4), 4.10 (dd, j = 8.6, 12.5 hz, 1h, h-7a), 4.094.06 (m, 1h, h-6), 4.034.01 (m, 1h, h-2), 3.89 (dd, j = 7.4, 10.0 hz, 1h, h-7a), 3.87 (dd, j = 1.5, 9.8 hz, 1h, h-5), 3.803.75 (m, 3h, h-7b, h-7b, h-3), 3.66 (bd, j = 9.4 hz, 1h, h-5), 3.28 (s, 3h, och3), 1.070.82 (m, 28h, tipds) ; c nmr (151 mhz, cdcl3) 165.6 (c = o), 139.2, 139.1, 139.0, 138.7, 138.57 (phc), 135.7 (d, jc, p = 7.7 hz, poch2phc1), 135.6 (d, jc, p = 6.6 hz, poch2phc1), 133.4, 130.0, 129.7, 128.6, 128.5, 128.42, 128.39, 128.37, 128.25, 128.1, 128.02, 128.00, 127.9, 127.2, 127.6, 127.53, 127.46, 127.4, 127.29, 127.27, 127.1, 127.05, 127.0, 126.8 (phc), 100.35 (c-1), 98.1 (c-1), 79.95 (c-3), 75.6 (c-6), 74.9 (c-2), 74.5 (dd, jc, p = 6.6 hz, c-4), 74.35 (c-3), 73.9 (c-4), 73.7 (ch2ph), 73.6 (c-6), 73.23 (c-5), 73.17 (ch2ph), 72.75 (ch2ph), 72.7 (c-2), 72.13 (ch2ph), 72.08 (d, jc, p = 5.5 hz, c-5), 71.6 (c-7), 71.4 (ch2ph), 69.4 (d, jc, p = 4.4 hz, poch2), 69.3 (d, jc, p = 5.5 hz, poch2), 68.2 (c-7), 55.1 (och3), 17.6, 17.5, 17.4, 17.3, 17.24, 17.21, 17.1 (8 tipds - ch3), 13.6, 13.55, 12.73, 12.71 (4 tipds - ch) ; p nmr (243 mhz, cdcl3) 2.97 ; hrms (esi - tof) m / z [m + nh4 ] calcd for c83h105no18p si2 1490.6602, found 1490.6596. a solution of 23 (89.5 mg, 66.1 mol) in dry dcm (3 ml) was treated with treat (216 l, 60 equiv of f) at 0 c for 2.5 h according to general procedure 1. the residue (87 mg) was purified by column chromatography (sio2 : 2 g cartridge, hexane / etoac 5:1 4:1 3:1) to give compound 26 (78.4 mg, 88%) as an oil : []d + 11.7 (c 1.0, chcl3) ; h nmr (600 mhz, cdcl3) 8.028.00 (m, 2h, bzh2/h6), 7.56 7.53 (m, 1h, bzh4), 7.387.34 (m, 2h, bzh3/h5), 7.307.27 (m, 4h, phh), 7.267.15 (m, 22h, phh), 7.127.08 (m, 2h, phh), 6.966.93 (m, 2h, phh), 5.38 (app t, j = 3.0 hz, 1h, h-2), 5.18 (d, j = 3.5 hz, 1h, h-1), 5.095.05 (m, 3h, poch2), 5.044.99 (m, 2h, h-4, poch2), 4.95 (d, j = 2.6 hz, 1h, h-1), 4.72 and 4.50 (2d, j = 12.2 hz, 2h, ch2ph), 4.684.63 (m, 3h, h-3, ch2ph), 4.67 and 4.35 (2 d, j = 11.25 hz, 2h, ch2ph), 4.524.47 (m, 2h, h-6, ch2ph), 4.45 and 4.21 (2 d, j = 12.1 hz, 2h, ch2ph), 4.17 (dd, j = 2.4, 9.6 hz, 1h, h-5), 3.90 (t, j = 9.4 hz, 1h, h-3), 3.783.73 (m, 2h, h-5, h-7a), 3.723.67 (m, 1h, h-7b), 3.61 (t, j = 9.5 hz, 1h, h-4), 3.47 (dd, j = 3.5, 9.8 hz, 1h, h-2), 3.41 (s, 3h, och3), 3.25 (dd, j = 2.7, 11.0 hz, 1h, h-6a), 3.11 (bd, j = 10.8 hz, 1h, h-6b), 1.060.92 (m, 28h, ftipds) ; c nmr (151 mhz, cdcl3) 165.8 (c = o), 138.85, 138.6, 138.4, 137.9, 135.9, 135.8 (2 poch2phc1), 133.2, 129.9, 129.6, 128.54, 128.48, 128.4, 128.3, 128.20, 128.16, 128.1, 128.01, 127.99, 127.96, 127.8, 127.6, 127.52, 127.4, 127.40, 127.29, 127.27 (phc), 98.2 (c-1), 97.9 (c-1), 81.3 (c-3), 79.7 (c-2), 77.2 (c-4 '), 76.2 (c-3), 75.2 (ch2ph), 74.7 (d, jc, p = 6.4 hz, c-4), 74.4 (ch2ph), 73.4 (ch2ph), 72.58 (c-2), 72.56 (ch2ph), 71.3 (c-5), 71.0 (bs, c-6), 70.2 (c-5), 69.6 (d, jc, p = 5.6 hz, poch2), 69.5 (d, jc, p = 5.3 hz, poch2), 67.9 (c-6), 63.4 (c-7), 55.4 (och3), 17.4, 17.3, 17.2, 17.1, 16.7, 16.6 (8 ftipds - ch3), 13.5, 13.1, 12.6, 12.5 (4 ftipds - ch) ; hrms (esi - tof) m / z [m + na ] calcd for c75h94fnao17psi2 1395.5643, found 1395.5638. acceptor 26 (33.2 mg, 24.2 mol) and donor 18 (30.2 mg, 36.0 mol, 1.5 equiv) were combined, coevaporated with dry toluene, dissolved in dry dcm (1.4 ml), and transferred into a flame - dried flask containing ground molecular sieves 4 (50 mg). the suspension was stirred under ar for 30 min at rt and was then cooled to 40 a solution of tmsotf (0.22 l, 1.2 mol, 0.05 equiv) in dry dcm (0.1 ml) was added dropwise, and the reaction mixture was stirred under ar while gradually raising the temperature to 35 c during 1 h. since tlc control (hexane / etoac 3:1) still indicated the presence of starting materials, additional tmsotf solution (0.11 l in 0.05 ml dcm) was added, and stirring was continued for 1 h. the suspension was warmed to 0 c, the addition of promotor was repeated, and stirring was continued for 1 h. the reaction was quenched by dropwise addition of a solution of et3n (6.7 l) in dcm (0.5 ml). the suspension was filtered through a plug of cotton, washed with dcm, and concentrated. purification of the residue was performed first on silica gel (sio2 : 2 g cartridge, hexane / etoac 6:1 4:1 3:1 0:1) to collect the trisaccharide - containing fraction (43.4 mg). final purification was achieved by preparative hplc (column a, hexane / etoac 5:1) to afford first the 7-o - tms disaccharide acceptor (2.5 mg, data not shown) followed by 27 (24.7 mg, 51%) and fractions containing anhydro derivative 25 and the -(17)-linked trisaccharide (8.9 mg, 18%, data not shown). analytical data for 27 : rf 0.55 (hexane / etoac 2:1) ; []d + 10.2 (c 1.1, chcl3) ; h nmr (600 mhz, cdcl3) 8.048.01 (m, 2h, bzh2/h6), 7.587.54 (m, 1h, bzh4), 7.387.31 (m, 6h, bzh3/h5, 4 phh), 7.297.13 (m, 47h, phh), 7.057.02 (m, 2h, phh), 6.946.90 (m, 2h, phh), 5.38 (dd, j = 3.4, 2.0 hz, 1h, h-2), 5.18 (d, j = 3.5 hz, 1h, h-1), 5.134.95 (m, 5h, h-4, 2 poch2), 5.05 (d, j = 1.7 hz, 1h, h-1), 4.904.86 (m, 2h, ch2ph), 4.81 (d, j = 1.6 hz, 1h, h-1), 4.824.77 (m, 1h, h-6), 4.734.63 (m, 4h, 4 ch2ph), 4.61 (dd, j = 3.3, 9.6 hz, 1h, h-3), 4.574.40 (m, 9h, 9 ch2ph), 4.354.31 (m, 2h, ch2ph), 4.23 (d, j = 12.0 hz, 1h, ch2ph), 4.21 (app t, j = 9.5 hz, 1h, h-4), 4.134.10 (m, 1h, h-6), 4.06 (br d, j = 9.9 hz, 1h, h-5), 3.933.86 (m, 4h, h-7a, h-3, h-3, h-7a), 3.83 (dd, j = 2.0, 2.7 hz, 1h, h-2), 3.783.73 (m, 3h, h-7b, h-5, h-5), 3.68 (dd, j = 5.7, 9.9 hz, 1h, h-7b), 3.60 (app t, j = 9.5 hz, 1h, h-4), 3.46 (dd, j = 3.5, 9.8 hz, 1h, h-2), 3.28 (s, 3h, och3), 3.263.23 (m, 1h, h-6a), 3.223.18 (m, 1h, h-6b), 1.130.79 (m, 28h, ftipds) ; c nmr (151 mhz, cdcl3) 166.0 (c = o), 138.93, 138.86, 138.8, 138.7, 138.6, 138.4, 138.3, 137.9 (phc), 136.0 (d, jc, p = 7.0 hz, poch2phc1), 135.9 (d, jc, p = 7.7 hz, poch2phc1), 133.2, 129.9, 129.6, 128.5, 128.45, 128.4, 128.34, 128.32, 128.25, 128.21, 128.18, 128.17, 128.1, 128.02, 127.97, 127.81, 127.76, 127.7, 127.62, 127.61, 127.56, 127.52, 127.48, 127.46, 127.45, 127.29, 127.28, 127.25, 127.21, 127.16 (phc), 98.6 (c-1), 98.1 (c-1), 97.6 (c-1), 81.2 (c-3), 80.5 (c-3), 79.5 (c-2), 77.2 (c-4), 76.7 (c-3), 75.3 (c-6), 75.15, 74.6 (2 ch2ph), 74.3 (c-2), 74.24 (ch2ph), 74.17 (c-4), 73.9 (d, j = 6.7 hz, c-4), 73.4, 73.3, 72.7 (3 ch2ph), 72.6 (c-2), 72.4 (ch2ph), 72.34 (c-5), 72.31, 71.65 (2 ch2ph), 71.5 (c-7), 71.4 (c-5), 69.7 (c-5), 69.4 (d, jc, p = 5.4 hz, poch2), 69.2 (d, jc, p = 4.9 hz, poch2), 68.65 (c-6), 67.9 (c-6), 66.8 (c-7), 55.6 (s, och3), 17.6, 17.5, 17.4, 17.3, 16.8, 16.7 (ftipds - ch3), 13.7, 13.4 (2 ftipds - ch), 12.6 (d, jf, c = 16.5 hz, ftipds - ch), 12.55 (d, jf, c = 16.9 hz, ftipds - ch) ; p nmr (243 mhz, cdcl3) 3.04 ; hrms (esi - tof) m / z [m + na ] calcd for c117h136fnao23psi2 2037.8625, found 2037.8564. compound 22 (38.5 mg, 25.8 mol) and donor 19 (55.0 mg, 77.3 mol, 3.0 equiv) were combined and coevaporated with dry toluene. the residue was dissolved in dry dcm (1.5 ml) and transferred into a flame - dried 10 ml flask containing ground molecular sieves 4 (50 mg). the suspension was stirred for 5 min at rt and then cooled to 22 c. a solution of tbdmsotf (0.29 l, 1.3 mol, 0.05 equiv) in dry dcm (0.1 ml) was added dropwise, and the reaction mixture was stirred under ar for 2 h. the reaction was quenched by dropwise addition of et3n (16 l, 26 mol) in dcm (0.5 ml) and warmed to rt. the suspension was filtered over a plug of cotton, washed with dcm, and concentrated. the crude material was passed through a short silica gel column (sio2 : 2 g, stepwise gradient of hexane / acetone 7:1 3:1) to afford a fraction containing the trisaccharides (50.7 mg). this material was further purified by preparative hplc (column a, flow rate 15 ml / min, hexane / etoac 7:1 6:1) to afford pure -anomer 27 (38.2 mg, 73.5%) as a colorless oil. continued elution afforded the -(13)-linked glucosyl isomer as colorless syrup (4.1 mg, 8%) : h nmr (600 mhz, cdcl3) 8.017.98 (m, 2h, bzh2/h6), 7.477.43 (m, 1h, bzh4), 7.387.35 (m, 2h, phh), 7.337.31 (m, 2h, phh), 7.287.10 (m, 46h, bzh3/h5, 44 phh), 7.097.04 (m, 3h, phh), 7.006.95 (m, 4h, phh), 5.55 (dd, j = 2.1, 3.0 hz, 1h, h-2), 5.23 (app q, j = 9.8 hz, 1h, h-4), 5.18 (dd, jh, p = 6.0, j = 12.2 hz, 1h, poch2), 5.03 (dd, jh, p = 7.1, j = 12.3 hz, 1h, poch2), 5.01 (d, j = 1.5 hz, 1h, h-1), 4.984.96 (m, 2h, poch2ph), 4.87 and 4.32 (2d, j = 11.2 hz, 2h, ch2ph), 4.85 (d, j = 11.9 hz, 1h, ch2ph), 4.814.76 (m, 2h, h-3, ch2ph), 4.75 and 4.60 (2 d, j = 11.2 hz, 2h, ch2ph), 4.69 and 4.65 (2 d, j = 12.3 hz, 2h, ch2ph), 4.65 (d, j = 1.7 hz, 1h, h-1), 4.63 and 4.33 (2 d, j = 11.0 hz, 2h, ch2ph), 4.61 (d, j = 7.7 hz, 1h, h-1), 4.554.50 (m, 3h, h-6, 2 ch2ph), 4.48 and 4.42 (2 d, j = 11.7 hz, 2h, ch2ph), 4.47 and 4.43 (2 d, j = 11.7 hz, 2h, ch2ph), 4.41 and 4.34 (2 d, j = 12.0 hz, 2h, ch2ph), 4.19 (app t, j = 9.6 hz, 1h, h-4), 4.104.07 (m, 1h, h-6), 3.91 (dd, j = 8.3, 9.5 hz, 1h, h-7a), 3.883.82 (m, 3h, h-5, h-7a, h-3), 3.773.74 (m, 2h, h-2, h-7b), 3.72 (dd, j = 1.4, 9.6 hz, 1h, h-5), 3.713.66 (m, 2h, h-7b, h-6a), 3.563.50 (m, 2h, h-6b, h-3), 3.463.43 (m, 1h, h-5), 3.32 (dd, j = 7.8, 9.2 hz, 1h, h-2), 3.21 (s, 3h, och3), 3.19 (app t, j = 9.5 hz, 1h, h-4);c nmr (151 mhz, cdcl3) 165.9 (c = o), 139.0, 138.82, 138.79, 138.6, 138.45, 138.24, 138.19, 138.1 (phc), 136.4 (d, j = 8.9 hz, poch2phc1), 136.0 (d, j = 7.7 hz, poch2phc1), 133.2, 130.0, 129.2, 128.5, 128.42, 128.36, 128.34, 128.29, 128.22, 128.17, 128.1, 127.9, 127.83, 127.80, 127.76, 127.74, 127.72, 127.64, 127.61, 127.59, 127.57, 127.56, 127.53, 127.52, 127.50, 127.39, 127.36, 127.31, 127.29, 127.22, 127.19, 126.75 (phc), 98.3 (d.i., c-1 and c-1), 98.2 (c-1), 84.3 (c-3), 81.6 (c-2), 80.1 (c-3), 77.5 (c-4), 75.18 (ch2ph), 75.16 (c-6), 74.8 (c-5), 74.75, 74.5 (2 ch2ph), 74.33 (c-2), 74.31 (ch2ph), 74.2 (c-4), 73.3, 73.2, 72.71 (3 ch2ph), 72.69 (c-4), 72.4 (ch2ph), 72.2 (c-5), 71.7 (ch2ph), 71.4 (c-3), 71.0 (c-7), 70.1 (c-5), 69.2 (c-6), 68.83 (d, jc, p = 5.6 hz, poch2), 68.77 (c-6), 68.75 (d, jc, p = 5.0 hz, poch2), 67.4 (c-2), 67.1 (c-7), 55.5 (s, och3), 17.70, 17.66, 17.5, 17.4, 16.8, 16.7 (ftipds - ch3), 13.9 (2 ftipds - ch), 13.3 (2 ftipds - ch), 12.59 and 12.57 (2 d, jf, c = 16.5 hz, ftipds - ch) ; p nmr (243 mhz, cdcl3) 2.69 ; hrms (esi - tof) m / z [m + na ] calcd for c117h136fnao23psi2 2037.8625, found 2037.8579. an aliquot of the hf pyridine solution (0.4 ml, 33 equiv of f) was added to a solution of 6 (112 mg, 0.120 mmol) in dry thf (3.4 ml) in a teflon vessel, and the solution was stirred at rt for 3 h. additional aliquots (0.1 and 0.285 ml) were added in two portions, and stirring was continued for 75 min. the solution was poured into a stirred mixture of cold 50% aq satd nahco3 and etoac. the aqueous layer was extracted twice with etoac, and the combined organic layers were washed with satd aq nahco3 and brine, dried (na2so4), and concentrated. the residue was coevaporated with toluene and submitted to column chromatography (sio2 : 2 g cartridge, tol / etoac 5:1 3:1 2:1 1:1 2:3) to give 28 (60 mg, 72.3%) as a glasslike solid. alternatively, heptoside 6 (35 mg, 37 mol) was treated according to general procedure 1. the crude material (39.1 mg) was purified by chromatography (sio2 : 2 g cartridge, hex / etoac 1:2 1:3 1:4 etoac) to give 28 (23.6 mg, 91%) as a glasslike solid : []d 80 (c 0.3, chcl3) ; h nmr (600 mhz, cdcl3) 8.088.06 (m, 2h, bzh2/h6), 7.857.83 (m, 2h, bzh2/h6), 7.647.61 (m, 1h, bzh4), 7.517.47 (m, 2h, bzh3/h5), 7.457.42 (m, 1h, bzh4), 7.317.27 (m, 3h, phh), 7.217.15 (m, 5h, bzh3/h5, 3 phh), 7.097.06 (m, 2h, phh), 6.896.87 (m, 2h, phh), 5.81 (dd, j = 9.8, 3.6 hz, 1h, h-3), 5.60 (dd, j = 3.5, 1.6 hz, 1h, h-2), 5.13 (app q, j = 9.7 hz, 1h, h-4), 4.92 (dd, j = 11.6, jh, p = 8.8 hz, 1h, poch2), 4.90 (d, j = 1.8 hz, 1h, h-1), 4.82 (dd, j = 11.7 hz, jp, h = 8.3 hz, 1h, poch2), 4.744.68 (m, 2h, ch2ph), 4.24 (d, j = 6.3 hz, 1h, 6-oh), 4.124.08 (m, 1h, h-6), 3.94 (dd, j = 11.1, 7.8 hz, 1h, h-7a), 3.90 (dd, j = 9.8, 1.5 hz, 1h, h-5), 3.703.65 (m, 1h, h-7b), 3.44 (s, 3h, och3), 2.12 (d, j = 8.7 hz, 1h, 7-oh) ; c nmr (151 mhz, cdcl3) 165.4 (c = o), 165.3 (c = o), 135.1 (d, jc, p = 6.5 hz, poch2phc1), 134.9 (d, jc, p = 5.7 hz, poch2phc1), 133.6, 133.1, 129.95, 129.8, 129.30, 129.26, 128.65, 128.54, 128.47, 128.38, 128.3, 127.9, 127.7 (phc), 98.8 (c-1), 71.6 (d, jc, p = 5.3 hz, c-4), 71.0 (d, jc, p = 3.7 hz, c-5), 70.6 (c-2), 70.1 (d, jc, p = 6.2 hz, poch2), 70.0 (d, jc, p = 4.2 hz, c-3), 69.8 (d, jc, p = 5.6 hz, poch2), 68.3 (c-7), 55.4 (och3) ; hrms (esi - tof) m / z [m + h ] calcd for c36h38o12p 693.2095, found 693.2101. a solution of dibenzylphosphate 28 (117 mg, 0.169 mol) in meoh (8 ml) was treated with 10% pd / c (23 mg) for 2 h according to general procedure 2. the filtrate was concentrated to give 29 (87 mg, quant) as a glasslike solid : []d 55.1 (c 0.25, meod) ; h nmr (600 mhz, meod) 8.078.04 (m, 2h, bzh2/6), 7.987.95 (m, 2h, bzh2/6), 7.657.60 (m, 1h, bzh4), 7.547.50 (m, 1h, bzh4), 7.507.45 (m, 2h, bzh3/5), 7.377.32 (m, 2h, bzh3/5), 5.625.60 (m, 2h, h-2, h-3), 5.06 (app q, j = 10.3 hz, 1h, h-4), 4.91 (bs, 1h, h-1), 4.17 (app t, j = 7.0 hz, 1h, h-6), 4.02 (bd, j = 9.8 hz, 1h, h-5), 3.79 (dd, j = 10.5, 6.8 hz, 1h, h-7a), 3.76 (dd, j = 10.5, 7.1 hz, 1h, h-7b), 3.50 (s, 3h, och3) ; c nmr (151 mhz, meod) 167.2, 166.9 (2 c = o), 134.7, 134.16, 131.1, 131.0, 130.7, 129.6, 129.2 (phc), 100.1 (c-1), 72.7 (d, jc, p = 2.2 hz, c-3), 71.6 (d, jc, p = 5.5 hz, c-5), 71.5 (c-2), 71.1 (d, jc, p = 4.9 hz, c-4), 69.8 (c-6), 63.3 (c-7), 55.8 (och3) ; hrms (esi - tof) m / z calcd for c22h25o12p [m h ] 511.1011, found 511.1013 ; hrms (esi - tof) m / z [m + h ] calcd for c22h26o12p 513.1156, found 513.1159. a solution of 29 (74 mg, 0.144 mmol) in meoh (5 ml) was treated with 0.1 m methanolic naome solution (13 ml). the solution (ph of 11) was stirred for 10 h at rt and made neutral by addition of cation - exchange resin (h - form). the residue was dissolved in water, extracted with et2o and the aqueous phase was passed through a pd-10 column (water). product containing fractions were pooled and lyophilized to give 47 mg (94%) of compound 30 : []d + 73.6 (c 0.35, h2o) [lit. []d + 60.1 (c 0.22, h2o) ; lit. []d + 30 (c 1.0, h2o) ] ; nmr data agree with ref (44). a different chemical shift of c-4 has been reported in ref (43) (72.39 ppm) : c nmr (151 mhz, d2o) 101.5 (c-1), 71.9 (c-3), 71.4 (d, jc, p = 6.9 hz, c-5), 70.35 (c-2), 69.9 (d, jc, p = 4.4 hz, c-4), 69.25 (c-6), 63.3 (c-7), 55.6 (och3) ; p nmr (243 mhz, d2o) 4.87 ; hrms (esi - tof) m / z calcd for c8h17o10p [m h ] 303.0487, found 303.0483 ; hrms (esi - tof) m / z [m + h ] calcd for c8h18o10p 305.0632, found 305.0634. disaccharide 20a (223.5 mg, 139.9 mol) was coevaporated with toluene, dissolved in dry dcm (6 ml), and transferred into a teflon - flask. pyridine 70% (188 l, 51 equiv of f) was added dropwise to the solution, and the mixture was vigorously stirred at rt for 3 h. the solution was added dropwise into a stirred mixture of ice - cold satd aq nahco3 (40 ml) and etoac (30 ml). the aqueous layer was extracted with etoac (30 ml), and the combined organic layer was washed with h2o and brine (10 ml), dried (na2so4), and concentrated. the residue (208 mg) was purified by chromatography (2 g cartridge, hexane / etoac 5:1 3:1 2:1) to afford compound 31 (150 mg, 80%) as a colorless oil : rf 0.27 (hexane / etoac 3:2) ; []d 19.5 (c 1.2, chcl3) ; h nmr (600 mhz, cdcl3) 8.088.06 (m, 2h, bzh2/h6), 7.877.84 (m, 2h, bz, h2/h6), 7.647.60 (m, 1h, bzh4), 7.507.46 (m, 2h, bzh3/h5), 7.467.42 (m, 1h, bzh4), 7.397.37 (m, 2h, phh), 7.357.32 (m, 2h, phh), 7.307.18 (m, 26h, bzh3/h5, phh), 7.187.16 (m, 1h, phh), 7.147.12 (m, 2h, phh), 7.097.05 (m, 2h, phh), 6.896.87 (m, 2h, phh), 5.78 (dd, j = 3.5, 9.9 hz, 1h, h-3), 5.55 (dd, j = 1.6, 3.4 hz, 1h, h-2), 5.12 (app q, j = 9.7 hz, 1h, h-4), 5.04 (d, j = 1.7 hz, 1h, h-1), 4.91 (d, j = 10.9 hz, 1h, ch2ph), 4.89 (dd, jh, p = 8.1 hz, j = 11.6 hz, 1h, poch2), 4.81 (d, j = 11.9 hz, 1h, ch2ph), 4.79 (dd, jh, p = 7.8 hz, j = 11.7 hz, 1h, poch2), 4.74 (d, j = 1.2 hz, 1h, h-1), 4.74 (d, j = 12.3 hz, 1h, ch2ph), 4.724.66 (m, 3h, poch2ph, ch2ph), 4.61 (d, j = 11.8 hz, 1h, ch2ph), 4.55 (d, j = 11.7 hz, 1h, ch2ph), 4.53 (d, j = 11.5 hz, 1h, ch2ph), 4.51 (bs, 2h, ch2ph), 4.36 (d, j = 10.7 hz, 1h, ch2ph), 4.21 (app t, j = 9.5 hz, 1h, h-4), 4.174.10 (m, 2h, h-6, h-6), 3.90 (dd, j = 3.0, 9.2 hz, 1h, h-3), 3.853.81 (m, 2h, h-2, h-7a), 3.803.73 (m, 4h, h-5, h-7b, h-7a, oh), 3.70 (dd, j = 1.3, 9.7 hz, 1h, h-5), 3.62 (dd, j = 7.3, 10.5 hz, 1h, h-7b), 3.25 (s, 3h, och3) ; c nmr (151 mhz, cdcl3) 165.4 (c = o), 165.3 (c = o), 138.9, 138.7, 138.34, 138.28, 138.1, 135.13 (d, jc, p = 7.4 hz, poch2phc1), 135.07 (d, jc, p = 6.6 hz, poch2phc1), 133.5, 133.1, 129.95, 129.8, 129.4, 129.3, 128.64, 128.61, 128.56, 128.44, 128.41, 128.38, 128.35, 128.29, 128.28, 128.23, 128.21, 127.8, 127.75, 127.73, 127.69, 127.64, 127.62, 127.5, 127.45, 127.41, 127.37, 127.3 (phc), 98.7 (c1), 98.4 (c-1), 80.2 (c-3), 74.9 (c-6), 74.5 (ch2ph), 74.2, 74.15 (c-2, c-4), 73.3 (ch2ph), 72.7 (ch2ph), 72.4 (ch2ph), 71.8 (ch2ph), 71.6 (c-5), 71.4 (d, jc, p = 5.2 hz, c-4), 70.6 (c-2), 70.4 (d, jc, p = 3.6 hz, c-5), 70.1 (c-7), 70.0 (d, jc, p = 3.6 hz, c-3), 69.9 (d, jc, p = 5.9 hz, poch2), 69.7 (d, jc, p = 5.6 hz, poch2), 67.4 (c-7), 66.5 (c-6), 55.5 (och3) ; p nmr (243 mhz, cdcl3) 0.34. hrms (esi - tof) m / z [m + nh4 ] calcd for c78h83o18np 1352.5342, found 1352.5315. a suspension of heptobioside 31 (147 mg, 0.110 mmol) in dry meoh (4.8 ml) and pd / c (14 mg) was treated for 7 h according to general procedure 2. the filtrate was treated with et3n (31 l, 0.22 mmol) and concentrated to afford the target compound as mono - et3n species 32 (84.1 mg, 95%) as a colorless oil : rf 0.1 (chcl3/meoh / h2o 14:6:1) ; []d 15.4 (c 0.5, meoh) ; h nmr (600 mhz, meod) = 8.068.04 (m, 2h, bzh2/h6), 8.027.99 (m, 2h, bzh2/h6), 7.637.59 (m, 1h, bzh4), 7.517.45 (m, 3h, bzh4, bzh3/h5), 7.357.31 (m, 2h, bzh3/h5), 5.60 (dd, j = 1.6, 3.4 hz, 1h, h-2), 5.54 (dd, j = 3.4, 9.9 hz, 1h, h-3), 4.93 (app q, j = 9.9 hz, 1h, h-4), 4.90 (d, j = 1.4 hz, 1h, h-1), 4.87 (d, j = 1.4 hz, 1h, h-1), 4.464.42 (m, 1h, h-6), 3.993.96 (m, 1h, h-6), 3.913.88 (m, 2h, h-5, h-7a), 3.86 (dd, j = 1.6, 3.2 hz, 1h, h-2), 3.85 (app t, j = 9.7 hz, 1h, h-4), 3.743.65 (m, 4h, h-3, h-7b, h-7a, h-7b), 3.63 (dd, j = 1.7, 9.7 hz, 1h, h-5), 3.50 (s, 3h, och3), 3.07 (q, j = 7.3 hz, 6h, nch2ch3), 1.25 (t, j = 7.3 hz, 9h, nch2ch3) ; c nmr (151 mhz, meod) 167.5 (c = o), 167.0 (c = o), 134.6, 134.0, 131.3, 131.1, 131.0, 130.8, 129.6, 129.15 (phc), 102.3 (c-1), 100.3 (c-1), 73.4 (d, jc, p = 3.1 hz, c-5), 73.04 (d, jc, p = 3.5 hz, c-3), 72.99 (c-5), 72.8 (c-3), 72.1 (c-2), 71.7 (c-2), 71.0 (c-6), 69.45 (d, jc, p = 5.6 hz, c-4), 68.8 (c-7), 68.1 (c-6), 67.9 (c-4), 64.7 (c-7), 55.85 (och3), 47.7 (nch2ch3), 9.55 (nch2ch3) ; p nmr (243 mhz, meod) 1.98 ; hrms (esi - tof) m / z [m h ] calcd for c29h36o18p 703.1645, found 703.1642. dibenzoate 32 (16.8 mg, 20.9 mol) was dissolved in dry meoh (1.0 ml) and 1 m methanolic naome (0.15 ml, 146 mol) was added, leading to the formation of a turbid emulsion. the reaction mixture was stirred for 19 h at rt and subjected to hplc additional reagent (0.15 ml, 146 mol) and meoh (0.5 ml) were added, and stirring was continued for 8 h. the ph of reaction mixture was adjusted to 5 by addition of cation - exchange resin (dowex, h - form). the resin was filtered off and washed with meoh, and the filtrate was made nearly neutral by adding 0.1 m naome to give a ph of78. the solution was concentrated, and the residue (13.4 mg) was dissolved in d2o (1 ml) and washed twice with chloroform (1 ml). the combined organic layers were re - extracted with d2o (0.8 ml), and the combined aqueous phases were concentrated to remove residual chcl3. a solution of 1 m methanolic naome (40 l) was added, and the mixture was stirred overnight at rt and processed as described above. the combined aqueous layers were neutralized by adding 0.1 m naome (ph 78), purged with a stream of argon, and lyophilized. since nmr analysis indicated 5% of residual sodium benzoate in the material, the extraction process was repeated to give pure compound 33 (10.2 mg, 94.4%) : []d + 71.2 (c 1.5, h2o) ; h nmr (600 mhz, d2o, ph 67) 4.91 (d, j = 1.7 hz, 1h, h-1), 4.75 (d, j = 1.6 hz, 1h, h-1), 4.294.26 (app q, j = 9.7 hz, 1h, h-4), 4.274.25 (m, 1h, h-6), 4.03 (ddd, j = 1.7, 5.5, 7.4 hz, 1h, h-6), 3.98 (dd, j = 1.6, 3.0 hz, 1h, h-2), 3.93 (dd, j = 1.7, 3.6 hz, 1h, h-2), 3.89 (dd, j = 3.5, 9.4 hz, 1h, h-3), 3.863.81 (m, 2h, h-3, h-4), 3.77 (dd, j = 4.2, 10.9 hz, 1h, h-7a), 3.73 (dd, j = 7.6, 11.2 hz, 1h, h-7a), 3.713.67 (m, 2h, h-7b, h-7b), 3.633.59 (m, 2h, h-5, h-5), 3.38 (s, 3h, och3) ; c nmr (151 mhz, d2o) 101.6 (c-1), 101.4 (c-1), 72.2 (c-5), 71.7 (d, jc, p = 7.0 hz, c-5), 71.64 (c-3), 71.57 (c-3), 70.8 (c-2), 70.4 (c-2), 70.3 (d, jc, p = 4.8 hz, c-4), 69.6 (c-6), 69.3 (c-7), 67.7 (c-6), 66.9 (c-4), 63.8 (c-7), 55.6 (och3) ; p nmr (243 mhz, d2o) 3.81 ; hrms (esi - tof) m / z [m - h ] calcd for c15h27o16p 495.1120, found 495.1122. compound 23 (24.6 mg, 18.2 mol) was treated and processed according to general procedure 1. the crude material was purified by column chromatography (sio2 : 2 g cartridge, stepwise gradient of hexane / etoac 1:11:3) to give pure 34 (16 mg, 79%) as a syrup : []d 12.0 (c 1.1, chcl3) ; h nmr (600 mhz, cdcl3) 8.027.99 (m, 2h, bzh2/h6), 7.57 (dd, j = 1.2, 7.5 hz, 1h, bzh4), 7.417.38 (m, 2h, bzh3/h5), 7.297.17 (m, 23h, phh), 7.137.07 (m, 3h, phh), 7.057.02 (m, 2h, phh), 6.946.91 (m, 2h, phh), 5.475.46 (m, 1h, h-2), 5.255.18 (m, 2h, poch2), 5.06 5.01 (m, 3h, h-4, h-1, poch2), 4.95 (dd, j = 7.7, 11.8 hz, 1h, poch2), 4.93 (d, j = 1.8 hz, 1h, h-1), 4.63 and 4.35 (2d, j = 11.2 hz, 2h, ch2ph), 4.62 and 4.58 (2 d, j = 12.0 hz, 2h, ch2ph), 4.53 (bd, j = 5.3 hz, 1h, 6-oh), 4.48 and 4.26 (2 d, j = 12.1 hz, 2h, ch2ph), 4.45 and 4.30 (2 d, j = 10.95 hz, 2h, ch2ph), 4.26 (dd, j = 3.0, 9.4 hz, 1h, h-3), 4.204.16 (m, 1h, h-6), 3.89 (br dd, j = 8.2, 10.8 hz, 1h, h-7a), 3.83 (dd, j = 8.8, 9.8 hz, 1h, h-3), 3.79 (dd, j = 1.5, 9.6 hz, 1h, h-5), 3.67 (dt, j = 2.1, 10.1 hz, 1h, h-5), 3.643.59 (m, 1h, h-7b), 3.62 (dd, j = 8.7, 10.0 hz, 1h, h-4), 3.50 (dd, j = 3.7, 9.8 hz, 1h, h-2), 3.39 (s, 3h, och3), 3.25 (dd, j = 2.5, 11.0 hz, 1h, h-6a), 3.21 (dd, j = 1.7, 10.9 hz, 1h, h-6b), 2.04 (bd, j = 8.5 hz, 1h, 7-oh) ; c nmr (151 mhz, cdcl3) 165.75 (c = o), 138.7, 138.55, 138.2, 137.85 (phc), 135.7 (d, jc, p = 6.2 hz, poch2phc1), 135.3 (d, jc, p = 7.1 hz, poch2phc1), 133.4, 129.8, 129.4, 128.62, 128.60, 128.56, 128.4, 128.23, 128.16, 128.1, 127.95, 127.9, 127.82, 127.78, 127.7, 127.5, 127.4, 127.34, 127.32, 127.30 (phc), 99.1 (c-1), 98.1 (c-1), 81.4 (c-3), 80.05 (c-2), 77.9 (d, j = 5.0 hz, c-3), 77.1 (c-4), 75.1 (ch2ph), 74.4 (ch2ph), 73.4 (ch2ph), 73.1 (ch2ph), 72.3 (c-2), 72.1 (d, jc, p = 5.1 hz, c-4), 71.53 (c-5), 71.47 (d, jc, p = 2.2 hz, c-5), 70.02, 69.99 (2 poch2), 68.3 (c-6), 67.8 (c-6), 63.15 (c-7), 55.4 (och3) ; p nmr (243 mhz, cdcl3) 0.60 ; hrms (esi - tof) m / z [m + h ] calcd for c63h68o16p 1111.4239, found 1111.4237. a suspension of disaccharide 34 (25 mg, 22.5 mol) and 10% pd / c (2.5 mg) was hydrogenated for 7 h at rt and processed as described in general procedure 2. a solution of et3n (4 l) in meoh (100 l) was added, and the filtrate was concentrated to afford compound 35 (14.3 mg, 95%) as a syrup : []d + 60.6 (c 1.4, meoh) ; h nmr (600 mhz, meod) 8.138.10 (m, 2h, bzh2/h6), 7.637.60 (m, 1h, bzh4), 7.507.46 (m, 2h, bzh3/h5), 5.39 (dd, j = 1.6, 3.3 hz, 1h, h-2), 5.13 (d, j = 4.0 hz, 1h, h-1), 4.78 (bs, 1h, h-1), 4.78 (app q, j = 10.1 hz, 1h, h-4), 4.21 (dt, j = 1.5, 6.8 hz, 1h, h-6), 4.16 (dd, j = 3.4, 9.8 hz, 1h, h-3), 3.80 (dd, j = 1.5, 9.9 hz, 1h, h-5), 3.73 (dd, j = 7.1, 10.8 hz, 1h, h7-a), 3.68 (dd, j = 6.8, 11.0 hz, 1h, h-7b), 3.66 (dd, j = 2.3, 12.2 hz, 1h, h-6a), 3.60 (dd, j = 3.7, 12.0 hz, 1h, h-6b), 3.56 (app t, j = 9.5 hz, 1h, h-3), 3.553.52 (m, 1h, h-5), 3.42 (s, 3h, och3), 3.37 (dd, j = 3.9, 9.8 hz, 1h, h-2), 3.34 (app t, j = 9.5 hz, 1h, h-4), 3.15 (q, j = 7.3 hz, 7h, nch2ch3), 1.29 (t, j = 7.3 hz, 11h, nch2ch3) ; c nmr (151 mhz, meod) 167.5 (c = o), 134.5, 131.13, 131.06, 129.6, 103.4 (c-1), 99.95 (c-1), 79.4 (d, jc, p = 2.5 hz, c-3), 74.5 (c-3), 74.4 (c-2), 74.1 (c-5), 73.8 (c-2), 72.7 (d, jc, p = 3.4 hz, c-5), 70.7 (c-4), 70.6 (d, jc, p = 4.9 hz, c-4), 69.9 (c-6), 63.1 (c-7), 61.8 (c-6), 55.6 (och3), 47.6 (nch2ch3), 9.3 (nch2ch3) ; p nmr (243 mhz, meod) 1.97. hrms (esi - tof) m / z [m h ] calcd for c21h30o16p 569.1277, found 569.1277. a solution of 35 (13.6 mg, 20.2 mol) in dry meoh (1.0 ml) was treated with 1 m naome (100 l, 100 mol) for 14 h at rt. the ph of the solution was adjusted to ph 78 by adding cation - exchange resin (h - form), the resin was filtered off, and the filtrate was concentrated. the residue was dissolved in d2o and was thoroughly washed with et2o. the filtrate was treated with 0.4 m methanolic naome (0.1 ml) and lyophilized to give 36 (10.7 mg, quant) as a colorless amorphous solid : []d + 87.5 (c 1.1, d2o) ; h nmr (600 mhz, d2o, ph 10) 5.19 (d, j = 3.9 hz, 1h, h-1), 4.67 (d, j = 1.2 hz, 1h, h-1), 4.32 (q, j = 10.1 hz, 1h, h-4), 4.12 (ddd, j = 1.9, 5.4, 7.5 hz, 1h, h-6), 4.02 (dd, j = 1.6, 3.2 hz, 1h, h-2), 3.87 (dd, j = 3.3, 9.9 hz, 1h, h-3), 3.813.77 (m, 3h, h-5, h-3, h-6a), 3.763.72 (m, 2h, h-6b, h-7a), 3.70 (dd, j = 5.2, 11.4 hz, 1h, h-7b), 3.64 (dd, j = 1.7, 10.0 hz, 1h, h-5), 3.38 (dd, j = 3.8, 9.8 hz, 1h, h-2), 3.35 (app t, j = 9.0, 1h, h-4), 3.34 (s, 3h, och3).c nmr (151 mhz, d2o) : 102.3 (c-1), 102.0 (c1), 80.1 (d, jc, p = 2.2 hz, c-3), 74.3 (c-3), 73.3 (c-5), 73.04 (d, jc, p = 3.4 hz, c-5), 72.96 (c-2), 71.6 (c-2), 70.5 (c-4), 69.4 (c-6), 68.5 (d, jc, p = 4.8 hz, c-4), 63.0 (c-7), 61.6 (c-6), 55.7 (och3) ; p nmr (243 mhz, d2o) 4.70 ; hrms (esi - tof) m / z [m h ] calcd for c14h26o15p 465.1015, found 465.1013. compound 24 (53 mg, 36.0 mol) was treated as described in general procedure 1. the crude material (45 mg) was purified by column chromatography (sio2 : 2 g cartridge, hex / etoac 1:3 1:4) to afford 37 (37.7 mg, 83.6%) as a colorless oil : rf 0.20 (hexane / etoac 1:2) ; []d 42.4 (c 1.2, chcl3) ; h nmr (600 mhz, cdcl3) 8.148.12 (m, 2h, bzh2/h6), 7.637.59 (m, 1h, bzh4), 7.497.45 (m, 2h, bzh3/h5), 7.407.37 (m, 2h, phh), 7.367.12 (m, 26h, phh), 7.077.01 (m, 5h, phh), 6.93 6.90 (m, 2h, phh), 5.54 (dd, j = 1.4, 3.1 hz, 1h, h-2), 5.32 (d, j = 1.4 hz, 1h, h-1), 5.08 (dd, jh, p = 8.7, 11.5 hz, 1h, poch2), 5.055.00 (m, 2h, 2 poch2), 4.92 (app q. j = 9.7 hz, 1h, h-4), 4.81 and 4.36 (2 d, j = 11.6 hz, 2h, ch2ph), 4.80 and 4.47 (2 d, j = 11.8 hz, 2h, ch2ph), 4.76 (dd, jh, p = 6.4, j = 11.9 hz, 1h, poch2), 4.69 (d, j = 1.3 hz, 1h, h-1), 4.66 and 4.55 (2 d, j = 12.2 hz, 2h, ch2ph), 4.54 (d, j = 5.7 hz, 1h, 6-oh), 4.444.40 (m, 3h, ch2ph, h-3), 4.17 (app t, j = 9.6, 1h, h-4), 4.174.15 (m, 1h, h-6), 4.08 (d, j = 11.5 hz, 1h, ch2ph), 4.084.04 (m, 1h, h-6), 4.02 (dd, j = 1.1, 9.8 hz, 1h, h-5), 3.963.90 (m, 2h, ch2ph, h-7a), 3.88 (dd, j = 6.7, 9.9 hz, 1h, h-7a), 3.83 (dd, j = 5.5, 9.8 hz, 1h, h-7b), 3.71 (dd, j = 1.2, 9.8 hz, 1h, h-5), 3.65 (dd, j = 2.9, 9.5 hz, 1h, h-3), 3.653.60 (m, 1h, h-7b), 3.59 (dd, j = 1.8, 2.8 hz, 1h, h-2), 2.06 (bd, j = 9.6 hz, 1h, 7-oh) ; c nmr (151 mhz, cdcl3) 165.1 (c = o), 139.1, 138.9, 138.8, 138.6, 138.5 (phc), 135.1 (d, jc, p = 7.7 hz, poch2phc1), 135.0 (d, jc, p = 6.6 hz, poch2phc1), 133.4, 130.0, 129.7, 128.9, 128.75, 128.65, 128.6, 128.3, 128.12, 128.09, 128.07, 128.03, 127.96, 127.55, 127.39, 127.36, 127.34, 127.31, 127.27, 127.23, 127.17, 127.1, 126.95, 126.8 (phc), 99.6 (c-1), 99.0 (c-1), 80.1 (c-3), 75.21 (c-6), 75.17 (c-2), 73.95 (d, jc, p = 5.5 hz, c-4), 73.83 (c-4), 73.78 (ch2ph), 73.2 (ch2ph), 72.90 (c-5), 72.88 (ch2ph), 72.3 (c-2), 72.1 (ch2ph), 71.8 (ch2ph), 71.7 (d, jc, p = 4.4 hz, c-3), 71.1 (d, jc, p = 3.3 hz, c-5), 71.0 (c-7), 70.3 (d, jc, p = 5.5 hz, poch2), 69.9 (dt, jc, p = 4.4 hz, poch2), 68.1 (c-6), 63.1 (c-7), 55.15 (och3) ; p nmr (243 mhz, cdcl3) 1.72 ; hrms (esi - tof) m / z [m + nh4 ] calcd for c71h79no17p 1248.5080, found 1248.5088. a suspension of heptoside 37 (36.5 mg, 29.6 mol) and pd / c (10 w%, 8.4 mg) in dry meoh (1.8 ml) was processed according to general procedure 2. the filtrate was concentrated to afford 38 as a syrup (18.3 mg, quant) : rf 0.13 (chcl3/meoh / h2o 10:5:1) ; h nmr (600 mhz, meod, ph 2) 8.128.09 (m, 2h, bzh2/h6), 7.647.60 (m, 1h, bzh4), 7.517.47 (m, 2h, bzh3/h5), 5.45 (dd, j = 1.7, 3.2 hz, 1h, h-2), 5.11 (s, 1h, h-1), 4.884.82 (m, h-4), 4.82 (d, j = 1.6 hz, 1h, h-1), 4.28 (dd, j = 3.3, 9.5 hz, 1h, h-3), 4.154.09 (m, 1h, h-6), 4.023.97 (m, 2h, h-6, h-2), 3.85 (bd, j = 9.8 hz, 1h, h-5), 3.84 (app t, j = 9.7 hz, 1h, h-4), 3.773.68 (m, 4h, h-7a, h-7b, h-7a, h-7b), 3.70 (dd, j = 1.5, 9.8 hz, 1h, h-5), 3.53 (dd, j = 3.2, 9.5 hz, 1h, h-3), 3.44 (s, 3h, och3) ; c nmr (151 mhz, meod) 167.1 (c = o), 134.55, 131.05, 131.0, 129.6, 103.85 (c-1), 100.1 (c-1), 75.2 (c-3), 74.2 (c-5), 73.83 (c-2), 73.80 (c-4), 72.4 (c-3), 71.8 (d, jc, p = 4.4 hz, c-5), 71.6 (c-2), 71.1 (c-6), 69.8 (c-6), 67.7 (c-4), 65.4, 63.2 (c-7, c-7), 55.7 (och3) ; p nmr (243 mhz, d2o) 0.80 ; hrms (esi - tof) m / z [m h ] calcd for c22h33o17p 599.1383, found 599.1382. a solution of 1 m methanolic naome (0.29 ml, 0.290 mmol) was added to a solution of 38 (17.6 mg, 29.3 mol) in dry meoh (1.8 ml) to give a ph 12. additional reagent (0.29 ml) and meoh (1.8 ml) were added, and stirring was continued for 24 h. another portion (4 ml meoh and 0.6 ml 1 m naome) was then added, and stirring was continued for 3 more days. the solution was acidified by adding cation - exchange resin (h - form), the resin was filtered off, washed with meoh, and the filtrate was concentrated. the aqueous layer was neutralized by addition of 1 m naome solution and submitted to lyophilization to yield a white powder (14.7 mg, 97%). since the product contained 1% of residual 38, hydrolysis of an aqueous solution was continued using 1 m naome in meoh (57 l) overnight at rt. workup as described afforded compound 39 (12.0 mg, 79%) as a colorless powder : []d + 86.3 (c 0.8, d2o) [lit. []d + 85 (c 0.8, h2o) ] ; nmr data agree with published values;p nmr (243 mhz, d2o) 2.71 ; hrms (esi - tof) m / z [m h ] calcd for c15h28o16p 495.1120, found 495.1122. pyridine 70% (26.4 l, 55 equiv of f) was added dropwise to a solution of trisaccharide 27 (37.3 mg, 18.5 mol, predried by coevaporation with toluene) in dry dcm (2.5 ml) in a teflon flask under vigorous stirring. the solution was stirred for 1 h at rt and was added dropwise into a stirred mixture of ice - cold satd aq nahco3 (15 ml) and etoac (30 ml). the aqueous layer was extracted with etoac (15 ml), and the combined organic layer was washed with brine (15 ml), dried (na2so4), and concentrated. the residue (37 mg) was purified by chromatography (sio2 : 2 g cartridge, hexane / etoac 2:1 3:2 1:1) to give compound 40 (26.5 mg, 82%) as a colorless oil : rf 0.25 (hexane / etoac 2:1) ; []d + 3.9 (c 1.3, chcl3) ; h nmr (600 mhz, cdcl3) 8.027.99 (m, 2h, bzh2/h6), 7.597.55 (m, 1h, bzh4), 7.417.35 (m, 4h, bzh3/h5, 2 phh), 7.347.14 (m, 46h, phh), 7.127.06 (m, 3h, phh), 7.057.01 (m, 2h, phh), 6.946.91 (m, 2h, phh), 5.44 (dd, j = 3.1, 1.9 hz, 1h, h-2), 5.22 (dd, j = 11.8 hz, jh, p = 7.8 hz, 1h, poch2), 5.19 (dd, j = 11.8 hz, jh, p = 9.0 hz, 1h, poch2), 5.075.01 (m, 4h, h-4, poch2, h-1, h-1), 4.94 (dd, j = 11.7 hz, jh, p = 7.1 hz, 1h, poch2), 4.90 (d, j = 11.2 hz, 1h, ch2ph), 4.82 and 4.53 (2 d, j = 11.95 hz, 2h, ch2ph), 4.82 (d, j = 1.7 hz, 1h, h-1), 4.73 and 4.67 (2 d, j = 12.35 hz, 2h, ch2ph), 4.64 and 4.58 (2 d, j = 12.0 hz, 2h, ch2ph), 4.63 (d, j = 11.2 hz, 1h, ch2ph), 4.59 and 4.54 (2 d, j = 11.75 hz, 2h, ch2ph), 4.524.47 (m, 3h, ch2ph), 4.45 and 4.30 (2 d, j = 10.95 hz, 2h, ch2ph), 4.384.33 (m, 2h, ch2ph), 4.284.15 (m, 5h, ch2ph, h-6, h-3, h-4, 6-oh), 4.134.10 (m, 1h, h-6), 3.91 (dd, j = 9.2, 3.1 hz, 1h, h-3), 3.863.82 (m, 3h, h-3, h-2, h-7a), 3.78 (dd, j = 9.7, 1.7 hz, 1h, h-5), 3.76 (dd, j = 9.8, 6.1 hz, 1h, h-7b), 3.73 (dd, j = 10.4, 5.6 hz, 1h, h-7a), 3.683.59 (m, 4h, h-5, h-5, h-4, h-7b), 3.50 (dd, j = 9.8, 3.6 hz, 1h, h-2), 3.25 (dd, j = 11.0, 2.3 hz, 1h, h-6a), 3.24 (s, 3h, och3), 3.22 (dd, j = 11.1, 1.7 hz, 1h, h-6b) ; c nmr (151 mhz, cdcl3) 165.7 (phc = o), 138.9, 138.7, 138.55, 138.37, 138.36, 138.21, 138.17, 137.8 (phc), 135.7 (d, j = 6.6 hz, poch2phc1), 135.3 (d, j = 7.7 hz, poch2phc1), 133.4, 129.8, 129.5, 128.62, 128.59, 128.56, 128.4, 128.3, 128.24, 128.21, 128.15, 128.1, 127.9, 127.81, 127.76, 127.71, 127.69, 127.53, 127.51, 127.48, 127.43, 127.42, 127.33, 127.30, 127.27 (phc), 99.15 (c-1), 98.4 (c-1), 98.0 (c-1), 81.4 (c-3), 80.4 (c-3), 79.9 (c-2), 77.9 (d, jp, c = 5.5 hz, c-3), 77.1 (c-4), 75.09 (ch2ph), 75.0 (c-6), 74.53 (ch2ph), 74.4 (ch2ph), 74.25 (c-4), 74.1 (c-2), 73.4 (ch2ph), 73.3 (ch2ph), 73.0 (ch2ph), 72.7 (ch2ph), 72.28 (c-2), 72.26 (ch2ph), 71.9 (d, jp, c = 4.4 hz, c-4), 71.8 (ch2ph), 71.7 (c-5), 71.5 (c-5), 71.1 (d, jp, c = 2.2 hz, c-5), 70.3 (c-7), 70.0 (d, jp, c = 5.8 hz, poch2), 69.9 (d, jp, c = 5.5 hz, poch2), 67.7 (c-6, c-7), 66.6 (c-6), 55.5 (och3) ; p nmr (243 mhz, cdcl3) 0.45 ; hrms (esi - tof) m / z [m + na ] calcd for c105h109nao22p 1775.704, found 1775.7015. a suspension of heptoside 40 (27.5 mg, 15.7 mol) and 10% pd / c (6.3 mg) in dry meoh (2.75 ml) was hydrogenated at atmospheric pressure for 5.5 h at rt. the suspension was filtered through celite and was washed repeatedly with meoh. triethylamine (2.6 l, 18.8 mol) was added to the filtrate, and the filtrate was concentrated to afford 41 (13.6 mg, quantitative) as a colorless oil : rf 0.32 (dcm / meoh / h2o 5:4:1) ; []d + 72.3 (c 1.4, meod) ; h nmr (600 mhz, meod) 8.128.09 (m, 2h, bzh2/h6), 7.637.60 (m, 1h, bzh4), 7.507.47 (m, 2h, bzh3/h5), 5.39 (dd, j = 3.3, 1.7 hz, 1h, h-2), 5.12 (d, j = 4.0 hz, 1h, h-1), 4.85 (d, j = 1.6 hz, 1h, h-1), 4.79 (d, j = 1.6 hz, 1h, h-1), 4.79 (app q, j = 10.2 hz, 1h, h-4), 4.384.33 (m, 1h, h-6), 4.16 (dd, j = 9.8, 3.4 hz, 1h, h-3), 3.95 (app td, j = 6.6, 1.4 hz, 1h, h-6), 3.853.80 (m, 3h, h-4, h-2, h-7a), 3.75 (dd, j = 10.0, 1.4 hz, 1h, h-5), 3.71 (dd, j = 9.6, 3.4 hz, 1h, h-3), 3.683.63 (m, 3h, h-7a, h-7b, h-6a), 3.633.58 (m, 3h, h-7b, h-5, h-6b), 3.56 (app t, j = 9.4 hz, 1h, h-3), 3.543.51 (m, 1h, h-5), 3.43 (s, 3h, och3), 3.37 (dd, j = 9.7, 3.9 hz, 1h, h-2), 3.33 (app t, j = 9.2 hz, 1h, h-4), 3.17 (q, j = 7.3 hz, 6h, nch2ch3), 1.30 (t, j = 7.3 hz, 9h, nch2ch3) ; c nmr (151 mhz, meod) 167.5 (phc = o), 134.5, 131.09, 131.06, 129.6, 103.5 (c-1), 102.3 (c-1), 99.95 (c-1), 79.3 (d, jp, c = 2.9 hz, c-3), 74.5 (c-3), 74.3 (c-2), 74.15 (c-5), 73.8 (c-2), 73.4 (d, jp, c = 3.8 hz, c-5), 72.9 (c-5), 72.8 (c-3), 72.1 (c-2), 70.9 (c-6), 70.7 (c-4), 70.5 (d, jp, c = 5.3 hz, c-4), 68.9 (c-7), 68.3 (c-6), 67.85 (c-4), 64.6 (c-7), 61.8 (c-6), 55.8 (och3), 47.75 (nch2ch3), 9.3 (nch2ch3) ; p nmr (243 mhz, meod) 1.82. hrms (esi - tof) m / z [m h ] calcd for c28h42o22p 761.1911, found 761.1917. a solution of benzoate 41 (13.6 mg, 15.7 mol) in dry meoh (2.7 ml) was stirred with 1 m methanolic naome (0.24 ml, 240 mol) for 30 min at rt. stirring was continued for 5.5 h, and the solution was made neutral (ph 7) by addition of cation - exchange resin (h form). the resin was filtered off and washed with meoh, and the filtrate was concentrated. the residue was dissolved in d2o and filtered over a bed of cation - exchange resin (h - form). the combined organic layers were extracted with d2o, and the aqueous layer was reextracted with et2o. the combined aqueous layers were neutralized by addition of 0.1 m naoh (ph 7) and lyophilized to give compound 42 (9.5 mg, 89%) as a white powder : rf 0.14 (dcm / meoh / h2o 5:4:1) ; []d + 98.6 (c 1.0, d2o) ; h nmr (600 mhz, d2o, ph 7) 5.19 (d, j = 3.9 hz, 1h, h-1), 4.88 (d, j = 1.6 hz, 1h, h-1), 4.68 (d, j = 1.5 hz, 1h, h-1), 4.32 (app q, j = 10.1 hz, 1h, h-4), 4.26 (ddd, j = 8.4, 3.9, 1.9 hz, 1h, h-6), 4.03 (dd, j = 3.3, 1.7 hz, 1h, h-2), 4.023.99 (m, 1h, h-6), 3.973.96 (m, 1h, h-2), 3.86 (dd, j = 9.9, 3.3 hz, 1h, h-3), 3.833.77 (m, 5h, h-4, h-6a, h-3, h-5, h-3), 3.763.69 (m, 3h, h-7a, h-6b, h-7a), 3.66 (dd, j = 11.3, 5.1 hz, 1h, h-7b), 3.65 (dd, j = 11.0, 8.5 hz, 1h, h-7b), 3.63 (dd, j = 9.9, 1.9 hz, 1h, h-5), 3.603.56 (m, 1h, h-5), 3.39 (dd, j = 9.9, 3.9 hz, 1h, h-2), 3.36 (app t, j = 9.5 hz, 1h, h-4), 3.35 (s, 3h, och3) ; c nmr (151 mhz, d2o) 102.3 (c-1), 102.0 (c-1), 101.5 (c-1), 80.1 (d, jp, c = 2.3 hz, c-3), 74.3 (c-3), 73.4 (d, jp, c = 3.3 hz, c-5), 73.3 (c-5), 72.95 (c-2), 72.4 (c-5), 71.8 (c-3), 71.5 (c-2), 70.9 (c-2), 70.5 (c-4), 69.9 (c-6), 68.9 (c-7), 68.5 (d, jp, c = 4.7 hz, c-4), 67.9 (c-6), 67.1 (c-4), 64.1 (c-7), 61.6 (c-6), 55.8 (och3) ; p nmr (243 mhz, d2o) 4.53 ; hrms (esi - tof) m / z [m h ] calcd for c21h38o21p 657.1649, found 657.1643. compound 6 (290 mg, 0.310 mmol) was dissolved in dry dcm (15 ml), transferred into a teflon flask, and cooled in an ice bath. pyridine reagent (88 l, 10 equiv of f) was added in four portions every 2 min to prevent local overheating. the reaction was quenched by dropping the solution into stirred ice - cold satd aq nahco3 (60 ml). the aqueous layer was extracted with dcm (3 30 ml), and the combined organic layers were washed with satd aq nahco3, and brine, dried (na2so4), and concentrated. the crude material (19.8 mg) was purified by column chromatography (sio2 : 17 g, toluene / etoac 2:3) to furnish 10 (245 mg, 83%). | the structurally conserved lipopolysaccharide core region of many gram - negative bacteria is composed of trisaccharides containing 4-o - phosphorylated l - glycero - d - manno - heptose (l, d - hep) units, which act as ligands for antibodies and lectins. the disaccharides glc-(13)-hep4p hep-(13)-hep4p and hep-(17)-hep4p and the branched trisaccharide glc-(13)-[hep-(17)]-hep4p, respectively, have been synthesized from a methyl heptopyranoside acceptor in less than 10 steps. the synthetic strategy was based on the early introduction of a phosphotriester at position 4 of heptose followed by a regioselective opening of a 6,7-o-(1,1,3,3-tetraisopropyl-1,3-disiloxane-1,3-diyl) group allowing for a straightforward access to glycosylation at position 7. perbenzylated n - phenyl trifluoroacetimidate glucosyl and heptosyl derivatives served as -selective glycosyl donors. |
ivermectin is an extremely safe drug when used in humans and other animals for the treatment and control of nematode and ectoparasite infections [1 - 3 ]. the destruction of nematodes in tissues, with a comparatively large mass of foreign material to remove, is a challenge to the host 's defense systems. indeed, it has been known for decades that anti - filarial chemotherapy is associated with excessive and sometimes fatal host reactions, the severity and clinical consequences of which depend on the therapeutic agent used. the use of ivermectin for the treatment and control of onchocerca volvulus infections is highly advantageous in this context, as this drug produces far fewer serious adverse reactions than the previous drug of choice, diethylcarbamazine (dec). there is a need to better understand the saes associated with anti - filarial chemotherapy, in particular the fatalities that have occurred in a small number of individuals who had recently been administered a standard dose of ivermectin ; these individuals had coincident high loads of circulating loa loa microfilariae (mf). these are people, for a large part, living in a specific region of cameroon and who died after falling into a coma within four days of treatment [5 - 7 ]. this unexpected and unprecedented situation has rightly caused much concern, both from the medical aspect and from a programmatic perspective. this drug has been a key to the success of the major global control program for onchocerciasis. the program has been in existence now for over fifteen years, and is now under threat of interruption in the many endemic areas because of this unexplained and extremely serious toxicity. any discussion of the pathogenesis of these saes must be prefaced by the fact that very little pathological material or data has been collected on these l. loa - ivermectin patients to date, and thus such a discussion must be made with a strong theoretical rather than factual basis. the presence of high loads of loa microfilariae strongly suggests that there is a possibility that the saes are associated with the destruction of these parasites in sensitive tissues such as the central nervous system (cns). it is pertinent therefore to first review what is known about the basic mechanisms of parasite death and removal of mf from the human host by way of introduction to mechanisms that may be involved in these adverse reactions. it is also important to compare the clinical events seen in cameroon with other medical events and conditions with a similar presentation or history ; this may be the only practical way to develop plausible theories about the pathogenesis of these specific adverse reactions. it is a remarkable biological phenomenon that they can reside in tissues, or circulate in the blood and lymph in large numbers, whilst only causing minimal clinical response and little or no apparent pathology. some filariae, such as mansonella perstans, despite their considerable prevalence, cause no significant pathology in contrast to the filariae that cause onchocerciasis and lymphatic filariasis ; the pathobiology underlying these differences remains poorly understood but nevertheless intriguing. the central pathogenic event in the development of clinical disease in onchocerciasis is thought to be the destruction of mf and the associated inflammatory events, such as local tissue damage and degradation of host structures (constitutive collagen fibers, pigmentary cells). the adult - containing nodules in themselves are usually more an aesthetic problem and cause no major or systemic clinical effects (although the presence of such large antigenic entities in the host in all likelihood has effects other than simply being the source of new mf). other phenomena, such as immune complex pathology, autoimmune disease and secondary infections, should be considered as possible factors in this disease complex. for example, there is a direct correlation between anti - collagen antibody levels and the development of chronic disease, and these levels of antibodies exceed those seen in major autoimmune diseases such as system lupus erythematosus (mackenzie, unpublished observations). the possibility that these pathogenic mechanisms may be involved can not be ignored ; obviously, much still remains to be understood. onchocerciasis patients are in a delicate immunological balance with their parasites, and host responses are clearly integral to the clinical outcome. many onchocerciasis patients carry > 100 million mf in the immunologically sensitive connective tissues of the skin, yet experience minimal discomfort or severe clinical effects, at least not in the short term ; this same phenomenon apparently exist with loa infections this phenomenon of tolerance contrasts with the situation occurring in onchocercal patients with " sowda " (reactive onchodermatitis) who appear to not be in this quiescent state and suffer tremendously from constant and extremely disquieting dermal pathology ; these patients also react severely to treatment with filariacides. furthermore, immune responses following administration of these drugs play a key role in parasite clearance (i.e., efficacy). the involvement of the immune system in the antifilarial efficacy of ivermectin has been raised repeatedly [11 - 13 ]. this phenomenon occurs with other filarial parasites as well, although not always as strikingly as in onchocerciasis. differences in the contribution of immune responses to chemotherapeutic success among the filariases may reflect the tissue or organs involved in the parasitism (and the ability of that tissue to handle dying mf), or perhaps a more complicated phenomenon involving the degree of adaptation of the parasite species to the host. the fundamental phenomenon of a balance between the host and the parasite maintained by parasite - derived immunosuppression and other mechanisms is central to the persistence of these infections in the untreated state. recently, it has also been proposed that endosymbiotic bacteria (wolbachia spp.) infecting filariae exacerbate the pathology of onchocerciasis. the literature concerning most human filariae, including o. volvulus, is replete with reports and details of anaphylactoid reactions known as mazzotti reactions (after the mexican luis mazzotti, who first described them) associated with chemotherapy. it is now thought possible that these reactions may be directed, at least in part, to bacterial rather than nematode antigens. it must be emphasized that it is the rule rather than the exception for a treated individual infected with onchocerciasis to experience some form of clinical discomfort or systemic change after treatment with antifilarial drugs (table 1). the frequency and severity of such reactions differ with the chemotherapeutic agent used, but all cause at least some undesirable effect. it is the degree of these effects that are of interest here, with special attention paid to the severe, sometimes fatal reactions that have occurred in one l. loa endemic area (cameroon). a key question is whether the pathogenesis of the severe cases simply reflects an unusual enhancement of commonly encountered processes, or involves new processes that are not seen in the vast majority of onchocerciasis patients. events surrounding the death and removal of mf are of primary importance in any discussion of the pathogenesis of saes encountered during the treatment of filarial infections. much of the information to date related to this phenomenon is taken from data collected with the treatment of o. volvulus and l. loa with older agents such as dec. many of the events following treatment with ivermectin are similar to those seen with dec although to lesser in intensity and rate. the body in most cases can, although with some discomfort, handle the events related to mf destruction and removal, and return to a state of relative normality within 2448 hr after anti - filarial drug administration. however, these reactions can exceed the capacity of the individual to manage them, for example, when topical dec is used. this latter treatment can lead to overwhelming local dermal reactions and associated systemic effects that are too severe for the patient to tolerate. this is an example of excessive and severe reactions in a tissue that is unable to absorb the changes and return to relative normality. in other words, there are situations in chemotherapy when the body can not easily handle the reactions that develop associated with the degenerating microfilariae. inflammation associated with mf destruction and removal probably includes all of the basic inflammatory phenomena, including cytokine release, immune complex formation, autoimmune responses and various physical events, all often resulting in tissue damage. high levels of immune responses are found in onchocerciasis patients, reflecting the heavy antigenic burden and very active antibody responses that are hallmarks of this infection ; their pathological significance remains unclear. neither peripheral lymphoid tissues, nor the liver or kidney (or other organs that might process such complexes) are notably compromised in these patients. thus, there is no definitive evidence for a central involvement of immune complexes in the pathogenesis of ivermectin - associated adverse reactions. that anti - inflammatory drugs (e.g. cortisone, antihistamines) can not completely suppress the immunological events associated with mf destruction emphasizes the complex balance among the various pathways activated. these microfilarial reactions have been investigated and described in most detail with dec treatment, but in all likelihood similar phenomena occur with reactions to other mf such as loa loa. it is a remarkable biological phenomenon that they can reside in tissues, or circulate in the blood and lymph in large numbers, whilst only causing minimal clinical response and little or no apparent pathology. some filariae, such as mansonella perstans, despite their considerable prevalence, cause no significant pathology in contrast to the filariae that cause onchocerciasis and lymphatic filariasis ; the pathobiology underlying these differences remains poorly understood but nevertheless intriguing. the central pathogenic event in the development of clinical disease in onchocerciasis is thought to be the destruction of mf and the associated inflammatory events, such as local tissue damage and degradation of host structures (constitutive collagen fibers, pigmentary cells). the adult - containing nodules in themselves are usually more an aesthetic problem and cause no major or systemic clinical effects (although the presence of such large antigenic entities in the host in all likelihood has effects other than simply being the source of new mf). other phenomena, such as immune complex pathology, autoimmune disease and secondary infections, should be considered as possible factors in this disease complex. for example, there is a direct correlation between anti - collagen antibody levels and the development of chronic disease, and these levels of antibodies exceed those seen in major autoimmune diseases such as system lupus erythematosus (mackenzie, unpublished observations). the possibility that these pathogenic mechanisms may be involved can not be ignored ; obviously, much still remains to be understood. onchocerciasis patients are in a delicate immunological balance with their parasites, and host responses are clearly integral to the clinical outcome. many onchocerciasis patients carry > 100 million mf in the immunologically sensitive connective tissues of the skin, yet experience minimal discomfort or severe clinical effects, at least not in the short term ; this same phenomenon apparently exist with loa infections this phenomenon of tolerance contrasts with the situation occurring in onchocercal patients with " sowda " (reactive onchodermatitis) who appear to not be in this quiescent state and suffer tremendously from constant and extremely disquieting dermal pathology ; these patients also react severely to treatment with filariacides. furthermore, immune responses following administration of these drugs play a key role in parasite clearance (i.e., efficacy). the involvement of the immune system in the antifilarial efficacy of ivermectin has been raised repeatedly [11 - 13 ]. this phenomenon occurs with other filarial parasites as well, although not always as strikingly as in onchocerciasis. differences in the contribution of immune responses to chemotherapeutic success among the filariases may reflect the tissue or organs involved in the parasitism (and the ability of that tissue to handle dying mf), or perhaps a more complicated phenomenon involving the degree of adaptation of the parasite species to the host. the fundamental phenomenon of a balance between the host and the parasite maintained by parasite - derived immunosuppression and other mechanisms is central to the persistence of these infections in the untreated state. recently, it has also been proposed that endosymbiotic bacteria (wolbachia spp.) infecting filariae exacerbate the pathology of onchocerciasis. the literature concerning most human filariae, including o. volvulus, is replete with reports and details of anaphylactoid reactions known as mazzotti reactions (after the mexican luis mazzotti, who first described them) associated with chemotherapy. it is now thought possible that these reactions may be directed, at least in part, to bacterial rather than nematode antigens. it must be emphasized that it is the rule rather than the exception for a treated individual infected with onchocerciasis to experience some form of clinical discomfort or systemic change after treatment with antifilarial drugs (table 1). the frequency and severity of such reactions differ with the chemotherapeutic agent used, but all cause at least some undesirable effect. it is the degree of these effects that are of interest here, with special attention paid to the severe, sometimes fatal reactions that have occurred in one l. loa endemic area (cameroon). a key question is whether the pathogenesis of the severe cases simply reflects an unusual enhancement of commonly encountered processes, or involves new processes that are not seen in the vast majority of onchocerciasis patients. events surrounding the death and removal of mf are of primary importance in any discussion of the pathogenesis of saes encountered during the treatment of filarial infections. much of the information to date related to this phenomenon is taken from data collected with the treatment of o. volvulus and l. loa with older agents such as dec. many of the events following treatment with ivermectin are similar to those seen with dec although to lesser in intensity and rate. the body in most cases can, although with some discomfort, handle the events related to mf destruction and removal, and return to a state of relative normality within 2448 hr after anti - filarial drug administration. however, these reactions can exceed the capacity of the individual to manage them, for example, when topical dec is used. this latter treatment can lead to overwhelming local dermal reactions and associated systemic effects that are too severe for the patient to tolerate. this is an example of excessive and severe reactions in a tissue that is unable to absorb the changes and return to relative normality. in other words, there are situations in chemotherapy when the body can not easily handle the reactions that develop associated with the degenerating microfilariae. inflammation associated with mf destruction and removal probably includes all of the basic inflammatory phenomena, including cytokine release, immune complex formation, autoimmune responses and various physical events, all often resulting in tissue damage. high levels of immune responses are found in onchocerciasis patients, reflecting the heavy antigenic burden and very active antibody responses that are hallmarks of this infection ; their pathological significance remains unclear. neither peripheral lymphoid tissues, nor the liver or kidney (or other organs that might process such complexes) are notably compromised in these patients. thus, there is no definitive evidence for a central involvement of immune complexes in the pathogenesis of ivermectin - associated adverse reactions. that anti - inflammatory drugs (e.g. cortisone, antihistamines) can not completely suppress the immunological events associated with mf destruction emphasizes the complex balance among the various pathways activated. these microfilarial reactions have been investigated and described in most detail with dec treatment, but in all likelihood similar phenomena occur with reactions to other mf such as loa loa. remarkably few serious reactions are reported with the use of ivermectin in humans or in most animals. in general, the signs and symptoms, which vary in duration (usually no longer than four days), include nausea, headache, minor fever and dermatological responses associated with the presence of dying mf in the skin ; pruritic injection of the conjunctiva has also been reported. these mazzotti - type reactions are similar in many respects to those seen with other microfilariacidal agents, such as dec, although lesser in extent and intensity than with the latter drug. the unacceptability of these reactions with dec, especially as associated with an adverse effect on ocular tissues (inducing loss of vision), and the severity of these dermal reactions in many people, drove the search for new and safer agents. ivermectin filled many of the requirements, including a lack of pathology in the ocular tissues, high microfilaricidal efficacy, long lasting mf suppression and much reduced severity of associated dermal reactions. most adverse reactions to treatment with antifilarial drugs occur in the period shortly after the administration of the drug, i.e. usually " discomforting " and are short lived. only when they persist, or are of severe intensity, do they become matters of medical concern. there have been one or two anecdotal reports of serious clinical responses that are consistent with classic allergic reactions to the drug itself. given the number of doses (tens of millions) that have been administered in the control programs as a whole, true ivermectin drug hypersensitivity cases are extremely rare. an apparent exception to the ivermectin success story has been the situation involving the exceptionally serious cns reactions observed in a small number of people, and proposed to be related to the presence of concurrent high burdens of loa loa mf. in 1996, cases of coma and death began to appear in cameroon [5 - 7,16,17 ].. the condition has probably been under - reported, with possibly up to 80 fatalities out of ~300 cases of the syndrome actually having occurred in cameroon (boussinesq, personal communication). limited medical information is available on most of the cases due to the rural location in which the cases occurred and the understandably minimal local medical support system at hand. the fatalities in cameroon occurred with patients who had high loads of circulating l. loa mf, and this association has raised great concern among local medical officials, program directors and the scientific community alike. these cases have been defined as " loa loa - associated adverse reactions " and have the characteristics of a gradually developing encephalopathy (table 3). it is important to note that co - infection with o. volvulus and l. loa is found in many other regions, and that these kinds of very severe reaction have only been reported from cameroon. the apparent geographic restriction of the phenomenon must be incorporated into any analysis of the pathology of this condition. serious cns pathology has been previously reported with dec treatment of patients with loa loa infection ; these have some clinical similarities as the current loa - associated cases treated with ivermectin. ducorps and colleagues reported significant adverse reactions in patients with loiasis, particularly those with > 30,000 mf / ml in their circulation ; these changes included fever, pruritus, headache, arthralgia, disturbed consciousness (obnubilation), as well as the more significant coma and renal impairment. the symptoms in these cases occurred a little earlier than those with ivermectin (i.e. within 2436 hr of dosing) but did show progressing neurological severity. other reports such as that of carme.,, which presents 5 cases showing fever and coma 918 days after dec treatment (with one at day 3) have distinct similarities to the present cases. thus there may be a similar etiologies in the pathology induced by these two drugs, i.e. the effects being primarily related to the death of microfilariae in cns tissue and ensuing inflammation. eye lesions have been reported after dec treatment of loiasis patients, as is seen in the current ivermectin cases in cameroon. toissant and danis found retinal hemorrhages and saccular microaneurysms associated with the degeneration of the associated cells such as pericytes. likewise, hemorrhages have been seen in the conjunctiva and the retinal fundus of the current ivermectin treated loa patients. importantly, these vascular lesions closely resemble ocular findings in malaria where it is believed that parasitic micro - emboli are an integral component of their pathogenesis. interestingly, mansonella perstans mf were seen to be associated with the presence of the lesions in the dec cases, but this observation requires substantiation. other records of cns and ocular pathology exist in relationship to the presence and treatment of loiasis. other reports describe various forms of neuropathology, including encephalitis, cerebral destruction with clinical hemiplegia, meningitis, and peripheral neuritis. in the current cases reactions in the cranial cavity to drug - damaged adult parasites should not be ruled out, as severe and fatal outcomes occur in other cns nematodiases, such as infections with angiostrongylus cantonensis. there is, however, little evidence for a short - term adulticidal activity for ivermectin. the similarities between these dec - induced microfilariae - related pathologies and those that are appearing in the patients with the present loa - associated syndrome support the hypothesis that the sae in the latter group involve pathology associated with microfilarial death and destruction. remarkably few serious reactions are reported with the use of ivermectin in humans or in most animals. in general, the signs and symptoms, which vary in duration (usually no longer than four days), include nausea, headache, minor fever and dermatological responses associated with the presence of dying mf in the skin ; pruritic injection of the conjunctiva has also been reported. these mazzotti - type reactions are similar in many respects to those seen with other microfilariacidal agents, such as dec, although lesser in extent and intensity than with the latter drug. the unacceptability of these reactions with dec, especially as associated with an adverse effect on ocular tissues (inducing loss of vision), and the severity of these dermal reactions in many people, drove the search for new and safer agents. ivermectin filled many of the requirements, including a lack of pathology in the ocular tissues, high microfilaricidal efficacy, long lasting mf suppression and much reduced severity of associated dermal reactions. most adverse reactions to treatment with antifilarial drugs occur in the period shortly after the administration of the drug, i.e. usually " discomforting " and are short lived. only when they persist, or are of severe intensity, do they become matters of medical concern. there have been one or two anecdotal reports of serious clinical responses that are consistent with classic allergic reactions to the drug itself. given the number of doses (tens of millions) that have been administered in the control programs as a whole, true ivermectin drug hypersensitivity cases are extremely rare. an apparent exception to the ivermectin success story has been the situation involving the exceptionally serious cns reactions observed in a small number of people, and proposed to be related to the presence of concurrent high burdens of loa loa mf. in 1996, cases of coma and death began to appear in cameroon [5 - 7,16,17 ]. to date, 46 cases of this syndrome have been recorded, with 22 fatalities. the condition has probably been under - reported, with possibly up to 80 fatalities out of ~300 cases of the syndrome actually having occurred in cameroon (boussinesq, personal communication). limited medical information is available on most of the cases due to the rural location in which the cases occurred and the understandably minimal local medical support system at hand. the fatalities in cameroon occurred with patients who had high loads of circulating l. loa mf, and this association has raised great concern among local medical officials, program directors and the scientific community alike. these cases have been defined as " loa loa - associated adverse reactions " and have the characteristics of a gradually developing encephalopathy (table 3). it is important to note that co - infection with o. volvulus and l. loa is found in many other regions, and that these kinds of very severe reaction have only been reported from cameroon. the apparent geographic restriction of the phenomenon must be incorporated into any analysis of the pathology of this condition. serious cns pathology has been previously reported with dec treatment of patients with loa loa infection ; these have some clinical similarities as the current loa - associated cases treated with ivermectin. ducorps and colleagues reported significant adverse reactions in patients with loiasis, particularly those with > 30,000 mf / ml in their circulation ; these changes included fever, pruritus, headache, arthralgia, disturbed consciousness (obnubilation), as well as the more significant coma and renal impairment. the symptoms in these cases occurred a little earlier than those with ivermectin (i.e. within 2436 hr of dosing) but did show progressing neurological severity. other reports such as that of carme.,, which presents 5 cases showing fever and coma 918 days after dec treatment (with one at day 3) have distinct similarities to the present cases. thus there may be a similar etiologies in the pathology induced by these two drugs, i.e. the effects being primarily related to the death of microfilariae in cns tissue and ensuing inflammation. eye lesions have been reported after dec treatment of loiasis patients, as is seen in the current ivermectin cases in cameroon. toissant and danis found retinal hemorrhages and saccular microaneurysms associated with the degeneration of the associated cells such as pericytes. likewise, hemorrhages have been seen in the conjunctiva and the retinal fundus of the current ivermectin treated loa patients. importantly, these vascular lesions closely resemble ocular findings in malaria where it is believed that parasitic micro - emboli are an integral component of their pathogenesis. interestingly, mansonella perstans mf were seen to be associated with the presence of the lesions in the dec cases, but this observation requires substantiation. other records of cns and ocular pathology exist in relationship to the presence and treatment of loiasis. other reports describe various forms of neuropathology, including encephalitis, cerebral destruction with clinical hemiplegia, meningitis, and peripheral neuritis. in the current cases reactions in the cranial cavity to drug - damaged adult parasites should not be ruled out, as severe and fatal outcomes occur in other cns nematodiases, such as infections with angiostrongylus cantonensis. there is, however, little evidence for a short - term adulticidal activity for ivermectin. the similarities between these dec - induced microfilariae - related pathologies and those that are appearing in the patients with the present loa - associated syndrome support the hypothesis that the sae in the latter group involve pathology associated with microfilarial death and destruction. most adverse reactions to treatment with antifilarial drugs occur in the period shortly after the administration of the drug, i.e. usually " discomforting " and are short lived. only when they persist, or are of severe intensity, do they become matters of medical concern. there have been one or two anecdotal reports of serious clinical responses that are consistent with classic allergic reactions to the drug itself. given the number of doses (tens of millions) that have been administered in the control programs as a whole, true ivermectin drug hypersensitivity cases are extremely rare. an apparent exception to the ivermectin success story has been the situation involving the exceptionally serious cns reactions observed in a small number of people, and proposed to be related to the presence of concurrent high burdens of loa loa mf. in 1996, cases of coma and death began to appear in cameroon [5 - 7,16,17 ]. to date, 46 cases of this syndrome have been recorded, with 22 fatalities. the condition has probably been under - reported, with possibly up to 80 fatalities out of ~300 cases of the syndrome actually having occurred in cameroon (boussinesq, personal communication). limited medical information is available on most of the cases due to the rural location in which the cases occurred and the understandably minimal local medical support system at hand. the fatalities in cameroon occurred with patients who had high loads of circulating l. loa mf, and this association has raised great concern among local medical officials, program directors and the scientific community alike. these cases have been defined as " loa loa - associated adverse reactions " and have the characteristics of a gradually developing encephalopathy (table 3). it is important to note that co - infection with o. volvulus and l. loa is found in many other regions, and that these kinds of very severe reaction have only been reported from cameroon. the apparent geographic restriction of the phenomenon must be incorporated into any analysis of the pathology of this condition. serious cns pathology has been previously reported with dec treatment of patients with loa loa infection ; these have some clinical similarities as the current loa - associated cases treated with ivermectin. ducorps and colleagues reported significant adverse reactions in patients with loiasis, particularly those with > 30,000 mf / ml in their circulation ; these changes included fever, pruritus, headache, arthralgia, disturbed consciousness (obnubilation), as well as the more significant coma and renal impairment. the symptoms in these cases occurred a little earlier than those with ivermectin (i.e. within 2436 hr of dosing) but did show progressing neurological severity.,, which presents 5 cases showing fever and coma 918 days after dec treatment (with one at day 3) have distinct similarities to the present cases. thus there may be a similar etiologies in the pathology induced by these two drugs, i.e. the effects being primarily related to the death of microfilariae in cns tissue and ensuing inflammation. eye lesions have been reported after dec treatment of loiasis patients, as is seen in the current ivermectin cases in cameroon. toissant and danis found retinal hemorrhages and saccular microaneurysms associated with the degeneration of the associated cells such as pericytes. likewise, hemorrhages have been seen in the conjunctiva and the retinal fundus of the current ivermectin treated loa patients. importantly, these vascular lesions closely resemble ocular findings in malaria where it is believed that parasitic micro - emboli are an integral component of their pathogenesis. interestingly, mansonella perstans mf were seen to be associated with the presence of the lesions in the dec cases, but this observation requires substantiation. other records of cns and ocular pathology exist in relationship to the presence and treatment of loiasis. other reports describe various forms of neuropathology, including encephalitis, cerebral destruction with clinical hemiplegia, meningitis, and peripheral neuritis. in the current cases reactions in the cranial cavity to drug - damaged adult parasites should not be ruled out, as severe and fatal outcomes occur in other cns nematodiases, such as infections with angiostrongylus cantonensis. there is, however, little evidence for a short - term adulticidal activity for ivermectin. the similarities between these dec - induced microfilariae - related pathologies and those that are appearing in the patients with the present loa - associated syndrome support the hypothesis that the sae in the latter group involve pathology associated with microfilarial death and destruction. in general the possible causes of the adverse reactions associated with antifilarial drugs can be considered in two major groups : pathological events and mechanisms involving a) the drug having a direct toxic effect on the host, and those involving b) the parasite - drug interaction effecting the host (table 4). ivermectin and its structural analogs have been given to tens of millions of people and hundreds of millions of animals, with remarkably few severe reactions. toxic consequences of administration of standard doses of a drug generally have two primary explanations : 1) the toxicity represents natural variation in sensitivity to any chemical compound, and represents a linear scale from trivial to serious. 2) alternatively, toxicity is unrelated to more common untoward effects, and reflects a unique pharmacological action of the drug that could not be anticipated as simply the outer fringe of the normal distribution of responses (either therapeutic or toxic). the serious clinical events observed in this cameroonian group represent a tiny percentage of the total treated population. this response could not have been easily anticipated since adverse neurological effects have not been described in treated populations. that is, we see no evidence for an escalating incidence of side effects, progressing from mild sleepiness or dizziness through slurred speech and ataxia to frank coma, in any population treated with ivermectin. therefore occurrence of coma in these patients likely reflects an idiosyncratic response, one that could not be anticipated from the more common side effects seen in other patients in this same population or in populations treated for similar infections (e.g., lymphatic filariasis). given that the mechanism of microfilaricidal activity of ivermectin requires the intervention of immune effectors, there are two possible explanations for an idiosyncratic response to the drug in dually infected patients : 1) the response has nothing whatsoever to do with parasitism, and would be observed in these patients even if they were free of filariae, or 2) the response reflects an atypical outcome of the immune response to mf in the presence of ivermectin. these possibilities must be evaluated in light of clinical experience obtained with ivermectin in veterinary and human medicine. the target for the antiparasitic effects of ivermectin is a group of glutamate - gated clchannels unique to invertebrates, which this drug profoundly and persistently opens. this action disrupts the regulation of transmembrane potential in invertebrate neuromuscular systems, leading to paralysis of critical muscles and the consequent demise of the organism. however, the drug also potently opens structurally related clchannels that are gated by gaba, which are broadly expressed in the mammalian cns. the remarkable therapeutic index of these compounds in mammals is due only in part because mammals completely lack glutamate - gated clchannels. instead, mammals are protected from ivermectin toxicity not by the absence of the molecular target, but by the presence of an effective blood - brain barrier that prevents access of the drug to a susceptible protein target in the cns [30 - 33 ]. work done in knock - out mice revealed (inadvertently) that exclusion of ivermectin from the cns is due solely to the function of a specific subtype of p - glycoprotein, a protein family that translocates hydrophobic compounds across cell membranes (associated in the past with resistance of tumor cells to anticancer drugs). disruption of the mdr1a locus in mice rendered them highly sensitive to ivermectin toxicity when the drug was given to control an outbreak of parasitism in the transgenic colony. the primary toxicity was coma. that a small percentage of certain breeds of dogs (some collies and shepherds) also became comatose when dosed with ivermectin for heartworm prevention recently, it has been shown conclusively that susceptible dogs have inactivating mutations in the mdr1a gene. the consequence of allowing ivermectin accumulation in the brain is a prolonged and progressive cns intoxication in both dogs and mice, resulting in coma. it would not be surprising to find that loss - of - function mdr1a mutations exist in humans, as they do in dogs. there is no evident phenotype for the mutation in dogs other than acute sensitivity to ivermectin and a few other drugs. it would also not be surprising to find that such a mutation would be geographically restricted, found perhaps at a detectable frequency in one or a few populations exposed to ivermectin (a rare event in wealthy countries). indeed, one might almost predict the eventual appearance of ivermectin toxicity in a human population from an analysis of the situation observed in canines. it would be relatively simple to rule out the possibility that loss - of - function mutations in the human mdr1a gene underlie ivermectin toxicity. the sequence of this gene is known and it could be cloned from peripheral lymphocytes obtained from survivors of ivermectin - induced coma or relatives of victims (the phenotype is probably recessive, so heterozygotes may be unaffected and would be more common in the local population). analysis of this locus will reveal whether lessons learned from dogs can be usefully applied to humans. given the background and overall resemblance of the syndrome in dogs and affected humans, the conservative approach is to first determine whether a common molecular basis exists. if this hypothesis proved to be correct, treatment programs could be continued without regard to coincident infections with l. loa. there is no simple and cheap diagnostic test that could pre - screen local populations that had not yet been dosed with ivermectin for evidence of mdr1a mutations. however, the phenotype is, on the evidence, exceedingly rare, and treatment could resume as long as the incidence of cns sequelae following treatment was closely monitored. it is conceivable that a cost - effective pcr - based diagnostic test could be implemented to identify individuals in villages where serious adverse reactions were observed who should be excluded from treatment. the possibility of the adverse reactions being directly related to microfilarial events, such as their death and destruction, has been proposed. in all likelihood antimicrofilarial agents such as dec and ivermectin function either in conjunction with, or at least enhanced by, host immunological or inflammatory components. it is important to note that neither dec nor ivermectin have detectable effects on microfilarial motility or viability in culture at pharmacologically relevant concentrations. understanding how these drugs work to stimulate immune - dependent killing of mf is an important step in clearly understanding the clinical reactions associated with them. conversely we should also understand how these relatively large parasites manage to survive in large numbers without attracting attention from the host immune system. the mechanisms by which these parasites suppress recognition and destruction by the host must be the targets for dec and ivermectin. the clinical presentation of these patients and a consideration of the literature points clearly to the involvement of an inflammatory process, this probably leading to the coma and possibly hepatic dysfunction. one line of investigation would be to determine if the individuals suffering from the adverse effects have a perturbed cytokine response that predisposes them to an excessive inflammation. these individuals could be typed for cytokine profiles ; this could be done retrospectively on survivors of adverse incidents. if predisposition was found to be the case then it might be possible to screen high loa loa microfilariae carriers to determine those at greatest risk ; however, this may not be practical in the field situation. the possibility exists that gene - controlled variation in specific immune response lies at the base of these idiosyncratic reactions to ivermectin. there are many factors that contribute to an individual 's response at a particular point in time : including the parasite load, the state of immune system (its immunosuppressive or stimulatory state), as well as the ability or inability to efficiently clear killed parasite material. however, a major factor that may be at play in these cases is the individual 's own genetic predisposition towards either th1 or th2 responses when challenged by stimuli, i.e. an individual responds with a predictable profile influenced or guided by their genetics. it could be important to determine which of the polymorphic alleles of several cytokine genes governs this phenomenon. it may be that the affected individuals in these cases have a predisposition to drive inflammatory responses to dying mf to clinically unacceptable levels in such sensitive target organs such as the cns. the fact that not all of the individuals with high microfilarial loads are affected by the encephalopathy is interesting in this light. there is a medical and moral need to move quickly to the next step of proposing actions regarding these saes. these actions include managerial and political activities, but there are also scientific questions that must be answered to provide a more rational approach to this problem. to put this problem in perspective it should be noted that in the drug industry, this level of adverse reactions (1 case in 12,000,000 administrations) would be regarded as being comparatively low. nevertheless, it is important consider the ramifications of the toxicity from the perspective of the state or community in which they occurred, and their immediate implications for onchocerciasis control programs there and elsewhere. there is an urgent need to proceed to a policy based on practical action to moves us ahead. what this action should be is open for discussion, but there a number of options and possibilities. the authors believe they must include research to better understand the scientific basis of the phenomenon, as well as steps that lead to responding to the field situation with appropriate levels of assessment and monitoring. it is important to discriminate among the possible explanations for the pathogenesis of the l. loa associated syndrome, whether or not further fatalities unfortunately occur. it would be straightforward to examine the possibility of genetic polymorphisms in mdr1a genes or inflammatory responses in a population sample obtained from the geographic locale of the main cluster of cases. careful prospective monitoring of the geographic distribution of the occurrence of cns reactions after ivermectin treatment in loiasis patients across the endemic areas will confirm or disprove the geographic restriction of the saes, pointing to human genetic variation as a key explanation of the syndrome. further work to characterize the possibility of a micro - embolic pathology similar to that seen in malaria is also important. direct examination of cns tissues will reveal crucial data ; core sampling of brain tissues after death, which requires minimal invasion of the body, is arguably the best approach. csf fluid should be monitored for cellular content, glucose content and proteins such c - reactive proteins. other potentially useful laboratory parameters include ivermectin concentration in csf, eosinophil products and indicators of fibrin deposition, fibrin degradation products (fdps), endothelial and platelet activation and hemorrhage. two major explanations for the fatal saes associated with l. loa infections in cameroon currently seem the most feasible. firstly, that the condition is directly related to loa microfilariae and the development of a cns centered micro - embolic vascular pathology with associated inflammatory reactions due to the parasite death and the intravascular clumping (supported by the clinical evidence of retinal and conjunctival hemorrhages) ; this has parallels with the micro - embolic events seen in malaria. such events may be more severe in patients with a predisposition to excessive inflammatory responses occurring with microfilarial death. there may be predisposition for l. loa mf to be trapped specifically in the cns vasculature, promoting problems in this organ when chemotherapy begins to destroy the parasites. increased migration of mf appears also to be an important event after therapy and this may enhance the formation of embolic inflammatory foci of the dying parasites. the timing of clinical manifestations of the ensuing inflammatory and anoxic pathology after the 3and 4days of treatment supports this theory. secondly, there may be a genetic susceptibility in the affected patients that allows ivermectin to accumulate in the cns, causing a toxicity similar to that seen in dogs and mice. the apparent geographic clustering supports this hypothesis of a genetic predisposition and one can not help but wonder if the human equivalent of the mdr-1 abnormality described in animals has now been found in humans. a genetically influenced inability to handle and absorb parasite induced - inflammation other mechanisms could very well be at play, but much more detailed clinical and pathological analyses is needed before definitive statements can be made about the cause of these fatal saes. confounding circumstances appear unlikely. the only parasitic infection so far correlated with the occurrence of the serious reactions is loiasis ; nonetheless, it would be wise to keep open the possibility of other co - infections. it has been suggested that alcohol intake may play a role in predisposing individuals to develop severe reactions to ivermectin by changing its pharmacokinetic behavior. however, this does not appear to be the case, as locally produced alcoholic beverages do not increase blood levels of ivermectin (homeida., unpublished). any research - oriented approaches to addressing this problem should be matched with actions at the community level. these should include the establishment of clear medical guidelines for the responding medical and support staff, improving procedures to screen for high mf load carriers, and developing better messages for the people living in the endemic areas where these fatalities occur. it is essential to address the medical, public and political challenges that the ivermectin - associated serious adverse reactions present, and to thus ensure the continuation of a medically sound chemotherapy program. author 2 (jg) and 3 (tgg) both contributed text and ideas. the types of reactions seen in the treatment of onchocerciasis with oral diethylcarbamazine, ivermectin and others ivermectin produces reactions significantly less in severity and duration. the major components associated with microfilarial destruction. major clinical characteristics of patients with loa - associated adverse reaction syndrome " loa encephalopathy " adapted from reference. | backgroundreactions are commonly associated with the chemotherapy of onchocerciasis. however unmanageable reactions are uncommon when ivermectin (mectizan) is used for the treatment of this infection, and this drug has proved to be a great improvement over previously used agents. serious adverse events (sae) nevertheless have occurred, and there is considerable concern about the negative effect such events may have on mass drug administration programs.this paper reviews the basic pathogenic mechanisms that can be involved in the destruction of microfilaria by chemotherapeutic agents. a central challenge to filarial chemotherapy is the need to remove parasites from biologically sensitive tissues, a more difficult medical challenge than eliminating nematodes from the gastrointestinal tract.explanations for the etiology of the serious adverse reactions occurring with ivermectin treatment in specific geographic areas where there is coincident heavy loa loa infections are hampered by a lack of specific pathological case material. ways to investigate these possibilities are reviewed. possible pathogenic mechanisms include embolic vascular pathology accompanied by local inflammation, blood brain barrier mdr1 abnormalities, and genetic predisposition to excessive inflammatory responses.conclusionit is important to keep ivermectin, and all its associated adverse clinical events, in perspective with the many other chemotherapeutic agents in general use many of which produce serious adverse events even more frequently than does ivermectin. currently available evidence indicates that the pathogenesis of the loa - associated adverse reactions are probably related to inflammatory responses to microfilariae in specific tissues. however, the possibility of genetic predispositions to pathology should also be considered. |
avaliar, em um modelo pulmonar simulando um paciente sob ventilao mecnica, a eficincia e a segurana da manobra de hiperinsuflao manual (hm) com o intuito de remover secreo pulmonar. oito fisioterapeutas utilizaram um ressuscitador manual autoinflvel para realizar hm com o objetivo de remover secrees, em duas condies : conforme rotineiramente aplicada durante sua prtica clnica, e aps receberem instrues verbais baseadas em recomendaes de especialistas. trs cenrios clnicos foram simulados : funo pulmonar normal, doena pulmonar restritiva e doena pulmonar obstrutiva. antes da instruo, o uso de duas compresses sequenciais do ressuscitador era comum, e a presso proximal (pprox) foi mais alta em relao obtida aps a instruo. entretanto, a presso alveolar (palv) nunca excedeu 42,5 cmh2o (mediana, 16,1 ; intervalo interquartil [iq ], 11,7 - 24,5), mesmo com valores de pprox de at 96,6 cmh2o (mediana, 36,7 ; iq, 22,9 - 49,4). o volume corrente (vc) gerado foi relativamente pequeno (mediana, 640 ml ; iq, 505 - 735) e o pico de fluxo inspiratrio (pfi) geralmente excedeu o pico de fluxo expiratrio (pfe) : 1,37 l / s (iq, 0,99 - 1,90) e 1,01 l / s (iq, 0,55 - 1,28), respectivamente. uma relao pfi / pfe < 0,9 (que teoricamente favorece a migrao do muco em direo s vias areas centrais) foi obtida em somente 16,7% das manobras. nas condies testadas, a hm gerou valores seguros de palv mesmo com altas pprox. entretanto, a hm foi comumente realizada de um modo que no favorecia a remoo de secreo (pfi excedendo pfe) mesmo aps a instruo. a relao pfi / pfe desfavorvel foi explicada pelas insuflaes rpidas e o baixo vc. manual hyperinflation (mh) is a proposed technique that is purported to promote secretion clearance and to re - expand areas of atelectasis, thereby improving lung compliance and oxygenation in patients on mechanical ventilation. despite a lack of scientific evidence confirming its benefits on clinical outcomes, mh the maneuver is widely accepted as effective and, in brazil, is largely embraced as a means of removing retained secretions. the rationale for the use of the technique as an aid for secretion removal is that it simulates a cough. when applied before suctioning, it theoretically moves secretions toward the central airways, thus increasing the efficacy of the suctioning procedure. it has been shown that the efficiency of the mh maneuver is affected by the operator and the type of manual resuscitator used, as well as by the resistance and compliance of the respiratory system, largely affecting the pressures and flows generated in the respiratory system. under certain conditions, the use of this technique can generate high peak airway pressures, increasing the risk of barotrauma. according to expert recommendations, in order to promote secretion clearance, mh should consist of a slow, deep inspiration, an inspiratory pause, and a quick release of the resuscitation bag to promote passive exhalation with high expiratory flow rates. however, the way in which the mh maneuver is performed can vary from country to country, impeding the understanding of its effects. since the 1980s, there has been increasing evidence that the peak expiratory flow to peak inspiratory flow (pef / pif) ratio is a critical factor for the removal of lung secretions, especially in heavily sedated or paralyzed patients. in fact, more than their ratio, the difference between the two, in terms of their absolute values, seems to be the major determinant : above a given threshold, whenever the pif exceeds the pef, secretions migrate deeper into the lung. intuitively, respiratory therapists (rts) have been promoting maneuvers to increase expiratory flows, analogous to those observed during coughing. however, relatively little attention has been given to their inspiratory counterpart, despite expert recommendations to apply a slow, deep inspiration during mh. the inspiratory phase of mh can be performed in many different ways, depending on the personal experience of the operator, and there is little evidence that the mh maneuver generates an adequate flow bias (i.e., with pef exceeding pif) or that the ultimate goal of the maneuver (i.e., improved secretion clearance) is achieved. the aim of this study was to evaluate the efficiency and safety of mh (as performed by rts in a lung model simulating a mechanically ventilated patient) as a means of increasing secretion clearance under two different conditions : mh performed in accordance with the routine clinical practice of rts ; and mh performed in accordance with expert recommendations. the efficiency of mh was determined through analysis of the volume and flow patterns generated in relation to the anticipated effects on secretion removal. the safety of mh was evaluated on the basis of the inspiratory pressures achieved (i.e., whether those pressures remained within safe limits during the maneuver). our ultimate objective was to obtain a qualitative analysis of how mh is performed by experienced professionals and not to address how it is performed in brazil. this was an experimental study conducted in the laboratory for medical research 09, specializing in pulmonology, at the university of so paulo school of medicine, in the city of so paulo, brazil. the study was approved by the research ethics committee of the university of so paulo school of medicine hospital das clnicas. the study comprised two phases, evaluating the effects of mh, as performed by rts, in promoting the clearance of pulmonary secretions. in the first phase (the pre - instruction phase), the rts were given no explicit verbal instructions on how to apply the maneuver. in the second phase, the rts were given instructions based on expert recommendations, as detailed below. the two phases are hereafter referred to as the pre- and post - instruction phases. all maneuvers were performed with self - inflating manual resuscitator (spur ; ambu, ballerup, denmark), with a capacity of 1,500 ml. the maneuver was applied by eight experienced rts (four males and four females), with an average of 2.6 years in practice (range, 2 - 4 years) in icus in the city of so paulo. five of the eight had graduated from universities located within the state of so paulo, and all had completed one of the one - year postgraduate training programs in respiratory therapy offered by university hospitals. the maneuver was applied to a mechanical model of the respiratory system known as a training and test lung (ttl 2600 ; michigan instruments, grand rapids, mi), connected to a tracheal tube (8.5 mm). the airway flow was measured proximally (between the manual resuscitator and proximal pressure sensor) by a pneumotach. two pressure transducers were connected to the model : one to measure proximal pressure (pprox - at the airway, between the flow sensor and tracheal tube) ; and another to record alveolar pressure (palv). study phases, we altered the resistance and compliance of the lung model in order to simulate three different clinical scenarios : a patient with obstructive lung disease (resistance at 20 cmh2o / l per second and compliance at 0.08 l / cmh2o) ; a normal patient (resistance at 5 cmh2o / l per second and compliance at 0.05 l / cmh2o) ; and a patient with restrictive lung disease (resistance at 5 cmh2o / l per second and compliance at 0.025 l / cmh2o). in the pre - instruction phase (as previously mentioned), the rts were instructed to perform mh to promote pulmonary secretion clearance in accordance with their routine clinical practice. the lung model was set according to those three clinical scenarios explained above and covered with a bed sheet, allowing rts to sense the movement of the bellows while remaining blinded to the clinical scenario selected. after approximately eight cycles of mh, the settings were changed to simulate the next scenario (in the sequence described above) until all three scenarios had been run. in the post - instruction phase, each rt received brief verbal instructions on how to perform mh according to expert recommendations, and all of the steps performed in the pre - instruction phase were repeated. the verbal instructions were given within the space of approximately 2 min, and the rts were not trained to follow a given pattern of insufflation. the instructions were always given by the same researcher, who instructed each rt as follows : " now you should perform mh with a slow inflation and a 2-s inspiratory pause, followed by rapid release of the bag ". while giving the instructions, the researcher demonstrated the maneuver with the manual resuscitator used in the study. the analog signals from the flow and pressure transducers were amplified, digitized, and recorded at 200 hz, using a data acquisition and off - line analysis system developed within the software laboratory virtual instrumentation and engineering workbench (labview ; national instruments, austin, tx, usa). for each experimental condition, the beginning of the inspiratory phase was defined as zero flow immediately before bagging, and the end of the respiratory cycle was defined as zero flow at the end of exhalation. the pprox was defined as the peak pressure value during the inspiratory phase, measured by the proximal pressure transducer. the pif was defined as the peak flow value during the inspiratory phase, measured by the flow transducer, and the pef was defined as the peak flow during expiratory phase. the palv was defined as the peak pressure value during the inspiratory phase, measured by the alveolar pressure transducer. inspiratory time (ti) was defined as the duration of the inflation plus the inspiratory pause. repeated measures anova was used to evaluate, for each variable, the following within - subject factors : the three clinical scenarios ; and the two phases (before and after instructions). inspiratory versus expiratory flow and alveolar versus proximal pressures were also tested as within - subject factors to compare peak flow and peak pressure variables, respectively. for each of the six experimental conditions (three clinical scenarios in each study phase), we analyzed three respiratory cycles, corresponding to eighteen respiratory cycles for each of the eight rts. therefore, we analyzed a total of 144 respiratory cycles. among all of the respiratory cycles analyzed, the maximum palv observed was 42.5 cmh2o (median, 16.1 cmh2o ; iqr, 11.7 - 24.5), despite the much higher pprox values (maximum, 96.6 cmh2o - median, 36.7 ; iqr, 22.9 - 49.4). the vt values were relatively low (maximum, 955 ml - median, 640 ; iqr, 505 - 735), and the ti was short (median, 1.29 s ; iqr, 0.95 - 1.72). a pif / pef ratio < 0.9, which theoretically favors mucus migration toward the central airways, was achieved in only 24 (16.7%) of the 144 maneuvers evaluated. this favorable ratio occurred primarily in the post - instruction phase (in 18 of the 24 maneuvers) and was strongly associated with just one of the rts tested (who was responsible for 12 of the 24 maneuvers). during the pre - instruction phase, six of the eight rts performed mh using two compressions of the resuscitator bag and produced a pif higher than that produced in the post - instruction phase (figure 2). figure 2differences in proximal pressures achieved by two different respiratory therapists (a and b) before and after explicit verbal instruction (routine clinical practice vs. expert recommendations - dashed lines and solid lines, respectively). table 1 shows a comparison between the pre- and post - instruction phases, in terms of the mechanical variables evaluated. after instruction, mh was performed in a slower manner, with a longer ti and a lower pif, producing a lower pprox. there were no statistically significant differences between the two phases in terms of the palv, vt and pef. figure 2 illustrates the difference between the two study phases and between two different rts. table 1mechanical variables before and after verbal instruction (routine clinical practice vs. expert recommendations).variablepre - instructionpost - instructionp proximal pressure (cmh2o)44.5 (33.4 - 63.4)26.8 (18.6 - 37.5)0.004 alveolar pressure (cmh2o)16.3 (11.6 - 25.8)14.8 (11.7 - 23.8)0.93 peak inspiratory flow (l / s)1.84 (1.28 - 2.19)1.14 (0.87 - 1.44)0.001 peak expiratory flow (l / s)1.04 (0.57 - 1.33)0.99 (0.55 - 1.27)0.28 tidal volume (ml)628 (497 - 699)647 (518 - 746)0.63 inspiratory time (s)0.95 (0.78 - 1.19)1.71 (1.44 - 2.13)<0.001 pif / pef ratio1.80 (1.29 - 2.34)1.15 (0.87 - 1.80)0.004values are expressed as median (25 - 75% interquartile range)pif : peak inspiratory flowpef : peak expiratory flow values are expressed as median (25 - 75% interquartile range) : peak inspiratory flow : peak expiratory flow comparing all three clinical scenarios tested and the two phases of the study, we found that pprox was markedly higher than was palv in all instances, with the exception of the restrictive lung disease scenario in the post - instruction phase (figure 3). in the pre - instruction phase, in the post - instruction phase, the pif / pef ratio was closer to 1:1. the only situation in which that ratio was consistently unfavorable (pif still far exceeding pef) was the obstructive lung disease scenario (figure 4). figure 3median values of proximal and alveolar pressures (interquartile ranges as error bars) obtained in the pre - instruction (routine clinical practice) and post - instruction (in accordance with expert recommendations) phases of the study. the dashed line indicates 30 cmh2o. p < 0.01 (difference between pre - instruction and post - instruction). # p < 0.05 (difference between proximal and alveolar pressure). figure 4median values for peak inspiratory and expiratory flows (interquartile ranges as error bars) obtained in the pre - instruction (routine clinical practice) and post - instruction (in accordance with expert recommendations) phases of the study. p < 0.01 (difference between pre - instruction and postinstruction). # p < 0.05 (difference between peak inspiratory and expiratory flows). the major finding of the present study was that, even after receiving explicit instructions, the rts evaluated here performed mh in a way that probably would not aid secretion removal, with pif commonly exceeding pef. compressing the resuscitator bag multiple times, in rapid succession, resulted in high values of pif and pprox. however, this finding was typically associated with a relatively low vt, probably because of short bag - compression times. therefore, the palv was often low at the start of exhalation, resulting in a low pef. both factors (rapid, multiple compressions and low palv) appear to be responsible for the unfavorable relationship between pif and pef. finally, although the mh maneuver might not promote secretion clearance, our results suggest that this maneuver, as performed in our study, is unlikely to cause barotrauma, a concern expressed by other authors because of the high pprox it generates. in the present study, however, the palv values - roughly represented by plateau pressures, which correlate better with barotrauma than do pprox values - were within the safe range. the mh maneuver was originally described as consisting of a slow, deep inspiration, with an inspiratory pause and rapid release of the resuscitator bag. since then, many different mh techniques have been described, including one providing a vt that is greater than the baseline vt for the patient in question ; one providing a vt that is 50% greater than that delivered by the ventilator ; and one consisting of a slow (3-s) inspiration to achieve a peak airway pressure of 40 cmh2o. in contrast, we found that rts working in the city of so paulo have customized the maneuver according to personal practice and bias, frequently applying two compressions of the resuscitator bag, without an inspiratory pause, resulting in pif being higher than pef. one possible explanation is that the maneuver applied in that way stimulates coughing and, consequently, improves secretion clearance, or at least gives the rt that impression. as observed in this study, however, applying the maneuver in that way might make it ineffective, with unfavorable relationships between inspiratory and expiratory flows, especially if the patient has a depressed cough reflex or is unable to cough efficiently. according to previous studies, above a given flow threshold, the direction of mucus transport (in or out of the respiratory system) is governed by the highest peak flow : a pef 10% higher than the pif (i.e., a pif / pef ratio < 0.9) will favor secretion movement from the distal to the central airways. a recent study evaluating the transport of artificial mucus in a test lung system reported that mucus transport is better explained by the absolute difference between pif and pef than by the pif / pef ratio. when pef is higher than pif, a greater difference between the two translates to better mucus transport. in the present study, the pif / pef ratio was below 0.9 in only 24 (16.7%) of the 144 maneuvers evaluated, most of those 24 being performed in the post - instruction phase. consequently, the median pif was much higher than was the median pef, which is a cause for great concern. this disappointing result is in agreement with the findings of some other studies in which self - inflating resuscitators were also used. the manual resuscitator tested in the present study had a self - inflating bag (the type of manual resuscitator most widely used in icus in brazil). such resuscitators usually generate a lower vt than that achieved with resuscitators that have flow - inflating bags. even after receiving explicit instructions, the rts produced vt values that were lower than those reported in other studies. that difference might be related solely to the size of the bag employed : 1.5 l in the present study, compared with 1.6 - 2.0 liters in the other studies cited. much more favorable pif / pef ratios have been reported when flow - inflating devices were used. the verbal instruction on how to perform the mh improved the performance of the rts in that the post - instruction maneuvers generated lower pifs and longer tis. however, those differences did little to improve the efficiency of the technique, because the pefs were still quite low. that is likely attributable to the fact that the palv was also quite low at the start of exhalation, which resulted in a low driving pressure for expiratory flow. other authors have reported great variability in the practical implementation of the mh maneuver, the execution of which rarely follows its original description. this makes it practically impossible to compare the effectiveness of mh across clinical studies. if we accept the concept that the relationship between pif and pef is responsible for the direction in which secretions move, we should question the use of an inspiratory pause on physiological grounds. because of stress relaxation, the effective driving pressure for flow after a pause will be always lower than immediately after end - inspiration. therefore the use of an inspiratory pause could decrease the pef and, consequently, impair the efficiency of the mh maneuver. the small number of rts participating in this study prevents us from generalizing our data for application in clinical practice. however, we believe that this qualitative analysis, performed with representative operators (of both genders, recruited from different hospitals from within the same city), illustrated scenarios that commonly occur in some icus. another limitation of this study was the use of a lung model, which can not be extrapolated to the complexity of human lungs. nevertheless, we believe that similar results - high pif / pef ratios, low vt, and high pprox - would also be obtained in human patients on mechanical ventilation, especially in those that are heavily sedated, and this should draw the attention of the rts to the way in which they perform the mh maneuver. whether applying high pif during mh will enhance secretion clearance in patients with preserved cough is a question that merits further investigation. that notwithstanding, the key message here is that performing mh with high pif / pef ratios in patients who are unable to cough will not aid secretion removal and might even contribute to mucus retention. another point that should be mentioned is that the mh maneuver can be performed in combination with expiratory chest compression in order to maximize the increase in the pef ; however we were not able to investigate the use of that combination, because of the experimental system used. in addition, as previously mentioned, we can not discard the possibility that the use of a different resuscitator device (a self - inflating resuscitator with larger internal volume bags or a flow - inflating resuscitator) might produce better results. given that, in brazil, mh is typically performed without pprox monitoring, the option of performing the maneuver with a mechanical ventilator, so that inspiratory flow, vt and pressure can be easily monitored and adjusted, should be considered. in studies comparing mh with a manual resuscitator and mh with a mechanical ventilator, no differences were found between the two modalities in terms of the amount of secretion removed, although the advantages of better monitoring with the mechanical ventilator were acknowledged. it is important to note that, although the mh maneuver was originally designed to reduce lung collapse and to improve oxygenation or lung compliance, those potential benefits were not tested here. in fact, we believe that mh should be used only as a secretion clearance technique. if the technique is applied to recruit collapsed lungs, its beneficial effects are going to be offset, at least in part, by the fact that the patient must be disconnected from the ventilator, thus exposing the lung to the lower pressure of the ambient air at the end of each compression. finally, we found no evidence that the mh maneuver, as performed here, increases the risk of barotrauma. the palv value is a result of the insufflating volume and lung compliance, pprox values that are higher than the palv although there have been anecdotal reports of high pprox during mh in adults, with volumes < 1 l, there is no hard evidence that the maneuver is dangerous. in conclusion, when asked to apply mh in accordance with their routine clinical practice, this small sample of rts performed the maneuver quite differently from what is recommended by experts, producing a concerning pattern of ventilation in terms of secretion clearance. the repetition of the maneuver after explicit instructions reduced the pprox but did not help much. alveolar pressures were usually low (because of the small generated vt) despite high proximal pressures and high inspiratory peak flows. as a result, pef was also low, far lower than the preceding pif, a condition theoretically impacting the secretions deeper into the lung. further studies are necessary in this area, especially focusing on the use of flow - inflating devices delivering higher tidal volumes. | objective : to evaluate, in a lung model simulating a mechanically ventilated patient, the efficiency and safety of the manual hyperinflation (mh) maneuver as a means of removing pulmonary secretions. methods : eight respiratory therapists (rts) were asked to use a self - inflating manual resuscitator on a lung model to perform mh as if to remove secretions, under two conditions : as routinely applied during their clinical practice ; and after receiving verbal instructions based on expert recommendations. in both conditions, three clinical scenarios were simulated : normal lung function, restrictive lung disease, and obstructive lung disease. results : before instruction, it was common for an rt to compress the resuscitator bag two times, in rapid succession. proximal pressure (pprox) was higher before instruction than after. however, alveolar pressure (palv) never exceeded 42.5 cmh2o (median, 16.1 ; interquartile range [iqr ], 11.7 - 24.5), despite pprox values as high as 96.6 cmh2o (median, 36.7 ; iqr, 22.9 - 49.4). the tidal volume (vt) generated was relatively low (median, 640 ml ; iqr, 505 - 735), and peak inspiratory flow (pif) often exceeded peak expiratory flow (pef), the median values being 1.37 l / s (iqr, 0.99 - 1.90) and 1.01 l / s (iqr, 0.55 - 1.28), respectively. a pif / pef ratio < 0.9 (which theoretically favors mucus migration toward the central airways) was achieved in only 16.7% of the maneuvers. conclusions : under the conditions tested, mh produced safe palv levels despite high pprox. however, the mh maneuver was often performed in a way that did not favor secretion removal (pif exceeding pef), even after instruction. the unfavorable pif / pef ratio was attributable to overly rapid inflations and low vt. |
the term inflammatory myofibroblastic tumor (imt) denotes a wide spectrum of pathological entities ranging from reactive to neoplastic lesions. though initially considered as a primary pathology of lung, these lesions can be found virtually at any anatomic site, most common extra pulmonary sites of involvement being liver and orbit. head and neck imts are rare and make up only around 14% of the extra pulmonary site. ever since the first report of oral lesion in 1981, until date only 25 cases had been published and none of these cases showed any evidence of recurrence, malignant transformation, metastasis or death. this is a rare case of imt showing sarcomatous change in a patient who presented with a swelling in the maxillary alveolar ridge following extraction of a mobile tooth. the present case report is about a 38-year - old female patient who presented with the complaint of a painless swelling of the upper left posterior alveolar ridge. she gave a history of extraction of a mobile upper left second molar tooth from that area 2 months ago, which had gingival swelling and was progressively mobile. on intra oral examination, a swelling of 3 cm 4 cm was noticed on the alveolar ridge at the site of extraction in 27 region. the swelling was normal in color with smooth surface except in one area where the indentation from the opposing tooth was noticed [figure 1 ]. the swelling started to increase following tooth extraction and started to impinge on lower tooth. frequent lacrimation from the left eye and difficulty in breathing through the left nostril were present since 3 months. extra oral examination revealed mild facial asymmetry due to the presence of a diffuse swelling in the left maxillary region. the orbit was pushed upwards and the left lateral ala of nose intra oral presentation of the tumor at the site of extraction facial asymmetry with raised left orbit orthopantamograph showed an ill - defined osteolytic lesion in the left posterior maxilla [figure 3 ]. computed tomography image revealed an irregular soft - tissue lesion in the left maxillary sinus causing expansion and thinning of medial wall and erosion of anterior, inferior and superior walls. intraorbital extension of the lesion was noticed with the involvement of inferior rectus muscle and there was extension of the tumor into the ethmoid sinus, nasal cavity, oral cavity, pterigopalatine fossa and infra temporal fossa [figure 4 ]. magnetic resonance imaging image showed intra orally the lesion extended into the left alveolus causing erosion and expansion of alveolus [figure 5 ]. based on the clinical presentation and imaging studies, the lesion was provisionally diagnosed as primary intra - alveolar carcinoma. orthopantamograph showing an ill - defined radiolucent lesion in the left posterior maxilla computed tomography scan showing a soft tissue mass in the maxillary sinus with extension of the tumor into the nasal cavity, ethmoid sinus and orbit magnetic resonance imaging t1-weighted image with contrast showing a soft - tissue lesion involving the left maxilla, nasal cavity, orbit and ethmoid sinus with extension into the alveolus and infra temporal fossa left side maxillectomy was performed retaining the orbit. the histological sections from the orbital margin showed sheets of atypical spindle cells with indistinct cytoplasm and large vesicular nuclei showing pleomorphism [figure 6 ]. the proliferating spindle cells were admixed with numerous inflammatory cells mainly neutrophils, lymphocytes and plasma cells [figure 7 ]. on immunohistochemical staining, the tumor cells were strongly positive for vimentin and smooth muscle actin (sma), but negative for cd 1a, s100, desmin, cd 34, activin receptor - like kinase (alk)-1 and mpo. nuclear positivity for the proliferative marker ki 67 was seen in 8 - 10% of the tumor cells. based on the clinical presentation, histopathological features and immunohistochemistry the lesion was diagnosed as imt with focal sarcomatous change. 6 months later the patient developed recurrence of the lesion and finally succumbed to death. the sections from the orbital margin displaying sheets of atypical spindle cells showing large, pleomorphic nuclei with prominent nucleoli (h and e, 40) fascicles of proliferating spindle cells admixed with numerous inflammatory cells orthopantamograph showed an ill - defined osteolytic lesion in the left posterior maxilla [figure 3 ]. computed tomography image revealed an irregular soft - tissue lesion in the left maxillary sinus causing expansion and thinning of medial wall and erosion of anterior, inferior and superior walls. intraorbital extension of the lesion was noticed with the involvement of inferior rectus muscle and there was extension of the tumor into the ethmoid sinus, nasal cavity, oral cavity, pterigopalatine fossa and infra temporal fossa [figure 4 ]. magnetic resonance imaging image showed intra orally the lesion extended into the left alveolus causing erosion and expansion of alveolus [figure 5 ]. based on the clinical presentation and imaging studies, orthopantamograph showing an ill - defined radiolucent lesion in the left posterior maxilla computed tomography scan showing a soft tissue mass in the maxillary sinus with extension of the tumor into the nasal cavity, ethmoid sinus and orbit magnetic resonance imaging t1-weighted image with contrast showing a soft - tissue lesion involving the left maxilla, nasal cavity, orbit and ethmoid sinus with extension into the alveolus and infra temporal fossa the histological sections from the orbital margin showed sheets of atypical spindle cells with indistinct cytoplasm and large vesicular nuclei showing pleomorphism [figure 6 ]. the proliferating spindle cells were admixed with numerous inflammatory cells mainly neutrophils, lymphocytes and plasma cells [figure 7 ]. on immunohistochemical staining, the tumor cells were strongly positive for vimentin and smooth muscle actin (sma), but negative for cd 1a, s100, desmin, cd 34, activin receptor - like kinase (alk)-1 and mpo. nuclear positivity for the proliferative marker ki 67 was seen in 8 - 10% of the tumor cells. based on the clinical presentation, histopathological features and immunohistochemistry the lesion was diagnosed as imt with focal sarcomatous change. following resection, patient underwent radiotherapy. at 6 months later the patient developed recurrence of the lesion and finally succumbed to death. the sections from the orbital margin displaying sheets of atypical spindle cells showing large, pleomorphic nuclei with prominent nucleoli (h and e, 40) fascicles of proliferating spindle cells admixed with numerous inflammatory cells imt represents a spectrum of myofibroblastic proliferation with diverse pathogenesis and clinical behaviors, varying from reactive to neoplastic processes. the controversies regarding the true nature of the lesion has led to the use of numerous terminologies such as pseudosarcomatous myofibroblastic proliferation, inflammatory sarcoma, plasma - cell granuloma, inflammatory pseudo tumor and inflammatory histiocytic proliferation to be used to denote this tumor. until 1998 imts were considered as indolent reactive lesions and were known as inflammatory pseudo tumors. an aberrant or exaggerated response to tissue injury without an established cause had been favored as the pathogenesis of imt. recently, the concept of imt being a benign reactive lesion has been challenged owing to the clinical demonstration of recurrences as high as 37%, the presence of regional metastases and cytogenetic evidence of acquired clonal chromosomal abnormality. it has also been suggested that reactive and neoplastic myofibroblastic lesions are two separate entities and the term imt need to be reserved for neoplastic lesions whereas the term inflammatory pseudotumor (ipt) be retained for reactive lesions. in the head and neck region, imts are more common in the upper respiratory tract (11% of extra pulmonary cases) and the remainder of the head and neck sites (including the orbit, paranasal sinuses, major salivary glands, thyroid and soft - tissues of the face and neck, in descending order of frequency) make up less than 5%. reviewed all the 22 published cases of oral imts diagnosed as ipt or imt including the one reported by them. they observed that the tumor mainly involved the soft - tissues of the oral cavity with majority of the cases occurring in the buccal mucosa. following their report, three additional cases of oral imts were documented thereby accounting for the total number of 25 published cases. intrabony imts are rare and a literature review on intrabony lesions in the head and neck region showed that majority of lesions occurred in the maxillary sinus. out of the 16 documented cases of imts involving maxillary sinus three cases showed extension of the lesion into the oral cavity presenting as intraoral tumors. histopathological resemblance of inflammatory fibroblastic lesions and spindle cell neoplasms makes the categorization of this tumor difficult and its diagnosis more challenging. the use of myofibroblastic markers seems to be promising but no consistency in the expressivity of these markers were noticed. however, in most cases there was strong diffuse staining of tumor cells with vimentin and less commonly with myogenic markers like sma, muscle specific actin, alk-1 and desmin. cd 68 positivity is noticed in 25% of the cases. in this case, the lesional tissue consisted of predominantly bland spindle cells which showed strong vimentin and sma positivity. the presence of chronic inflammatory infiltrate consisting of neutrophils, lymphocytes, plasma cells and cd68 positive macrophages admixed with tumor cells was a consistent finding. a very unusual finding was the cytological atypia with nuclear pleomorphism and increased mitotic activity consistent with malignant transformation seen in the lesion, which otherwise appeared to be reactive in origin. positivity for the nuclear protein ki 67 also indicated the aggressive nature of this tumor. though malignant transformation in imts had been observed in other regions no evidence of tumor recurrence or malignant transformation in the present case, the lesion was noticed at the site of extraction in the maxillary alveolar ridge. taking into consideration the role played by myofibroblast in the formation of granulation tissue and wound healing, the occurrence of imts at extraction site however, it is more likely that mobility in the tooth which was extracted resulted from the destruction of alveolar bone due to the preexisting tumor and the trauma of extraction would have accelerated the growth of the tumor leading to the sudden clinical manifestation. in our patient the tumor of maxillary sinus showed extension into the nasal cavity, orbit, ethmoid sinus, infratemporal fossa, pterigopalatine fossa and oral cavity. the clinical symptoms like epiphora, nasal congestion and difficulty in breathing seen in this patient could be attributed to the extension of tumor into the orbit and nasal cavity. tooth mobility invariably result from weakening of tooth supporting periodontal tissues, but it need not always be a resultant of diseases of periodontium. this present case is a clear example of rare diseases that involves tooth supporting areas as well as the basal bone that could manifest as mobility of involved tooth. a tooth presenting with grade 3 mobility is generally assumed to be periodontally weak and is considered for extraction. here, the mobility of tooth and the trauma of extraction are suspected to have contributed to the accelerated growth of this reactive tumor. this emphasizes the need of investigation of unexplainable periodontal disease manifestation like tooth mobility, beyond the level of oral cavity. | inflammatory myofibroblastic tumor (imt) is a rare tumor of uncertain origin with variable biological behavior ranging from reactive lesions to highly aggressive malignancy. oral imts are extremely rare and only 25 cases had been reported so far. a case of imt with sarcomatous transformation in an extraction site with a history of tooth extraction following tooth mobility of an upper left molar tooth is presented here. the tooth was extracted following a complaint of gingival swelling and mobility of tooth. though malignant transformation in imts had been documented in the extra oral sites, wide search of associated literature suggests, this is the first case of oral imt showing malignant change associated with gingiva. the case report attempts to highlight the variant possibilities of tooth mobility other than periodontitis and the importance of assessing the primary cause of such conditions. |
participants for this study included 57 children (25 girls) obtained from a larger ongoing longitudinal study which was approved by the governing institutional review board. four hundred and forty seven participants were initially recruited at two years of age through child care centers, the county health department, and the local women, infants, and children program. further details about the recruitment may be found in smith, calkins, keane, anastopoulos, and shelton (43). the recruitment sample was diverse with 67% percent of the children classified as european american, 27% were african american, 4% were biracial, and 2% were hispanic. at age 2, the children were primarily from intact families (77%), and families were economically diverse, with hollingshead (1975) scores ranging from 14 to 66 (m = 39.56). there were no significant differences between families who did and did not participate at 5.5-years in terms of gender, race, and ses. the current study focused on a subgroup of children for whom laboratory measures and height / weight measurements were obtained. complete data was available on 57 children who were racially (65% caucasian) and economically diverse (hollingshead scores ranging from 1461, m = 38.91). additionally, complete data, other than 2yr height / weight, were available on 204 children. there were no significant differences in terms of gender, race, or ses between children with complete versus partial data nor were there any differences between this study 's sample and the original recruitment sample. when the children were 2 years of age, mothers brought their children to the laboratory and were videotaped during several tasks. the order of the tasks were standardized and children were given small breaks at the end of each task to ensure that there were no carry over effects from one task to another. the first task was a sustained attention task in which children were instructed to watch a 5 minute segment of the videotape following this task, children engaged in various mother - child interaction tasks including a teaching task, a free - play session, a compliance task, and a puzzle task. once the mother - child interaction tasks were completed, children participated in two tasks designed to elicit emotion regulation. the prize in a box task, where a desirable toy (puppet) was placed in a clear box that the child was unable to open for 2 minutes, and a high chair task, where the child was placed in a high chair without any toys for 5 minutes were used to code observed emotion regulation and emotional reactivity (lab - tab, 44). the tasks were ended early if the child was highly distressed / cried hard for more than 30 seconds. following a more extensive break (5 min) children came back to the laboratory and participated in a delay of gratification task aimed at measuring children 's reward sensitivity and to a certain degree their inhibitory control skills. while in the laboratory, mothers completed various questionnaires. weight and height measures were collected on the entire sample during the 5.5-year visit with a subgroup of children (n = 57) also having weight / height measures at the 2-year visit. trained research assistants measured children 's height and weight during their 2-year and 5.5-year laboratory visit. degree of overweight was calculated based on age norms from the centers for disease control (45). prior research has shown relations between emotion regulation and emotion reactivity measures where reactivity is a part of the response to the contextual demands that require regulatory strategies to adjust for this change in reactivity (46). thus, both degree of distress and specific regulatory behaviors are considered evidence of emotion regulation processes. consequently, both emotional reactivity and regulation were coded from videotapes of the frustration tasks (prize in the box and high chair). for reactivity, distress was defined as when the child whined, fussed, cried, or tantrummed. a global measure of negative reactivity was coded on a scale from 0, meaning no negative response, to 4, meaning task ended with the child in extreme distress. regulation was defined as the overall effectiveness of using various strategies (e.g., self - stimulation, self - soothing, distraction). a global measure of regulation was coded on a scale from 0, meaning dysregulated or no control of distress, to 4, when the child seemed to completely regulate their distress during most of the task. the reactivity and regulation codes were averaged across tasks to produce a separate mean score for each. as expected the measures of emotion regulation and emotional reactivity consequently, these constructs were combined by creating z scores of both variables and averaging these standardized scores to create a single measure of emotion regulation. the overall duration - proportion of time the child spent looking at the video - was used as this study 's laboratory measure of sustained attention. the reliability among coders for the overall duration was excellent (r =.98). children participated in a delay of gratification task in which they were presented with an appealing gift wrapped box and told there was a gift inside for them but that they could not open it for 2 minutes. the overall total time touching gift - combined time the child was in contact with the box - was used as this study 's measure of inhibitory control / reward sensitivity. this overall time was reversed score with higher numbers indicating better inhibitory control skills or lower reward sensitivity. the reliability among coders for the overall time was excellent (r =.99). mother - reported 2-year total behavior problems measured by the child behavior checklist (cbcl 23 ; 47) was used as a control variable. the cbcl has been widely used by researchers studying early social adjustment and has adequate reliability and validity (49, 50). participants for this study included 57 children (25 girls) obtained from a larger ongoing longitudinal study which was approved by the governing institutional review board. four hundred and forty seven participants were initially recruited at two years of age through child care centers, the county health department, and the local women, infants, and children program. further details about the recruitment may be found in smith, calkins, keane, anastopoulos, and shelton (43). the recruitment sample was diverse with 67% percent of the children classified as european american, 27% were african american, 4% were biracial, and 2% were hispanic. at age 2, the children were primarily from intact families (77%), and families were economically diverse, with hollingshead (1975) scores ranging from 14 to 66 (m = 39.56). there were no significant differences between families who did and did not participate at 5.5-years in terms of gender, race, and ses. the current study focused on a subgroup of children for whom laboratory measures and height / weight measurements were obtained. complete data was available on 57 children who were racially (65% caucasian) and economically diverse (hollingshead scores ranging from 1461, m = 38.91). additionally, complete data, other than 2yr height / weight, were available on 204 children. there were no significant differences in terms of gender, race, or ses between children with complete versus partial data nor were there any differences between this study 's sample and the original recruitment sample. when the children were 2 years of age, mothers brought their children to the laboratory and were videotaped during several tasks. the order of the tasks were standardized and children were given small breaks at the end of each task to ensure that there were no carry over effects from one task to another. the first task was a sustained attention task in which children were instructed to watch a 5 minute segment of the videotape spot, a short story about a puppy that explores its neighborhood. following this task, children engaged in various mother - child interaction tasks including a teaching task, a free - play session, a compliance task, and a puzzle task. once the mother - child interaction tasks were completed, children participated in two tasks designed to elicit emotion regulation. the prize in a box task, where a desirable toy (puppet) was placed in a clear box that the child was unable to open for 2 minutes, and a high chair task, where the child was placed in a high chair without any toys for 5 minutes were used to code observed emotion regulation and emotional reactivity (lab - tab, 44). the tasks were ended early if the child was highly distressed / cried hard for more than 30 seconds. following a more extensive break (5 min) children came back to the laboratory and participated in a delay of gratification task aimed at measuring children 's reward sensitivity and to a certain degree their inhibitory control skills. while in the laboratory, mothers completed various questionnaires. weight and height measures were collected on the entire sample during the 5.5-year visit with a subgroup of children (n = 57) also having weight / height measures at the 2-year visit. trained research assistants measured children 's height and weight during their 2-year and 5.5-year laboratory visit. degree of overweight was calculated based on age norms from the centers for disease control (45). prior research has shown relations between emotion regulation and emotion reactivity measures where reactivity is a part of the response to the contextual demands that require regulatory strategies to adjust for this change in reactivity (46). thus, both degree of distress and specific regulatory behaviors are considered evidence of emotion regulation processes. consequently, both emotional reactivity and regulation were coded from videotapes of the frustration tasks (prize in the box and high chair). for reactivity, distress was defined as when the child whined, fussed, cried, or tantrummed. a global measure of negative reactivity was coded on a scale from 0, meaning no negative response, to 4, meaning task ended with the child in extreme distress. regulation was defined as the overall effectiveness of using various strategies (e.g., self - stimulation, self - soothing, distraction). a global measure of regulation was coded on a scale from 0, meaning dysregulated or no control of distress, to 4, when the child seemed to completely regulate their distress during most of the task. the reactivity and regulation codes were averaged across tasks to produce a separate mean score for each. as expected the measures of emotion regulation and emotional reactivity consequently, these constructs were combined by creating z scores of both variables and averaging these standardized scores to create a single measure of emotion regulation. the overall duration - proportion of time the child spent looking at the video - was used as this study 's laboratory measure of sustained attention. the reliability among coders for the overall duration was excellent (r =.98). children participated in a delay of gratification task in which they were presented with an appealing gift wrapped box and told there was a gift inside for them but that they could not open it for 2 minutes. the overall total time touching gift - combined time the child was in contact with the box - was used as this study 's measure of inhibitory control / reward sensitivity. this overall time was reversed score with higher numbers indicating better inhibitory control skills or lower reward sensitivity. the reliability among coders for the overall time was excellent (r =.99). mother - reported 2-year total behavior problems measured by the child behavior checklist (cbcl 23 ; 47) was used as a control variable. the cbcl has been widely used by researchers studying early social adjustment and has adequate reliability and validity (49, 50). trained research assistants measured children 's height and weight during their 2-year and 5.5-year laboratory visit. degree of overweight was calculated based on age norms from the centers for disease control (45). prior research has shown relations between emotion regulation and emotion reactivity measures where reactivity is a part of the response to the contextual demands that require regulatory strategies to adjust for this change in reactivity (46). thus, both degree of distress and specific regulatory behaviors are considered evidence of emotion regulation processes. consequently, both emotional reactivity and regulation were coded from videotapes of the frustration tasks (prize in the box and high chair). for reactivity, distress was defined as when the child whined, fussed, cried, or tantrummed. a global measure of negative reactivity was coded on a scale from 0, meaning no negative response, to 4, meaning task ended with the child in extreme distress. regulation was defined as the overall effectiveness of using various strategies (e.g., self - stimulation, self - soothing, distraction). a global measure of regulation was coded on a scale from 0, meaning dysregulated or no control of distress, to 4, when the child seemed to completely regulate their distress during most of the task. the reactivity and regulation codes were averaged across tasks to produce a separate mean score for each. as expected the measures of emotion regulation and emotional reactivity consequently, these constructs were combined by creating z scores of both variables and averaging these standardized scores to create a single measure of emotion regulation. children were instructed to watch a 5-minute segment of the videotape spot, a short story about a puppy exploring a neighborhood. the overall duration - proportion of time the child spent looking at the video - was used as this study 's laboratory measure of sustained attention. the reliability among coders for the overall duration was excellent (r =.98). children participated in a delay of gratification task in which they were presented with an appealing gift wrapped box and told there was a gift inside for them but that they could not open it for 2 minutes. the overall total time touching gift - combined time the child was in contact with the box - was used as this study 's measure of inhibitory control / reward sensitivity. this overall time was reversed score with higher numbers indicating better inhibitory control skills or lower reward sensitivity. the reliability among coders for the overall time was excellent (r =.99). mother - reported 2-year total behavior problems measured by the child behavior checklist (cbcl 23 ; 47) was used as a control variable. the cbcl has been widely used by researchers studying early social adjustment and has adequate reliability and validity (49, 50). first, it was important to determine if any of the demographic variables related to children 's 2yr and 5.5yr body mass index (bmi). these analyses revealed no significant association between gender f(2, 59) =.572, p>.05, race f(4, 116) = 1.08, p>.05, or ses (r =.05 and.02, p>.05) or children 's 2yr and 5.5yr bmi. however, there was a positive association between children 's overall behavior problems at age 2 and their 2yr bmi (r =.27, p.05). children 's emotion regulation skills were positively associated with sustained attention (r =.18, p.05, indicating that both children classified as overweight / at - risk or normal weight at age 5.5 had similar 2yr bmi levels. however, there was a significant main effect for the weight groups, f (3, 51) = 2.97, p.05. of note, the significant main effect for weight group on early self - regulation skills occurred even when we did not control for 2yr bmi, f (3, 193) = 4.86, p.05, race f(4, 116) = 1.08, p>.05, or ses (r =.05 and.02, p>.05) or children 's 2yr and 5.5yr bmi. however, there was a positive association between children 's overall behavior problems at age 2 and their 2yr bmi (r =.27, p.05). children 's emotion regulation skills were positively associated with sustained attention (r =.18, p.05, race f(4, 116) = 1.08, p>.05, or ses (r =.05 and.02, p>.05) or children 's 2yr and 5.5yr bmi. however, there was a positive association between children 's overall behavior problems at age 2 and their 2yr bmi (r =.27, p.05). children 's emotion regulation skills were positively associated with sustained attention (r =.18, p.05, indicating that both children classified as overweight / at - risk or normal weight at age 5.5 had similar 2yr bmi levels. however, there was a significant main effect for the weight groups, f (3, 51) = 2.97, p.05. of note, the significant main effect for weight group on early self - regulation skills occurred even when we did not control for 2yr bmi, f (3, 193) = 4.86, p <.01, partial eta - squared =.07. in addition, all three self - regulation variables significantly differentiated the weight groups, p<.05. this study examined the role of early self - regulation skills in the development of pediatric obesity. first, it is important to note that as early as 5.5 years of age, 31% of the children in this study were found to be either overweight / at - risk for being overweight. this prevalence rate is relatively similar to those found in previous studies with older children (49), suggesting that pediatric obesity may be identified as early as kindergarten. entrance to kindergarten marks a time when parents and teachers assess children 's cognitive and socio - emotional abilities as they relate to the child 's readiness to learn (20). the current study suggests that it may also be important to assess children 's physical functioning at this time, as a significant portion of children are already classified as overweight / at - risk for being overweight by kindergarten. second, given the theoretical notion that self - regulation skills consist of multi - faceted control systems, the current study examined multiple aspects of toddler 's self - regulation skills. results revealed positive associations between children 's emotion regulation skills, sustained attention, and inhibitory control / reward sensitivity. these results are consistent with the notion that self - regulation skills become more integrated and sophisticated through development, as toddlers ' abilities to control their affective state, attention, and behavioral impulsivity were positively related to one another. these associations are consistent with previous research linking attentional control processes to affect regulation, especially within the ad / hd (19) and aggression literature (50). consistent with this study 's hypotheses, self - regulation skills in toddlerhood were predictive of both normal variations in bmi development and pediatric obesity. specifically, emotion regulation appears to be the primary self - regulation skill involved in predicting normative changes in bmi as no effects were found for sustained attention or inhibitory control / reward sensitivity. however, both emotion regulation and inhibitory control / reward sensitivity predicted more extreme weight problems, even after controlling for 2yr bmi. thus, toddlers with poorer emotion regulation skills and lower inhibitory control skills / higher reward sensitivity were more likely to be classified as overweight / at - risk for being overweight in early childhood. self - regulation / executive functioning difficulties in overweight / obese individuals have been recently documented within the adult (41) and adolescent literature (33, 34). the few self - regulation / executive functioning studies conducted with children have mainly been cross - sectional and have focused on school - age children (27, 29, 30) or were part of an intervention (25, 28). the current longitudinal study extends such findings to the toddlerhood and early childhood period and suggests that early self - regulation skills constitute an important individual factor to consider when examining both normal variations of bmi and the development of obesity. in addition, early self - regulation skills were stronger predictors of which children were classified as overweight / at - risk at age 5.5 compared to 2yr bmi. such finding highlights the importance of early self - regulation skills and that early deficits serve as significant risk factors for the development of obesity. multiple measures of self - regulation, outside the context of eating, were found to predict children overweight / at - risk for being overweight. these results extend previous self - regulation studies by birch and colleagues (8, 14) in a two major ways. first, it demonstrates the association between self - regulation and obesity outside the context of eating highlighting the complexity of such association and indicating that overweight children may have more general self - regulation deficits. second, it also demonstrates the multiple domains (i.e., behavioral and emotional) in which regulatory functions may contribute to the development of obesity. this finding is counter to a previous study indicating that school age children with adhd had higher body mass indexes compared to age - adapted reference values (25). it may be the case that it is only the behavioral disinhibition / impulsivity aspect of adhd, not the inattention, which is associated with obesity. the current study extends the literature by demonstrating that higher reward sensitivity, indicative of lower inhibitory control skills, as early as during the toddlerhood period represents a significant risk factor in the development of pediatric obesity. children with higher levels of reward sensitivity are more likely to engage in impulsive behaviors. while binge eating episodes have mostly been documented within the adult or adolescent literature (13), children may also engage in impulsive eating behaviors. in addition to impulsive eating, children with higher levels of reward sensitivity may end up preferring food that are sweet and fat, which tend to be reported as tastier compared to healthier / bland food (51). while a causal link between impulsivity and overeating has been suggested by experimental manipulation designs (52) along with a link between reward sensitivity and food choices (51), the results of our study suggest that toddlerhood may be an important period for future studies to examine these key questions. the current study also found an important link between the self - regulation of affective processes and both normal variations in bmi development and pediatric obesity. past research had established a strong link between emotion dysregulation and overeating in the adult and adolescent population (13, 33). the current findings extend this link to the child population and suggest that poor emotion regulation skills may be an important risk factor for the development of obesity and not merely a consequence of it. although the current study can not address the mechanisms by which poor emotion regulation skills contribute to obesity, adult studies have suggested an important physiological link via the vagus nerve (39). specifically, the vagal pathways have been shown to have a crucial role in facilitating metabolic resources in the service of coping, as well as in satiety and short - term regulation of food intake (15, 38). thus, it may be that children with poor emotion regulation have a difficult time assessing whether they are satiated. children 's abilities to determine whether they are satiated are especially important given that the portion size of the average meal has increased dramatically over the last two decades (53). given that the vagal pathways show considerable growth from birth to the early childhood years (54), it will be important for future research to examine the link between the development of physiological regulation and children 's eating behaviors. the other potential mechanism by which emotion regulation relates to obesity is via the concept of eating as a way to cope with stressors / negative emotions. emotional eating has been clearly documented within the adult / adolescent literature (32) and a recent study documented emotional eating in children as young as 9 years of age (55). however, it remains unclear whether young children engage in emotional eating as a coping strategy. given that young children do experience various stressors as they enter school, such as peer victimization and learning difficulties (56), it is important for future studies to assess whether children are using eating as an emotion regulation strategy. in summary, the current study found that toddlers ' self - regulation skills across behavioral and emotional domains significantly differentiated which children at age 5.5 were classified as overweight / at - risk of being overweight compared to normal weight children. that toddlers ' self - regulation skills were measured in laboratory tasks represents a significant strength of this study as the majority of past studies rely on parental report of children 's functioning. in terms of this study 's limitations, it is important to point out that we did not have information on either child eating behaviors or information on parental weight as this was not the primary aim of the study design. given that parental weight status is one of the biggest predictors of child weight status (5), it is difficult to know whether self - regulation skills would continue to predict children 's weight status above and beyond the shared genetic effects of their parents ' weight status. however, parental weight status does not only predict children 's weight status via genetics, but also influences children 's eating behavior (6). thus, it may be that the socialization of eating behavior that occurs between an overweight parent and his / her child is mediated through regulatory processes. for example, children may watch their overweight parents eat to cope with stress or watch their parents consistently eat large portions. in turn, children may engage in similar behaviors and their regulatory abilities to detect when they are satiated may worsen. the effects of parental behaviors on regulatory processes are well documented within the human (26) and animal literature (57). future research should examine whether parental eating behavior has an effect on children 's early regulatory skills. lastly, it is important to point out that despite our longitudinal design and attempts in controlling early weight status, we can not affirm a causal link between self - regulation and the development of obesity. nevertheless, our results do provide initial evidence for the importance of examining toddlers ' early self - regulation skills as risk factors for the development of obesity. for example, in addition to helping children and their families make lifestyle changes in terms of dieting / exercise, future treatment may want to teach children how to better monitor their emotional state as it relates to eating behavior, how to identify when they are feeling satiated, and reinforce them for inhibiting additional eating. | objectiveto investigate the role of early self - regulation skills, including emotion regulation, sustained attention, and inhibitory control / reward sensitivity, in predicting pediatric obesity in early childhood.methodparticipants for this study included 57 children (25 girls) obtained from three different cohorts participating in a larger ongoing longitudinal study. at 2 years of age, participants participated in several laboratory tasks designed to assess their self - regulation abilities. height and weight measures were collected when children were 2 and 5.5 years of age.resultsself-regulation skills in toddlerhood were predictive of both normal variations in bmi development and pediatric obesity. specifically, emotion regulation was the primary self - regulation skill involved in predicting normative changes in bmi as no effects were found for sustained attention or inhibitory control / reward sensitivity. however, both emotion regulation and inhibitory control / reward sensitivity predicted more extreme weight problems (i.e., pediatric obesity), even after controlling for 2yr bmi. thus, toddlers with poorer emotion regulation skills and lower inhibitory control skills / higher reward sensitivity were more likely to be classified as overweight / at risk at 5.5 years of age.conclusionearly self - regulation difficulties across domains (i.e., behavioral, and emotional) represent significant individual risk factors for the development of pediatric obesity. mechanisms by which early self - regulation skills may contribute to the development of pediatric obesity are discussed. |
let g be a finitely generated group and k a (not necessarily finitely generated) subgroup of g. we can choose a finite set ag of generators such that every element of g is of the form g = g1gn, where n0 and g1,,gna. we shall say that (g, k) is context - free, if loosely spoken the language of all words over a that represent an element of k is context - free. let be a finite alphabet and :g be a (not necessarily injective) mapping such that a=() satisfies the above finite generation property for g. then has a unique extension, also denoted, as a monoid homomorphism :g. recall that consists of all words w = a1an, where n0 and a1,,an (repetitions allowed). the number n is the length |w| of w. if n=0 this means that w=, the empty word. this is the neutral element of, and is a free monoid with the binary operation of concatenation of words. the extension of is of course given by (a1an)=(a1)(an), where the product on the right hand side is taken in g. given these ingredients, we shall say that :g is a semigroup presentation of g, referring to the fact that a generates g as a semigroup. a language over is a non - empty subset of. definition 1.1the word problem of (g, k) with respect to is the language l(g, k,)={w:(w)k}. we say that the triple (g, k,) is context - free, if l(g, k,) is a context - free language. the word problem of (g, k) with respect to is the language l(g, k,)={w:(w)k}. we say that the triple (g, k,) is context - free, if l(g, k,) is a context - free language. a context - free grammar is a quadruple c=(v,,p, s), where v is a finite set of variables, disjoint from the finite alphabet (the terminal symbols), the variable s is the start symbol, and pv(v) is a finite set of production rules. we write tu or (tu)p if (t, u)p. for v, w(v), we write vw if v = v1tv2 and w = v1uv2, where u, v1,v2(v) and tu. this is a single derivation step, and it is called rightmost, if v2. a derivation is a sequence v = w0,w1, the succession of steps of any derivation tw can be reordered so that it becomes a rightmost derivation. for tv, we consider the language lt={w:tw}. the language generated by c is l(c)=ls. a context - free language is a language generated by a context - free grammar. as a basic reference for language and automata theory, the above definition of a context - free pair, or rather triple, (g, k,) makes sense when g is a finitely generated monoid and k is a sub - monoid, but here we are interested in groups. when in addition k={1 g }, this leads to the notion of g being a context - free group. in two celebrated papers, muller and schupp, have carried out a detailed study of context - free groups and more generally, context - free graphs. in particular, context - freeness of a group is independent of the particular choice of the generating set a of g. the main result of, in combination with a fundamental theorem of dunwoody, is that a finitely generated group is context - free if and only if it is virtually free, that is, it contains a free subgroup of finite index. (in, it is assumed that a = a1 and that :a=() is one - to - one, but the results carry over immediately to the more general setting where those two properties are not required.) previously, anisimov had shown that the groups whose word problem l(g,{1g},) is regular (see section 2 for the definition) are precisely the finite groups. the above mentioned context - free graphs are labelled, rooted graphs with finitely many isomorphism classes of cones. the latter are the connected components of the graph that remain after removing a ball around the root with arbitrary radius., there is a natural correspondence between such graphs and pushdown automata, which are another tool for generating context - free languages ; see section 3. among subsequent work, we mention plecq and snizergues, who studied actions on, resp have introduced and studied co - context - free groups, which are such that the complement of l(g,{1g},) is context - free, see also lehnert and schweitzer. this concept has an obvious extension to co - context - free pairs of groups, resp. graphs, on whose examination we do not (yet) embark. in the present notes, we collect properties and examples of context - free pairs of groups (g, k). the language l(g, k,) is regular if and only if the index [g : k ] of k in g is finite (proposition 2.4).the property that l(g, k,) is context - free does not depend on the specific choice of the semigroup presentation, so that context - freeness is just a property of the pair (g, k), a consequence of lemma 3.1.if (g, k) is context - free then l(g, k,) is a deterministic context - free language (see section 3 for the definition) for any semigroup presentation :g (corollary 4.8.a).if (g, k) is context - free and h is a finitely generated subgroup of g, then the pair (h, kh) is context - free (lemma 3.1).if [g : h]m.construction of d(c). we define d(c) as the subgraph of x induced by all vertices yx with d(o, x)=d(o, y)+d(x, y)andd(x, y)mfor some xc. in particular, y lies on some geodesic path from o to c.now let x1,x2c, and consider some path x1,x2(c) (i.e., it lies in c). choose a geodesic path 1 from o to x1 and a geodesic path 2 from x2 to o. then we can concatenate the three paths to a single path 12o, o. its label is the word w=(1)()(2)lo, o. set n=|w| and write w=(a1ak)(ak+1ank)(ank+1an) where the 3 pieces in the parentheses are (in order) (1), () and (2). the words (1), () and (2)s are the labels of three consecutive arcs that fill the boundary of the polygon p(w). (to be precise, along the last edge of the 3rd arc, we are reading the label s in the reversed direction.) by [11, lemma 5 ], its triangulation has a triangle which meets each of those arcs. (it may also occur that one corner of the triangle meets two arcs.) thus, there are i{0,,k } and i{k,,nk } such that the vertices ti and ti of p(w) lie on that triangle. they correspond to the vertices y1=oa1ai and y=oa1ai of x. we either have ii1, or else a diagonal (ti, u, ti) is a side of our triangle. by lemma 4.5, we get d(y1,y)m(u)m. thus kid(o, y)i+m, that is, ikm>0. in particular, ti does not lie on the third arc. in the same way, there is j{nk,,nk+m } (and not larger) such that tj is a corner of our triangle. the points y1 and y2 are in d(c), and by lemma 4.4, tv1()v2.[it is here that we can see lemma 4.3, since we deduced that d(x1,x2)3 m for all x1,x2c.]by lemma 4.4, we also have sa1aitaj+1an, so that vlt implies a1aivaj+1anlo, o and consequently vly1,y2, that is, y1v = y2.we now insert into d(c) the additional labelled edge (y1,v1tv2,y2), whose label is the word v1tv2v. we insert all diagonals of the same type that can be obtained in the same way, and write d(c) for the resulting edge - enrichment of d(c).subsuming, we have an edge (y1,v1tv2,y2) in d(c) if and only if the following properties hold. |vi|m (i=1,2) and tv,the path with label v1 starting at y1 and ending at x1=y1v1c is part of a geodesic from o to x1,the path with label v2 starting at x2=y2v2c and ending at y2 is part of a geodesic from x2 to o, andthere is a path in c from x1 to x2 such that tv1()v2,if tv then v is the label of a path in y1,y2.now, there are only finitely many cones c with respect to o with d(c, o)m. on the other hand, for all cones c with d(c, o)m, there is a bound on the number of vertices of d(c), as well as on the number of possible labels on its edges. in particular, there are only finitely many possible isomorphism types of the labelled graphs (d(c),c) with marked boundary cd(c).we now suppose that c and c are two cones at distance m from o, such that (d(c),c) and (d(c),c) are isomorphic. we claim that c and c are isomorphic, and this will conclude the proof that there are only finitely many isomorphism types of cones with respect to o.let :d(c)d(c) be an isomorphism with (c)=c, and its inverse mapping. we extend to a mapping from c to c, also denoted.claim 1let xc and v+ such that the path x(v) lies in c and meets c only in its initial point x. then the path x(v) lies in c and meets c only in its initial point x=(x)c.proofif a is the initial letter of v then (always using the notation of definition 1.1) the first edge of x(v) is (x, a, xa). we now consider the path x(v) with label v starting at xc. we first claim that the latter lies in c and only its initial point x is in c. let (x,a,(x)a) be the first edge of the path. then (x)a can not lie in d(c), since otherwise (x, a, xa)=((x),a,(x)a) would be an edge in d(c), a contradiction. thus, the path x(v) goes at least initially into cc.so now suppose that x(v) ever returns to c, and let be its initial part up to the first return. then v=(x(v)) is an initial part of v with |v|2, and is a path within c from x1=x to x2=(x)vc. but then, by construction, d(c) must contain an edge (y1,v1tv2,y2) such that x1=(y1)v1, y2=(x2)v2, and tv1vv2. using the isomorphism :d(c)d(c), we set yi=(yi), i=1,2, and x2=(x2)c. but this contradicts the fact that x(v) meets c only in its initial point. we conclude that also the path x(v) lies in c and meets c only in its initial point, and claim 1 is verified. then there are xc and v+ such that z = xv and the path x(v) from x to z meets c only in its initial point x. by claim 1, the analogous statement holds for the path x(v) in c, where x=(x). this will follow from the next claim.claim 2let x1,x2c, v, w+ such that the paths x1(v) and x2(w) lie in c, meet c only in their initial points and end at the same point of cc. then, setting xi=(xi), also x1(v) and x2(w) end at the same point of cc.prooflet w1 be the inverse of w, as defined in definition 1.1. then x2w1=x2w, and vw1 is the label of the path from x1 to x2 that we obtain by first following x1(v) and then the inverse of x2(w). it lies entirely in c, and only its endpoints are in c. by construction, d(c) therefore v1vw1v2ly1,y2. but this implies that vw1 is the label of a path from x1 to x2, and we know from claim 1 that it lies in c and has only its endpoints in c. thus, is well defined, and the same works of course also for by exchanging the roles of cand c.claim 3the map :cc is bijective.proofwe know that :cc is bijective and that (cc)cc. let zcc, and let xc, v+ such that x(v) is a path from x to z that intersects c only at the initial point. setting x=(x), z=(z), we know from the construction of and claim 1 that x(v) is a path in c from x to z that meets c only in its initial point. now therefore is the identity on c. exchanging roles, we also get the is the identity on c. this proves claim 3. it is now immediate from the construction that also preserves the edges and their labels, so that it is indeed an isomorphism between the labelled graphs c and c that sends c to c. this concludes the proof of theorem 4.6. 2.7 ] says that if a symmetric labelled graph is context - free with respect to one root o, then it is context - free with respect to any other vertex chosen as the root x. in view of theorem 4.2, theorem 4.6, this is also obtained from the following, when the graph is fully deterministic. there is a reduced grammar c=(v,,p, s) in cnf that generates lo, o. each of the languages lt, tv, is non - empty, only ls contains, and we define (3)m = max{m(t):tv }, where m(t) is as in (2). we define d(c) as the subgraph of x induced by all vertices yx with d(o, x)=d(o, y)+d(x, y)andd(x, y)mfor some xc. now let x1,x2c, and consider some path x1,x2(c) (i.e., it lies in c). choose a geodesic path 1 from o to x1 and a geodesic path 2 from x2 to o. then we can concatenate the three paths to a single path 12o, o. its label is the word w=(1)()(2)lo, o. set n=|w| and write w=(a1ak)(ak+1ank)(ank+1an) where the 3 pieces in the parentheses are (in order) (1), () and (2). the words (1), () and (2)s are the labels of three consecutive arcs that fill the boundary of the polygon p(w). (to be precise, along the last edge of the 3rd arc, we are reading the label s in the reversed direction.) by [11, lemma 5 ], its triangulation has a triangle which meets each of those arcs. (it may also occur that one corner of the triangle meets two arcs.) thus, there are i{0,,k } and i{k,,nk } such that the vertices ti and ti of p(w) lie on that triangle. they correspond to the vertices y1=oa1ai and y=oa1ai of x. we either have ii1, or else a diagonal (ti, u, ti) is a side of our triangle. by lemma 4.5, we get d(y1,y)m(u)m. thus kid(o, y)i+m, that is, ikm>0. in particular, ti does not lie on the third arc. in the same way, there is j{nk,,nk+m } (and not larger) such that tj is a corner of our triangle. the points y1 and y2 are in d(c), and by lemma 4.4, tv1()v2. [it is here that we can see lemma 4.3, since we deduced that d(x1,x2)3 m for all x1,x2c. ] by lemma 4.4, we also have sa1aitaj+1an, so that vlt implies a1aivaj+1anlo, o and consequently vly1,y2, that is, y1v = y2. we now insert into d(c) the additional labelled edge (y1,v1tv2,y2), whose label is the word v1tv2v. we insert all diagonals of the same type that can be obtained in the same way, and write d(c) for the resulting edge - enrichment of d(c). subsuming, we have an edge (y1,v1tv2,y2) in d(c) if and only if the following properties hold. |vi|m (i=1,2) and tv,the path with label v1 starting at y1 and ending at x1=y1v1c is part of a geodesic from o to x1,the path with label v2 starting at x2=y2v2c and ending at y2 is part of a geodesic from x2 to o, andthere is a path in c from x1 to x2 such that tv1()v2,if tv then v is the label of a path in y1,y2. |vi|m (i=1,2) and tv, the path with label v1 starting at y1 and ending at x1=y1v1c is part of a geodesic from o to x1, the path with label v2 starting at x2=y2v2c and ending at y2 is part of a geodesic from x2 to o, and there is a path in c from x1 to x2 such that tv1()v2, if tv then v is the label of a path in y1,y2. now, there are only finitely many cones c with respect to o with d(c, o)m. on the other hand, for all cones c with d(c, o)m, there is a bound on the number of vertices of d(c), as well as on the number of possible labels on its edges. in particular, there are only finitely many possible isomorphism types of the labelled graphs (d(c),c) with marked we now suppose that c and c are two cones at distance m from o, such that (d(c),c) and (d(c),c) are isomorphic. we claim that c and c are isomorphic, and this will conclude the proof that there are only finitely many isomorphism types of cones with respect to o. let :d(c)d(c) be an isomorphism with (c)=c, and its inverse mapping. we extend to a mapping from c to c, also denoted. claim 1let xc and v+ such that the path x(v) lies in c and meets c only in its initial point x. then the path x(v) lies in c and meets c only in its initial point x=(x)c. let xc and v+ such that the path x(v) lies in c and meets c only in its initial point x. then the path x(v) lies in c and meets c only in its initial point x=(x)c. proofif a is the initial letter of v then (always using the notation of definition 1.1) the first edge of x(v) is (x, a, xa). we now consider the path x(v) with label v starting at xc. we first claim that the latter lies in c and only its initial point x is in c. let (x,a,(x)a) be the first edge of the path. then (x)a can not lie in d(c), since otherwise (x, a, xa)=((x),a,(x)a) would be an edge in d(c), a contradiction. thus, the path x(v) goes at least initially into cc.so now suppose that x(v) ever returns to c, and let be its initial part up to the first return. then v=(x(v)) is an initial part of v with |v|2, and is a path within c from x1=x to x2=(x)vc. but then, by construction, d(c) must contain an edge (y1,v1tv2,y2) such that x1=(y1)v1, y2=(x2)v2, and tv1vv2. using the isomorphism :d(c)d(c), we set yi=(yi), i=1,2, and x2=(x2)c.. but this contradicts the fact that x(v) meets c only in its initial point. we conclude that also the path x(v) lies in c and meets c only in its initial point, and claim 1 is verified. if a is the initial letter of v then (always using the notation of definition 1.1) the first edge of x(v) is (x, a, xa). we now consider the path x(v) with label v starting at xc. we first claim that the latter lies in c and only its initial point x is in c. let (x,a,(x)a) be the first edge of the path. then (x)a can not lie in d(c), since otherwise (x, a, xa)=((x),a,(x)a) would be an edge in d(c), a contradiction. so now suppose that x(v) ever returns to c, and let be its initial part up to the first return. then v=(x(v)) is an initial part of v with |v|2, and is a path within c from x1=x to x2=(x)vc. but then, by construction, d(c) must contain an edge (y1,v1tv2,y2) such that x1=(y1)v1, y2=(x2)v2, and tv1vv2. using the isomorphism :d(c)d(c), we set yi=(yi), i=1,2, and x2=(x2)c.. but this contradicts the fact that x(v) meets c only in its initial point. we conclude that also the path x(v) lies in c and meets c only in its initial point, and claim 1 is verified. then there are xc and v+ such that z = xv and the path x(v) from x to z meets c only in its initial point x. by claim 1, the analogous statement holds for the path x(v) in c, where x=(x). claim 2let x1,x2c, v, w+ such that the paths x1(v) and x2(w) lie in c, meet c only in their initial points and end at the same point of cc. then, setting xi=(xi), also x1(v) and x2(w) end at the same point of cc. let x1,x2c, v, w+ such that the paths x1(v) and x2(w) lie in c, meet c only in their initial points and end at the same point of cc. then, setting xi=(xi), also x1(v) and x2(w) end at the same point of cc. prooflet w1 be the then x2w1=x2w, and vw1 is the label of the path from x1 to x2 that we obtain by first following x1(v) and then the inverse of x2(w). it lies entirely in c, and only its endpoints are in c. by construction, d(c) therefore v1vw1v2ly1,y2. but this implies that vw1 is the label of a path from x1 to x2, and we know from claim 1 that it lies in c and has only its endpoints in c. then x2w1=x2w, and vw1 is the label of the path from x1 to x2 that we obtain by first following x1(v) and then the inverse of x2(w). it lies entirely in c, and only its endpoints are in c. by construction, d(c) therefore v1vw1v2ly1,y2. but this implies that vw1 is the label of a path from x1 to x2, and we know from claim 1 that it lies in c and has only its endpoints in c. thus, is well defined, and the same works of course also for by exchanging the roles of cand c. claim 3the map :cc is bijective. let zcc, and let xc, v+ such that x(v) is a path from x to z that intersects c only at the initial point. setting x=(x), z=(z), we know from the construction of and claim 1 that x(v) is a path in c from x to z that meets c only in its initial point. now therefore is the identity on c. exchanging roles, we also get the is the identity on c. this proves claim 3. let zcc, and let xc, v+ such that x(v) is a path from x to z that intersects c only at the initial point. setting x=(x), z=(z), we know from the construction of and claim 1 that x(v) is a path in c from x to z that meets c only in its initial point. now therefore is the identity on c. exchanging roles, we also get the is the identity on c. this proves claim 3. it is now immediate from the construction that also preserves the edges and their labels, so that it is indeed an isomorphism between the labelled graphs c and c that sends c to c. this concludes the proof of theorem 4.6. corollary 4.7let (x, e,) be a fully deterministic, strongly connected graph with label alphabet. if lo, o is context - free then lx, y is deterministic context - free for all x, yx. let (x, e,) be a fully deterministic, strongly connected graph with label alphabet. if lo, o is context - free then lx, y is deterministic context - free for all x, yx. theorem 4.2, theorem 4.6, together with lemma 3.1 also imply the following. corollary 4.8let g be a finitely generated group and k a subgroup.(a)the pair (g, k) is context - free if and only if for any symmetric :g, the schreier graph x(g, k,) is a context - free graph. in this case, the language l(g, k,) is deterministic for every (not necessarily symmetric) semigroup presentation :g.(b)if (g, k) is context - free, then also (g, g1 kg) is context - free for every gg. let g be a finitely generated group and k a subgroup.(a)the pair (g, k) is context - free if and only if for any symmetric :g, the schreier graph x(g, k,) is a context - free graph. in this case, the language l(g, k,) is deterministic for every (not necessarily symmetric) semigroup presentation :g.(b)if (g, k) is context - free, then also (g, g1 kg) is context - free for every gg. the pair (g, k) is context - free if and only if for any symmetric :g, the schreier graph x(g, k,) is a context - free graph. in this case, the language l(g, k,) is deterministic for every (not necessarily symmetric) semigroup presentation :g. if (g, k) is context - free, then also (g, g1 kg) is context - free for every gg. regarding (b), for the schreier graph x(g, k,), we have l(g, k,)=lo, o and l(g, g1kg,)=lx, x with x = kg, gg. regarding (b), for the schreier graph x(g, k,), we have l(g, k,)=lo, o and l(g, g1kg,)=lx, x with x = kg, gg. lemma 4.9let g be a finitely generated group and k, h be subgroups with kh and [h : k]n is a rooted, labelled tree that is isomorphic to one of the cones of t. thus, the schreier graph, resp. it now follows from proposition 3.3 and lemma 4.9 that also (g, k) is context - free. then k = kf is a free subgroup of k with [k : k]n is a rooted, labelled tree that is isomorphic to one of the cones of t. thus, the schreier graph, resp. it now follows from proposition 3.3 and lemma 4.9 that also (g, k) is context - free. we remark here that one can always reduce the study of context - free pairs to free groups and their subgroups. given (g, k), let f be a finitely generated free group that maps by a homomorphism onto g. let k be the preimage of k under that homomorphism. then clearly (g, k) is context - free if and only (f, k) has this property. of course, there are context - free pairs with g free beyond the situation of corollary 5.3. example 5.4consider the free group f=a, b and the subgroup k with the infinite set of free generators { akblablak : k, lz, l0}. the associated schreier graph with respect to { a1,b1 } is the comb lattice.its vertex set is the set of integer points in the plane. the edges labelled by a are along the x - axis, from (k,0) to (k+1,0), and there is a loop with label a at each point (k, l) with l0. the edges labelled by b are all the upward edges of the grid, that is, all edges from (k, l) to (k, l+1), where (k, l)z2. to these, we have to add the oppositely oriented edges whose labels are the respective inverses (in fig. 3, consider the free group f=a, b and the subgroup k with the infinite set of free generators { akblablak : k, lz, l0}. the associated schreier graph with respect to { a1,b1 } is the comb lattice. the edges labelled by a are along the x - axis, from (k,0) to (k+1,0), and there is a loop with label a at each point (k, l) with l0. the edges labelled by b are all the upward edges of the grid, that is, all edges from (k, l) to (k, l+1), where (k, l)z2. to these, we have to add the oppositely oriented edges whose labels are the respective inverses (in fig. 3, it is very easy to see that context - freeness is not transitive in the following sense : if (g, h) and (h, k) are context - free (with g, h finitely generated and khg) then in general (g, k) will not be context - free. example 5.5let g = z2, h = z{0}z and k={(0,0)}. then h (i.e., (h, k)) is context - free. of course, this also holds for (g, h), whose schreier graphs are just the cayley graphs of z. but z2 (i.e., (g, k)) is not context - free. let g = z2, h = z{0}z and k={(0,0)}. then h (i.e., (h, k)) is context - free. of course, this also holds for (g, h), whose schreier graphs are just the cayley graphs of z. but z2 (i.e., (g, k)) is not context - free. this also shows that the converse of lemma 3.1 does not hold in general (while we know that it does hold when [g : h]<). finally, we construct examples of three groups khg, where (g, h) is context - free, [h : k]<, and (g, k) is not context - free. example 5.6we construct a family of fully deterministic, symmetric labelled graphs xw, wz(non - empty), and one such graph y, so that y is the factor graph with respect to the action of a 2-element group of automorphisms of each of the labelled graphs xw. while y will be a context - free graph, many of the graphs xw in our family are not context - free. we then translate this back into the setting of pairs of groups.the vertex set of xw is z{0,1}. the set of labels is ={a, b, a1,b1}. the edges are as follows : ((k,0),a,(k+1,0))and((k,1),a,(k+1,1))for allkz,((k,0),b,(k+1,0))and((k,1),b,(k+1,1))for allkzw, and((k,0),b,(k+1,1))and((k,1),b,(k+1,0))for allkw. these reversed edges together with the corresponding labels are omitted for simplicity). since w0, there is at least one of the crosses (pair of the third type of edges). therefore xw is connected. in general, it does not have finitely many cone types, i.e., it is not context - free. for example, it is not context - free when w={k(|k|+1):kz}for arbitrary w, the two - element group that exchanges each (k,0) with (k,1) acts on xw by label preserving graph automorphisms.. 5) has vertex set z and edges (k, a, k+1)and(k, b, k+1)for allkz, plus the associated reversed edges (in fig. 5, these edges together with the corresponding labels are omitted for simplicity). it is clearly a context - free graph.now let f = f be the free group (universal cover of xw and y), and for given w, let kw be the fundamental group of xw at the vertex (0,0). then it is straightforward that kw has index 2 in k. the mapping is the embedding of into f, as above. we construct a family of fully deterministic, symmetric labelled graphs xw, wz(non - empty), and one such graph y, so that y is the factor graph with respect to the action of a 2-element group of automorphisms of each of the labelled graphs xw. while y will be a context - free graph, many of the graphs xw in our family are not context - free. the vertex set of xw is z{0,1}. the set of labels is ={a, b, a1,b1}. the edges are as follows : ((k,0),a,(k+1,0))and((k,1),a,(k+1,1))for allkz,((k,0),b,(k+1,0))and((k,1),b,(k+1,1))for allkzw, and((k,0),b,(k+1,1))and((k,1),b,(k+1,0))for allkw. these reversed edges together with the corresponding labels are omitted for simplicity). since w0, there is at least one of the crosses (pair of the third type of edges). therefore xw is connected. in general, it does not have finitely many cone types, i.e., it is not context - free. for example, it is not context - free when w={k(|k|+1):kz } for arbitrary w, the two - element group that exchanges each (k,0) with (k,1) acts on xw by label preserving graph automorphisms. the factor graph y (see fig. 5) has vertex set z and edges (k, a, k+1)and(k, b, k+1)for allkz, plus the associated reversed edges (in fig. 5, these edges together with the corresponding labels are omitted for simplicity). now let f = f be the free group (universal cover of xw and y), and for given w, let kw be the fundamental group of xw at the vertex (0,0). then it is straightforward that kw has index 2 in k. the mapping is the embedding of into f, as above. we then have y = x(f, k,) and xw = x(f, kw,), providing the required example. example 5.7at the end of the introduction, we mentioned the possible interplay with ends. the number of ends e(x) of a symmetric, connected graph is the supremum of the number of connected components of the complement of any finite subgraph. via stallings celebrated structure theorem, ends of groups (i.e., ends of cayley graphs) are closely related with amalgamated free products and hnn - extensions. thus, it is natural to ask the following question.let (g1,k) and (g2,k) be two context - free pairs of groups sharing the same subgroup k. let g = g1kg2 be the amalgamated free product of g1 and g2 over the group k. is it then true that (g, k) is context - free ? when k is finite, the answer is of course yes, because then g1,g2 and g are virtually free. here is a brief outline.let g=a1,a2,b1,b2[a1,b1][a2,b2] be the fundamental group of an orientable surface of genus 2. let k be the infinite cyclic subgroup generated by the commutator [a1,b1]=[a2,b2]1, and for i=1,2, let gi be the free group with free generators ai and bi. then g is the amalgamated free product of g1 and g2 over k.by corollary 5.3, the pairs (g1,k) and (g2,k) are context - free. but (g, k) is not context - free. indeed, let x be the schreier graph of (g, k) with respect to the above generators and their inverses. thus, there is a finite subgraph f of x such that xb(f, n) has exactly two infinite cones for any n. if x were context - free, then the finite upper bound on the number of boundary elements of any cone would yield that x has linear growth, that is |b(f, n)|cn for all n. this contradicts the fact that g, as well as the schreier graphs of (g1,k) and (g2,k), have exponential growth. at the end of the introduction the number of ends e(x) of a symmetric, connected graph is the supremum of the number of connected components of the complement of any finite subgraph. via stallings celebrated structure theorem, ends of groups (i.e., ends of cayley graphs) are closely related with amalgamated free products and hnn - extensions. let (g1,k) and (g2,k) be two context - free pairs of groups sharing the same subgroup k. let g = g1kg2 be the amalgamated free product of g1 and g2 over the group k. is it then true that (g, k) is context - free ? when k is finite, the answer is of course yes, because then g1,g2 and g are virtually free. let k be the infinite cyclic subgroup generated by the commutator [a1,b1]=[a2,b2]1, and for i=1,2, let gi be the free group with free generators ai and bi. then g is the amalgamated free product of g1 and g2 over k. by corollary 5.3, the pairs (g1,k) and (g2,k) are context - free. but indeed, let x be the schreier graph of (g, k) with respect to the above generators and their inverses. thus, there is a finite subgraph f of x such that xb(f, n) has exactly two infinite cones for any n. if x were context - free, then the finite upper bound on the number of boundary elements of any cone would yield that x has linear growth, that is |b(f, n)|cn for all n. this contradicts the fact that g, as well as the schreier graphs of (g1,k) and (g2,k), have exponential growth. | let g be a finitely generated group, a a finite set of generators and k a subgroup of g. we define what it means for (g, k) to be a context - free pair ; when k is trivial, this specializes to the standard definition of g to be a context - free group.we derive some basic properties of such group pairs. context - freeness is independent of the choice of the generating set. it is preserved under finite index modifications of g and finite index enlargements of k. if g is virtually free and k is finitely generated then (g, k) is context - free. a basic tool is the following : (g, k) is context - free if and only if the schreier graph of (g, k) with respect to a is a context - free graph. |
in nursing education, incivility is referred to as unreflected destructive behaviors leading to physical or emotional distress in the people involved in education. the consequences of disruptive behaviors are interfering with learning atmosphere, especially in learning teams and collaborative learning. today s students are our future colleagues and if their incivility are not recognized and managed, they will change to uncivilized personnel. if the students are trained weakly in terms of personality, an irreversible damage will occur, such as interactional problems with colleagues, patients, and caregivers. hazards for patients safety, absence from workplace, changing the workplace, and personnel s physical and emotional health are secondary expenses. nursing education incivilities include interruption in class, disrespectful behavior toward teacher or other students, lack of students attention to class, delayed arrival and early leaving the class, lack of academic honesty, and bullying. incivilities lead to poor learning atmosphere, poor behaviors in the workplace, and violence. students incivility may cause negative physical, emotional and psychological effects on faculty members, such as stress, anxiety, illness, job dissatisfaction, behavioral changes, as well as harmful impacts on work setting. in addition, the students incivility may negatively impact the functions of college authorities. on the other hand, violent behavior that nursing education has experienced in recent years colleges, as a part of community, are not immune from incivility and the complications of academic incivility. in clinical setting more than 50% of nurses experience uncivilized behaviors, and more than 90% of them witness abusive behaviors toward others. few studies are published about nursing students incivility management worldwide, particularly in iran. moreover, in today s world with shortage of nurses, lack of competent nurses is a major concern, so it is necessary to recognize the causes of incivility and revise them. since the universal aim of education is improving civility and respect, the role of higher education is training researchers, professionals and useful citizens. medical sciences students should not only be expert in sciences and skills, but also acquire high standards of morality and humanity. unprofessional behaviors have occurred both in teachers and students and studied in nursing, dentistry and pharmacology ; however, no study has been conducted on the causes of incivility. as recognizing the causes of incivility is necessary to prevent and treat the students incivility, so there is a need to conduct this research. lack of rules and regulations for managing the students behaviors makes them think they are not responsible as to incivility. therefore, researchers focused on documenting rules and regulations for incivilities and necessity of familiarization of teachers with them. if managers and teachers recognize the cause of incivility, they can provide a sounder atmosphere for improving communication between students and teachers as well as training committed and expert people. when the variables of a concern such as causes of students incivility are unclear, a qualitative approach is most revealing than quantitative methods. thus, a qualitative approach was used to take a holistic perspective about the causes of incivility. in addition, no study has been done in iran to explore the causes of students incivility ; therefore, a qualitative study was applied using open ended questions in order to explore the causes of incivility in nursing students from both educators and students points of view. considering the aim of the research, i.e. exploring the cause of incivility, we used the qualitative approach to understand human feelings and discover the latent meaning in daily experiences ; also, to understand the factors which play a role in forming uncivil behaviors, we used qualitative content analysis. this method was applied for extracting the main themes and exploring the existent patterns in the data. it is also an appropriate method for achieving valid and dynamic results affecting deep understanding of contextual data in order to form new knowledge, and present reality and practical guide for practice. indeed, this method with qualitative reduction and categorization of data attempts to understand the meaning of data. participants were nursing students and teachers who were either working or studying in khorasan razavi universities of medical sciences. since we used individual interviews for data gathering, the participants who had encountered with uncivil behaviors and y could explain their experience in more detail were selected. for this purpose, questions were asked about the colleges officials, educators, and participants (snowball sampling). then, it continued regarding maximum diversity and stopped with arriving data saturation ; in this step, we found that further interviews do nt provide different information to develop a new category about the causes of students incivility. therefore, saturation of data was confirmed with no new data achieved in the three last interviews. in order to maximize the variation in participants selection, the researchers considered factors such as age, gender, educational degree, teaching experience, type of university, and having an organizational position in the university. inclusion criteria for the teachers were at least one year history of teaching either in the classroom or in clinical settings and those for the students were a history of 2-terms of study in the university. also, they participated in the study and interview voluntarily and were able to present their experiences. students were 18 - 33 years old and the teachers were 30 - 55. in qualitative studies, sample size is not fixed, so the researcher should continue the sampling process until no new idea is acquired. in the present study, the samples were 17 teachers : 8 males and 9 females and 9 students : 4 males and 5 females (tables 1, 2). the interviews were started with questions such as : what do you think about the students or teachers incivility ?, how is incivility practiced ?, what are the conditions leading to incivility ? they were continued with probing questions such as would you please explain more ? the participants were interviewed by the first author for 30 - 90 min. all interviews were recorded and listened several times ; then, they were typed in word 2007. they were analyzed by qualitative content analysis which is applied for subjective interpretation of the textual data. in this method content analysis is far from extracting objective content of the text ; it is applied for extracting hidden themes and patterns from the participants data. after several listening, transcribing, reading and immersing session in the data, a big picture of data was resulted ; meanings were extracted, key ideas were bolded and the codes were categorized based on their relations. different strategies for credibility of the data analysis such as peer check techniques and member check were also applied. in member checking, codes of interview were returned to the participants, and they confirmed the researchers representing ideas. for peer checking, the researchers coded and categorized the data independently and where there was a disagreement, discussions and clarifications were done in order to reach a consensus. faculty members demographic characteristics students demographic characteristics mashhad university of medical sciences (mums) ethics committee approved the proposal (approval code 921903/1/114). they were insured that they could stop their participation in any stage of the study. all of them were informed of the confidentiality of data and kept them anonymous ; then they gave their written consent. analysis of the interview data led to emergence of 900 primary codes, reduced to 42 sub - subcategories, 10 subcategories, and eventually 3 main categories were developed. in this study 3 causes of nursing students incivility : categories and subcategories educational engagement : one of the main causes of incivility was non - educational engagement such as matrimony, distance of living place, and involvement in other activities except studying. these factors led to lack of discipline and behaviors such as delay, absence, and making teachers present intensive courses ; thus, tiredness and drowsiness are the consequence. due to the matrimony of some students and being away from their home town, they make other students and teachers have an intensive course and also they were absent in some classes. not holding certain classes, they plan not come to class and at the end they want to stop the class early, go home and do home care. they were absent from the beginning of the educational term. because of this they hold us and the teachers under pressure (student 4, 23 years old, male). attracting attention : one of the other causes of incivility is attracting attentions which could interfere with class discipline and mental distraction of the teacher and other students. these students ask some unnecessary questions, come late to class and leave early, talk, sit or behave differently teacher 7 stated : i felt this question is not related to class and students would like to present themselves in the class (51 years old, female). lack of motivation : lack of motivation is a result of lack of interest, disappointment about future career, lack of a role model in nurses and feeling of inferiority compared to other students such as medical students. low scores, coming late to class and leaving early, lack of interest and lack of attention to educational issues are signs of lack of motivation and presenting incivility. student 5 said : but unfortunately our faculty students are really limited, i.e. they would not like to learn even the information they are presented to or they study just at the night before the exam (20 years old, male). student personal quality : low understanding, unfairness, insistence on incivility and student s gender are the other causes of incivility in this category based on the participants experiences. they believe that students have not acquired an acceptable level of sociocognitive development ; therefore, incivility appears. i have taught students whose behaviors could not be comparable between the years of studies and some years later. (teacher 2, 38 years old, female) lack of experience : lack of insight, learning incivility from others, misunderstanding the teachers behaviors and words as well as lack of following rules and regulations of the class and clinical setting are signs of the students lack of experience. lack of experience causes less insight and some destructive behaviors towards students, teachers and the educational system. lack of insight causes students to ignore their lesson plan and lessons presented to them as well as not trying to be professional and not to do their homework. teacher 10 : the foresighted students value the present opportunities ; of course, such students are less found in bsc programs (34 years old, male). lack of expertise : class management is one of the necessary skills for teachers. it is both scientific ability and communication with students during the class as well as control on class atmosphere. content should be rich, because if it is poor, students will get tired, class will be monotonous and unattractive for students. inability to control the class due to lack of expertise causes less control on class, leading to high incidence of incivility. student 2 said : if a teacher could not manage his class and present his / her lessons effectively, students have to listen to his hands - free (22 years old, male). personality : the sub - category of teachers personality is breaking boundaries by teachers ; prejudices ; lack of self - confidence ; contradiction between words and behavior ; and lack of tolerance, control on themselves, sympathy and grooming. according to the participants experience, teachers specially novice ones and in some cases expert teachers had not enough self confidence. they reported some feelings such as fear of conflict ; avoidance of reminding, feeling of worthlessness, incompetency, and lack of respect from others and feeling of useless effort. teacher 5 : some time, a student had a disrespectful behavior and i tried to be silent based on my experience and i left the situation. indeed, i had no contradiction with him (35 years old, female). teachers uncivil behavior : teachers are role models of students. if they behave disrespectfully, students are to be allowed to have uncivilized behave and cause furiosity and several reactions of students which are uncivilized as well. some teachers incivility occurs when teachers do not follow islamic rules, are not updated scientifically, do not try properly to train students, have unclear objective, do not guide the students, do not rust students, do not assess students fairly, do not involve students in learning, would like to prove themselves, focus on personnel s words, stigmatize the students, speak badly, scrutinize and behave stereotypically. in an islamic society if they do not follow the islamic rules, students will not also be sensitive to follow them. student 6 talked about lack of following these rules in performing nursing procedure by his teacher. on the day 2, she said all the patients were females. we were four single male nursing students ; it is not correct to change the abdominal dressing of a young even middle age female. (student 6, 22 years old, male) subcategories are lack of assessment system for teachers, lack of understanding rules and regulations and organizational atmosphere. lack of assessment system for teachers : teachers, as the staff for training students, should evaluate scientific issues, morality and their performance both at their entrance and during their work. incivility of students could be the result of increasing faculty and lack of competent teachers, lack of motivation in teachers and a lack of integrity for observing rules and regulations and making subjective and personal judgments for incivility or even no reply to incivility. if teachers are not evaluated properly, their attention diverts from education to other aims. one of the issues for ignoring education is concentration of universities on research in the faculty evaluation system. teacher 11 said : when we talk to teachers, they say the ranking is determined based on research. therefore, it makes no difference for them to do their educational activities, get a high score from students, spend time for in the class or clinical setting or merely do research (35 years old, female). lack of understanding rules and regulations : the organization and faculty members could effectively prevent incivility by informing the newcomer students about the rules and regulations. if students do not know the educational rules, students do not know them even after the occurrence of the behavior. i had the worst experience in the previous term ; i lost 2 sessions during 12 work days. i do not know the allowed number of sessions for being absent (20 years old, male). organizational atmosphere : different organizations behave differently toward their students and teachers based on their policies. some educational organizations follow regulations strictly so students freedom is restricted and incivility will be less. on the contrary, the other organizations had a student - centered system ; this freed atmosphere and let the students behave uncivilized. teacher 5 said : in that university with student - centered system, the teachers were afraid of expressing students behavior, but in this university students get direct warning easily (35 years old, female). educational engagement : one of the main causes of incivility was non - educational engagement such as matrimony, distance of living place, and involvement in other activities except studying. these factors led to lack of discipline and behaviors such as delay, absence, and making teachers present intensive courses ; thus, tiredness and drowsiness are the consequence. due to the matrimony of some students and being away from their home town, they make other students and teachers have an intensive course and also they were absent in some classes. not holding certain classes, they plan not come to class and at the end they want to stop the class early, go home and do home care. because of this they hold us and the teachers under pressure (student 4, 23 years old, male). attracting attention : one of the other causes of incivility is attracting attentions which could interfere with class discipline and mental distraction of the teacher and other students. these students ask some unnecessary questions, come late to class and leave early, talk, sit or behave differently teacher 7 stated : i felt this question is not related to class and students would like to present themselves in the class (51 years old, female). lack of motivation : lack of motivation is a result of lack of interest, disappointment about future career, lack of a role model in nurses and feeling of inferiority compared to other students such as medical students. low scores, coming late to class and leaving early, lack of interest and lack of attention to educational issues are signs of lack of motivation and presenting incivility. student 5 said : but unfortunately our faculty students are really limited, i.e. they would not like to learn even the information they are presented to or they study just at the night before the exam (20 years old, male). student personal quality : low understanding, unfairness, insistence on incivility and student s gender are the other causes of incivility in this category based on the participants experiences. they believe that students have not acquired an acceptable level of sociocognitive development ; therefore, incivility appears. i have taught students whose behaviors could not be comparable between the years of studies and some years later. (teacher 2, 38 years old, female) lack of experience : lack of insight, learning incivility from others, misunderstanding the teachers behaviors and words as well as lack of following rules and regulations of the class and clinical setting are signs of the students lack of experience. lack of experience causes less insight and some destructive behaviors towards students, teachers and the educational system. lack of insight causes students to ignore their lesson plan and lessons presented to them as well as not trying to be professional and not to do their homework. teacher 10 : the foresighted students value the present opportunities ; of course, such students are less found in bsc programs (34 years old, male). lack of expertise : class management is one of the necessary skills for teachers. it is both scientific ability and communication with students during the class as well as control on class atmosphere. content should be rich, because if it is poor, students will get tired, class will be monotonous and unattractive for students. inability to control the class due to lack of expertise causes less control on class, leading to high incidence of incivility. student 2 said : if a teacher could not manage his class and present his / her lessons effectively, students have to listen to his hands - free (22 years old, male). personality : the sub - category of teachers personality is breaking boundaries by teachers ; prejudices ; lack of self - confidence ; contradiction between words and behavior ; and lack of tolerance, control on themselves, sympathy and grooming. according to the participants experience, teachers specially novice ones and in some cases expert teachers had not enough self confidence. they reported some feelings such as fear of conflict ; avoidance of reminding, feeling of worthlessness, incompetency, and lack of respect from others and feeling of useless effort. teacher 5 : some time, a student had a disrespectful behavior and i tried to be silent based on my experience and i left the situation. indeed, i had no contradiction with him (35 years old, female). if they behave disrespectfully, students are to be allowed to have uncivilized behave and cause furiosity and several reactions of students which are uncivilized as well. some teachers incivility occurs when teachers do not follow islamic rules, are not updated scientifically, do not try properly to train students, have unclear objective, do not guide the students, do not rust students, do not assess students fairly, do not involve students in learning, would like to prove themselves, focus on personnel s words, stigmatize the students, speak badly, scrutinize and behave stereotypically. in an islamic society if they do not follow the islamic rules, students will not also be sensitive to follow them. student 6 talked about lack of following these rules in performing nursing procedure by his teacher. on the day 2, she said we were four single male nursing students ; it is not correct to change the abdominal dressing of a young even middle age female. subcategories are lack of assessment system for teachers, lack of understanding rules and regulations and organizational atmosphere. lack of assessment system for teachers : teachers, as the staff for training students, should evaluate scientific issues, morality and their performance both at their entrance and during their work. incivility of students could be the result of increasing faculty and lack of competent teachers, lack of motivation in teachers and a lack of integrity for observing rules and regulations and making subjective and personal judgments for incivility or even no reply to incivility. if teachers are not evaluated properly, their attention diverts from education to other aims. one of the issues for ignoring education is concentration of universities on research in the faculty evaluation system. teacher 11 said : when we talk to teachers, they say the ranking is determined based on research. therefore, it makes no difference for them to do their educational activities, get a high score from students, spend time for in the class or clinical setting or merely do research (35 years old, female). lack of understanding rules and regulations : the organization and faculty members could effectively prevent incivility by informing the newcomer students about the rules and regulations. if students do not know the educational rules, students do not know them even after the occurrence of the behavior. i had the worst experience in the previous term ; i lost 2 sessions during 12 work days. i do not know the allowed number of sessions for being absent (20 years old, male). organizational atmosphere : different organizations behave differently toward their students and teachers based on their policies. some educational organizations follow regulations strictly so students freedom is restricted and incivility will be less. on the contrary, the other organizations had a student - centered system ; this freed atmosphere and let the students behave uncivilized. teacher 5 said : in that university with student - centered system, the teachers were afraid of expressing students behavior, but in this university students get direct warning easily (35 years old, female). according to the findings, incivility can be seen in students, teachers, and organizations. there are a few findings related to study s direct causes of incivility in nursing students. so, the findings were compared to the results of other studies. in the present study, causes of incivility were non - educational engagement ; attracting attention, lack of motivation, students personality, and lack of experience were student - related factors for incivility. in a study about student stressors these include obligations of the university and family, financial pressures, time management, lack of support from teachers, incivility of teachers, mental problems and personal issues. as the above study showed non - educational engagements for reasons such as family and financial pressures were the factors related to the students incivility ; this is in agreement with the present study. lack of teachers support and incivility of teachers were the other factors that led to the students incivility ; in comparison with our study that showed lack of teachers skill in management of the students behaviors including inability to communicate with students, and inability to support the students was in line with the results of the present study. students personality was one of the influential factors in incivility ; the results of the present study confirmed this. lack of motivation was of the incivility causes which was in agreement with the results of clark s study, i.e. she showed lack of interest and lack of preparedness as the signs of lack of experience influencing incivility ; it was reported that students experience high pressure and high workload which causes incivility. it seems that it is due to lack of strictness for bsc students in iran. lack of expertise, teachers personal qualities, lack of experience, and teacher s incivility were the teacher - related factors for emerging incivility. in the present study, breaking boundaries by teachers, prejudice, lack of self confidence, contradiction between words and behavior, lack of sympathy, lack of tolerance, lack of self control, and lack of sincerity were found. in a study, the feeling of superiority of the teacher was reported as a violation of moral principles which is in agreement with the findings of the present study. the important incivilities on the side of the teachers were lack of following islamic rules, lack of updating, unclear objectives, no guide for students, lack of trust on students, unfair evaluation, discrimination and humiliation of the students, threatening students, lack of attention to students, lack of attention to boundaries, trying to prove themselves, focusing on personal words, stigmatizing the students, using bad words and intonation, stereotypic behavior and lack of involving students in learning ; these were different to some extent with clark s study. in his study, incivility of the teachers from students point of view were humiliating the students, weak teaching method, poor communication skills, arrogant behavior, misusing students in the presence of colleagues, and threatening the students for drop out were mentioned. the difference was related to lack of following islamic rules and lack of students involvement in learning which could be related to cultural differences between iranian and western societies. another study supported the findings and indicated that the teachers do not know that their incivility could lead to students incivility and they make hostility between students and teachers by humiliating their students inadvertently. another study reported that in nursing education, students paid attention to teachers incivility and when teachers behave in an uncivil manner, they are affected negatively ; they get angry and unsatisfied, their communication breaks, some problems arise in their learning atmosphere and level of stress increases both in students and teachers. in another study, unclear expectations and objectives of students and teachers were reported as causes of incivility ; this was in agreement with the findings of the present study. reaction of students towards teachers incivility was feeling of injury, hopelessness and weakness ; eventually, the students experienced emotional strain and disrespectful behaviors towards teachers formed because nothing was worthwhile for them. students with higher levels of stress have negative coping strategies and experience higher burn out. lack of assessment system for teachers, and lack of understanding rules and organizational atmosphere were organization - related factors. organizational atmosphere which supports the students and teachers in preventing incivility is in agreement with hunt s study in which it is reported that organizational support causes people to become secured in organizational norms and effectively brings about organizational success. the finding of lack of understanding the rules is in agreement with the above study. it is believed that lack of rules and regulations for management of the students behavior could produce incivility in students. it should be emphasized in documenting the rules and regulations for incivility ; it is necessary to inform the teachers of these behaviors and their causes. lack of teacher evaluation as the cause of incivility was not found in other studies. in a study, it was shown that the students incivility has been attributed to causes such as the sense of entitlement among students, being unprepared for class, distrust of the faculty member as the director of the classroom. these findings show that students are not familiar with the rules of the colleges and these results are in agreement with those of the present study. limitation of this study was disregarding the grounding causes of incivility due to family and society because we assessed the incivility causes only from the teachers and students point of view. several factors contribute to students incivility. managers and teachers should recognize aforementioned factors enhancing the students incivility and try to prevent them. in addition, according to the results it is suggested that faculty members should involve the students in the education, and they should not demonstrate uncivil behaviors. also, the organization must clarify the rules and regulations ; and all of three groups including students, faculty members, and managers should be sensitive to incivility. so, it is suggested that sufficient educational programs should be held, via workshops, about the causes of these behaviors for the faculty members and managers in order to increase their knowledge about the students incivility and promote their civil behaviors. | background : incivility among nursing students is a common academic problem. knowing the causes of students incivility will enable the faculty members and academic institutions to select correct strategies to deal with this problem. this study was conducted to explore the causes of incivility among nursing students from both educators and students points of view.methods:gthis qualitative content analysis study was applied in order to explore experiences and insights of 17 nursing lecturers and 9 nursing students who were selected through purposeful sampling and interviewed on the causes of incivility. participants were selected among students and lecturers of nursing schools in khorasan razavi. the inclusion criteria for the students were having passed one educational term and for the lecturers having one year experience of teaching respectively. data gathering was done using deep semi - structured interviews starting from march 2014 to march 2015.results:three main categories extracted from the data were student related factors, teacher related factors, and organizational factors. non - educational engagement, attracting attentions, lack of motivation, students personality, and lack of experience were the subcategories of student related factors. subcategories of teacher related factors included lack of skills, teachers personal qualities, lack of experience, and incivility of teachers. finally, the subcategories of organizational factors included no evaluation system for teachers and lack of understanding the organizational rules and regulations.conclusion:the results of this study indicated that factors related to students, teachers, and organization may lead to nursing students incivility and clarified its dimensions. in order to develop a civil environment in nursing college, managers and educators awareness should be promoted via various ways such as workshops. |
several modalities for the treatment of patients with prostate cancer have become well established, including radical prostatectomy, radiation therapy and androgen deprivation therapy. both radical prostatectomy and external - beam radiation therapy are considered equivalent options in patients with non - metastatic disease, with different risk profiles (1, 2). the prognostic risk group, based on tumor staging, pre - treatment prostate - specific antigen (psa) levels and gleason score, has been shown to predict survival and treatment outcomes (3). for patients with low - risk localized disease, the 10-year mortality may be as low as 2%. however, patients with intermediate- and high - risk disease have a worse prognosis, with mortality rates ranging from 12% to 40% (3, 4). adjuvant and/or neoadjuvant androgenic suppression has been studied in an attempt to improve outcomes in intermediate- and high - risk non - metastatic prostate cancer (5). several randomized controlled trials (58) and systematic reviews (9, 10) have reported an improved disease - free and overall survival for radiotherapy combined with androgen blockade compared to radiotherapy alone. combined treatment with androgen deprivation and radiotherapy has also been shown to be superior to hormonal blockade alone (11, 12). the type of hormonal blockade (central, peripheral, or combined) combined to radiation therapy ; the timing of treatment (neoadjuvant, adjuvant or both) and duration of deprivation have all varied among trials. while hormonal blockade with gonadotro - pin - releasing hormone (gnrh) agonists has been shown to improve outcomes (9), peripheral blockade with bicalutamide has not (13). there is evidence that short - term androgen deprivation may also have some benefit in the neoadjuvant setting, both reducing positive margins in patients treated with radical prostatectomy and improving survival in patients treated with radiation therapy (10). androgen deprivation with gonadotropin releasing agents is already a standard of care for intermediate and high - risk localized or locally advanced prostate cancer treated with radiation therapy, but the optimal duration of hormonal deprivation is yet to be defined (10). establishing a standardized treatment may be important because while hormone deprivation improves survival, it is also associated with aes like loss of bone density, erectile dysfunction, hot flashes, and increased cardiovascular risk (14, 15). two large randomized controlled trials (16, 17) have suggested that a longer course of androgen deprivation (2 to 3 years) may improve outcomes in high - risk patients compared to 6-month adjuvant therapy. however, there is no consensus of how much longer the adjuvant treatment should be, with a recent trial suggesting that adjuvant hormonal treatment could be safely reduced from 36 months to 18 months (18). considering the conflicting conclusions of individual trials, we conducted a systematic review with meta - analysis to critically evaluate the existing evidence to support an indication of an optimal strategy for androgen deprivation to combine with external beam radiation therapy in patients with intermediate- or high - risk non - metastatic prostate cancer. a systematic search was performed in electronic databases, including pubmed / medline, embase, lilacs, clinicaltrials.gov, the cochrane library, and asco meetings abstracts. reports of results from prospective randomized clinical trials comparing different durations of androgen blockade were selected for the meta analysis. studies evaluating only radiotherapy plus hormonal blockade versus radiotherapy alone were excluded, even if subgroup analysis data comparing different androgen deprivation durations has been published. trials comparing hormonal blockade alone versus radiation therapy (with or without associated androgen deprivation) were also excluded. the shorter duration of hormonal therapy in each trial was considered its control arm, regardless of what arm was considered control in the original report. disagreements were resolved by discussion and consensus. overall survival (os), disease - free survival (dfs), disease - specific survival (dss), and toxicity were the outcomes of interest. the reported hazard ratios (hr) were used as a measure of survival benefit. for the articles in which the hr was not reported, we calculated estimates by transcription of the survival curves presented as figures in original articles and calculation with a spreadsheet developed by tierney. information about randomization, blinding, allocation concealment, drop - outs, intention - to - treat (itt) analysis, and funding source was especially evaluated. eligibility criteria for the studies were also assessed for differences in patient populations among the trials. meta - analyses were conducted with revman 5.0 software (cochrane collaboration 's information management system). analyses of data consisted of the hr for time - to - event outcomes, considering a random effect model. the 95% con - fidence intervals (ci) were calculated and presented in forest plots. the diamond at the bottom of the plot summarizes the best estimate results (with the width representing its corresponding 95% ci). statistical heterogeneity was evaluated with the chi - square test, and expressed using the i index (20). if significant heterogeneity (i index > 50%) was identified, a possible explanation was investigated. differences in eligibility criteria, patient population, and treatment delivered were assessed and discussed. from 127 potential studies identified through the search in databases, 11 articles met the inclusion criteria. of these, all but one (21) clearly reported pre - established sample size calculation, with alpha and beta errors defined. one trial performed conventional external beam radiation therapy, while all the others used 3d conformal radiation. four studies recruited patients with both intermediate- and high - risk non - metastatic prostate cancer ; one study also recruited low - risk patients ; and one recruited only high - risk prostate cancer patients. two studies recruited patients in the neoadjuvant setting, while four trials were performed only in the adjuvant indication. the eortc 22961 (bolla.) performed androgen deprivation with triptorelin, while all other trials used goserelin. hormone suppression duration was highly variable among the trials, both in the control and experimental arms. for that reason, we clas - sified the trials in three subgroups, according to the duration of blockade. two studies compared a short - term blockade of less than 12 months with a long - term blockade of more than 12 months --these studies were classified as the long vs. short blockade subgroups. in two studies, both arms had duration of less than 12 months (subgroup short vs. shorter), while one study had both arms longer than 12 months blockade (subgroup longer vs. long). two studies (21, 22) reported dfs data separately for intermediate- and high - risk disease. pooled data from these two studies, stratified by risk group, is described below. five studies reported data on os (16, 17, 22, 23). only two trials presented statistically significant results favoring a longer androgen blockade. however, in our pooled analysis, we observed a significant os benefit favoring longer blockade duration, with a hazard ratio of 0.84 (95% ci, 0.74 0.96), with low heterogeneity (i = 23%). this benefit was not different among the three subgroups analyzed (long vs. short, short vs. shorter, and longer vs. long), with a negative test for subgroup differences (p=0.96). five studies reported dfs. the pooled analysis for this endpoint resulted in a statistically significant benefit favoring the longer blockade, with a hr 0.74 (95% ci, 0.62 - 0.89). this heterogeneity was mainly due to a difference in study subgroups, with a positive test for interaction (p=0.002). in the long vs short subgroup, there was a statistically significant benefit favoring the extended blockade treatment arm with a hr 0.63 (95% ci, 0.56 - 0.71) and low heterogeneity within the subgroup (i=11%). in the short vs. shorter only a non - significant trend favoring the extended hormone deprivation was observed (hr 0.86 ; 95% ci, 0.76 - 1.02), also with low internal heterogeneity (i=2%). it is important to note that the two studies held in the neoadjuvant setting demonstrated no dfs benefit, with a hr 0.96 (95% ci, 0.75 - 1.24) and no heterogeneity (i=0%). a subgroup analysis by risk stratification was performed using data from the two studies that reported dfs results separately for intermediate- and high - risk disease. pooled data from these two studies shows a non - significant bene - fit for high - risk prostate cancer patients (hr 0.8 ; 95% ci 0.56 - 1.15), with no heterogeneity between trials (i=0%). for intermediate - risk neoplasms, pooled data from these two studies also showed a non - significant trend favoring the longer blockade (hr 0.74 ; 95% ci 0.36 - 1.55). for this subgroup of patients, however, heterogeneity between these two trials results was high (i=73%). no obvious reasons were found to clearly justify this high heterogeneity between trials. disease - specific survival (dss) was also reported in four trials (1618, 23). pooled data from these have shown a statistically significant benefit for the longer hormone deprivation with a hr 0.73 (95% ci, 0.62 - 0.85). four studies reported information on toxicity (16, 17, 22, 23). none of them reported systematically the number of occurrences for each ae. therefore, a pooled analysis was not possible. also, authors of different studies reported the observed aes in different ways. data regarding cardiovascular events was reported in the rtog 92.02 and eortc 22961 trials, and neither found a statistically significant difference in cardiac events. five studies reported data on os (16, 17, 22, 23). only two trials presented statistically significant results favoring a longer androgen blockade. however, in our pooled analysis, we observed a significant os benefit favoring longer blockade duration, with a hazard ratio of 0.84 (95% ci, 0.74 0.96), with low heterogeneity (i = 23%). this benefit was not different among the three subgroups analyzed (long vs. short, short vs. shorter, and longer vs. long), with a negative test for subgroup differences (p=0.96). the pooled analysis for this endpoint resulted in a statistically significant benefit favoring the longer blockade, with a hr 0.74 (95% ci, 0.62 - 0.89). this heterogeneity was mainly due to a difference in study subgroups, with a positive test for interaction (p=0.002). in the long vs short subgroup, there was a statistically significant benefit favoring the extended blockade treatment arm with a hr 0.63 (95% ci, 0.56 - 0.71) and low heterogeneity within the subgroup (i=11%). in the short vs. shorter only a non - significant trend favoring the extended hormone deprivation was observed (hr 0.86 ; 95% ci, 0.76 - 1.02), also with low internal heterogeneity (i=2%). it is important to note that the two studies held in the neoadjuvant setting demonstrated no dfs benefit, with a hr 0.96 (95% ci, 0.75 - 1.24) and no heterogeneity (i=0%). a subgroup analysis by risk stratification was performed using data from the two studies that reported dfs results separately for intermediate- and high - risk disease. pooled data from these two studies shows a non - significant bene - fit for high - risk prostate cancer patients (hr 0.8 ; 95% ci 0.56 - 1.15), with no heterogeneity between trials (i=0%). for intermediate - risk neoplasms, pooled data from these two studies also showed a non - significant trend favoring the longer blockade (hr 0.74 ; 95% ci 0.36 - 1.55). for this subgroup of patients, however, heterogeneity between these two trials results was high (i=73%). no obvious reasons were found to clearly justify this high heterogeneity between trials. disease - specific survival (dss) pooled data from these have shown a statistically significant benefit for the longer hormone deprivation with a hr 0.73 (95% ci, 0.62 - 0.85). four studies reported information on toxicity (16, 17, 22, 23). none of them reported systematically the number of occurrences for each ae. therefore, a pooled analysis was not possible. also, authors of different studies reported the observed aes in different ways. data regarding cardiovascular events was reported in the rtog 92.02 and eortc 22961 trials, and neither found a statistically significant difference in cardiac events. despite the marked differences in andro - gen blockade duration evaluated in the trials, our pooled analysis showed an os benefit for longer androgen deprivation. such benefit was consistent between study subgroups. when considering dfs, however, the benefit observed in the pooled analysis was mostly due to the studies in the long vs short subgroup, while the short vs. shorter subgroup demonstrated only a non - significant trend toward an advantage. the unique study that compared two arms with longer - than - one - year hormone deprivation did not show any statistically significant difference in os or dss survival. however, with a hr 0.87 for os favoring the longer blockade, its results were consistent with the other studies, and contributed to a statistically significant benefit in the pooled analysis. also, the trial did not have a non - inferiority design, with no pre - specified boundary for assuming the hr as clinically irrelevant. therefore, it may not yet be considered safe to reduce the standard treatment duration from 2 - 3 years to 18 months in current clinical practice. a large, randomized, non - inferiority trial should be performed to test this approach. the two larger studies (16, 17) demonstrated quite different results with respect to os. while bolla and colleagues have reported an important benefit for the longer blockade in terms of os in the european eortc 22961 trial, horwitz and colleagues found no statistically significant difference in the american rtog 92.02 study. inclusion criteria, radiation dose, and patient characteristics were similar between the two trials, so there is no obvious explanation for such a discrepancy between their results. one should note that the european trial used a longer androgen deprivation (36 months) than the american study (24 months). patients with recurrent prostate cancer can have a relatively good overall survival, especially when compared to other recurrent or metastatic neoplasms (24). also, prostate cancer patients tend to be elderly and many of them have important comorbidities and competitive risks of death (25). for these reasons, a benefit in terms of dfs or dss may not necessarily translate into an os benefit. when considering the two larger randomized trials, it is important to observe that they are consistent in showing a dfs and dss benefit, despite their discordant results with regard to os. in fact, dss was the endpoint with the most consistent positive results among all trials evaluated. regarding comparison between shorter duration of androgen deprivation, two studies, the trog 96.01 by denham. and the irish clinical oncology group 97 - 01 by armstrong and colleagues, reported os data from randomized trials. they demonstrated an important discrepancy in results : denham and colleagues demonstrated a dfs and os benefits while armstrong and colleagues demonstrated no benefit in any endpoint. there were, however, important differences between these trial designs. in the trog trial, patients received hormone blockade after radiation therapy, while in the irish trial, hormonal deprivation was performed in the neoadjuvant setting. though previous trials have shown benefit of hormone blockade prior to radiation therapy versus radiation therapy alone (10), there is no previous evidence suggesting that a longer neo - adjuvant blockade bears additional benefit. data from pooled analysis of the studies by crook and colleagues and armstrong and colleagues, both performed in the neoadjuvant scene, demonstrated no dfs benefit for the longer androgen deprivation. so, extending hormonal therapy before local control may not be recommended, once long - term therapy can be delivered after prostate irradiation and a longer neoadjuvant therapy could delay local control (26). the unique study that included there is no consistent evidence that adjuvant hormone therapy provides benefit in such a good prognosis subgroup, so including those patients in long - term blockade trials may not be recommended (3). most other trials have included both intermediate- and high - risk cancer patients, so it is not yet possible to establish a difference in the recommended treatment strategy between these groups. data on subgroups of the two largest trials evaluated in this systematic review (rtog 92 - 02 and eortc 22961) has not been published, leading to a reduction in the power of this analysis. the pooled data from the irish and canadian trials may not be enough to establish if intermediate - risk patients benefit from treatment as much as high - risk ones do. no prospective randomized trial has addressed the specific question of whether hormone deprivation therapy duration could be reduced in the intermediate - risk group. however, there is some evidence from subgroup analysis and retrospective studies that extending androgen blockade bears a greater benefit for high - risk patients than for the intermediate - risk ones. androgen deprivation is associated with important aes like loss of bone density, erectile dysfunction, and hot flushes (14, 27). however, hormonal therapy has been associated with increased cardiovascular risks in observational studies, including an increased risk of obesity, diabetes, myocardial infarction, and cardiac sudden death (15). however, it has not been demonstrated that hormone deprivation significan - tly increases mortality from cardiac causes, with reports from several trials demonstrating no difference in risk of cardiovascular death (8, 28, 29). in this systematic review we did n't find any evidence suggesting an increase in cardiovascular risk for patients treated with long - term adjuvant hormone deprivation. given the os benefit and generally favorable toxicity profile, extended duration adjuvant hormone blockade may be recommended after radiation therapy for intermediate- and high - risk non - metastatic prostate cancer. however, it is important to note that the evidence is based on trials using older radiation techniques. there is a need of new studies, with non - inferiority design, evaluating different durations of androgen deprivation in patients with intermediate- and high - risk non - metastatic prostate cancer receiving modern radiation therapy. | abstractobjectives : to investigate current evidence on the optimal duration of adjuvant hormone deprivation for prostate cancer treated with radiation therapy with curative intent.materials and methods : a systematic search was performed in electronic databases. data from randomized trials comparing different durations of hormone blockade was collected for pooled analysis. overall survival, disease - free survival, disease - specific survival and toxicity were the outcomes of interest. meta - analyses were performed using random - effects model.results:six studies met the eligibility criteria. for overall survival, the pooled data from the studies demonstrated a statistically significant benefit for longer hormone deprivation (hazard ratio 0.84 ; 95% ci 0.74 0.96). a statistically significant benefit was also found for disease - free survival (hazard ratio 0.74 ; 95% ci 0.62 0.89), and disease - specific survival (hazard ratio 0.73 ; 95% ci 0.62 0.85). studies with longer blockade duration arm demonstrated greater benefit. toxicity was low, with no increase in cardiovascular events.conclusions:longer duration of androgen deprivation combined to radiotherapy prolongs os, dfs and dss in patients with intermediate and high - risk non - metastatic prostate cancer. however, this evidence is based on trials using older radiation techniques, and further research of combination of androgen deprivation and new rt technologies may be warranted. |
the incidence of the coarctation of the aorta (coa) in children with congenital heart disease is 5 to 8%. most frequently, it is associated with a bicuspid aortic valve, different levels of aortic stenosis, and congenital mitral valve stenosis. the significant after - load increase affects the left ventricle (lv) in the coa, resulting in increased wall stress, compensatory lv hypertrophy, and finally lv dysfunction. life expectancy will be reduced in patients without correction due to accelerated coronary artery disease, stroke, and congestive heart failure. in adult life, some cases of the coa have been treated during childhood, some others are re - coarctations following previous transcatheter or surgical therapy, and others are missed cases of native coarctations. diagnosis is made by clinical suspicion and physical findings such as blood pressure difference between the upper and lower extremities, pulse delay in the femoral artery, and systolic murmur over the thoracic spine. different methods are employed for the treatment of the coa in adults, including surgical or percutaneous balloon angioplasty with or without stent placement. today, transcatheter approaches have been increasingly utilized, because of improved balloon and stent technology, which confers improved safety and success of these procedures. a 26-year - old male with a severe coa diagnosed by computed tomography angiography referred to our center for an attempted stent implantation. cardiac catheterization and aortography revealed a long coa after the origin of the left subclavian artery with a 60 mmhg gradient. moreover, there was a large aneurysm in the site of the coarctation (figure 1). under general anesthesia and fluoroscopic guidance, two balloon - expandable covered cheatham - platinum (cp) stents (size 18 in 44 millimeters and size 18 in 50 millimeters) were successfully implanted across the coa. a post - procedural aortography showed an excellent result, and the gradient had decreased to 8 mmhg. on follow - up multi - slice computed tomography, performed before discharge and 4 weeks after the procedure, there were no complications (figure 2). aortography, showing a long coarctation after the left subclavian artery origin with a large aneurysm multi - slice computed tomography after the procedure, illustrating the successful deployment of the stents and no residual aneurysm or coarctation the coa in adulthood is challenging and different complications even with proper treatment can occur. in these patients, associated aneurysm, near - atretic aortic isthmus, and recurrent pathology need special attention. in complex cases, surgical procedures may not be as effective as those for simple childhood coarctations. furthermore, controlling the hemostasis of large intercostal arteries is important, especially when an aneurysm is present. percutaneous techniques are promising, and their limitations are vessel dissection or aneurysm formation at the time of stent deployment and risk of restenosis. these limitations were relatively overcome with the use of covered cp stents. with the introduction of cp stents, balloon angioplasty has been mostly replaced by stenting. also, bare metal stents are less popular due to the risk of stent fracture and aneurysm formation. cp stents have rounded edges with safer placement and less injury to the native vessels, reducing risk of dissection. covered stents are indicated in concomitant aneurysm, older age, and tight coarctation and their use even in simple cases is increasing. however, a potential concern in using covered stents is related to distal embolization of the stent. with a bare stent, the problem is easier to address because the stent can be dilated without occluding any side branch in the abdominal or thoracic aorta, whereas with covered stents, important arteries may be closed by the polytetrafluoroethylene (eptfe) coverage. another issue is the need for a larger diameter sheath compared to a similar - sized bare stent. nonetheless, there are no reports of access - related complications in the previous studies. in the case presented herein, the use of a covered stent within the aneurysm was safe and effective. recent reports of patients treated with stents have demonstrated significantly lower acute complications compared to surgery and balloon angioplasty methods and better hemodynamic and imaging outcome on intermediate follow - up, although there has been more planned reintervention required. the coa in adulthood and in patients with associated aneurysm is challenging, and different complications even with proper treatment can occur. covered stents are indicated in concomitant aneurysm, older age, and tight coarctation. in this case, the use of a covered stent within the aneurysm proved safe and effective. | abstractthe coarctation of the aorta (coa) is rare in adulthood. diagnosis is made by clinical suspicion and physical findings such as blood pressure difference between the upper and lower extremities, pulse delay in the femoral artery, and systolic murmur over the thoracic spine. the coa in adulthood and in patients with associated aneurysm is challenging and different complications even with proper treatment can occur. covered stents are indicated in concomitant aneurysm, older age, and tight coarctation.a 26-year - old male with resistant hypertension due to a coa diagnosed by computed tomography angiography referred to our center for an attempted stent implantation. cardiac catheterization and aortography revealed a long coa after the origin of the left subclavian artery with a 60 mmhg gradient. moreover, there was a large aneurysm in the site of the coarctation. under general anesthesia and fluoroscopic guidance, two balloon - expandable covered cheatham - platinum stents (size 18 in 44 millimeters and size 18 in 50 millimeters) were successfully implanted across the coa with no residual gradient. on 2 years ' follow - up, the patient had no symptoms except for mild hypertension. in this patient, the use of a covered stent within the aneurysm was safe and effective. |
chronic obstructive pulmonary disease (copd) is a common chronic respiratory disease, and its prevalence rate and mortality are both increasing. the number of patients with a copd diagnosis in japan is about 210,000 ; however, there may be 5,300,000 persons with copd according to the nippon copd epidemiology study1. according to the global initiative for chronic obstructive lung disease guideline2, the objective of copd management is to relieve symptoms, prevent and treat exacerbations, improve the health status and exercise tolerance of patients, and prevent comorbidities. dyspnea is the main symptom of a chronic respiratory disease such as copd, and the activities of daily living (adl) are gradually compromised by the progression of dyspnea. patients can fall into a vicious circle in which physical activity declines, disuse syndrome occurs, and exacerbation of dyspnea follows. many persons with copd also have psychological symptoms such as depression and insecurity4,5,6. these psychological symptoms also have a marked influence on the quality of life (qol) of patients with chronic respiratory disease. therefore, the target of rehabilitation is to free patients from the vicious circle by maintaining and improving their adl, qol, and physical activity. many studies have shown that improvement in exercise tolerance, adl, and qol can be achieved by means of respiratory rehabilitation, and it is assigned a high level of evidence in copd guidelines2, 3. however, the effect of long - term respiratory rehabilitation on copd is still controversial, as the duration of intervention has generally been limited to 612 weeks in studies of rehabilitation by physiotherapists7,8,9,10. accordingly, we examined the effect of long - term rehabilitation that was performed more than once a month for 12 months by physiotherapists in patients with chronic respiratory disease. twelve patients with chronic respiratory disease were recruited from the rehabilitation program by physiotherapists at our hospital and participated in this study. nine patients had copd, two had asthma, and one had interstitial pneumonia. the patients comprised of 10 men and 2 women, with an average age of 69.8 8.1 years. lung function tests showed the following results : vital capacity (vc), 3.4 1.2 l ; % vc, 101.7 20.0% ; forced expiratory volume (fev) 1.0%, 55.6 32.7% ; and % fev in 1 s, 48.3 25.0%. all participants signed a consent form after the study procedures were explained to them in detail. this study was carried out in accordance with the declaration of helsinki and was approved by the ethics committee of jobu hospital for respiratory disease (approval no. the following outcome measures and tests were investigated and conducted : height, weight, body mass index (bmi), medical research council dyspnea scale, isometric knee extension strength, strength to body weight - bearing index (wbi), 6-min walking test, nagasaki university respiratory adl questionnaire (nradl), st george s respiratory questionnaire (sgrq), and copd assessment test (cat). the isometric knee extension strength was measured by using a handheld dynamometer (hhd ; anima corp, -tas f-1). the subjects were tested while sitting on a table with the trunk upright and the knee joint in 90 flexion. they were instructed to push against a force plate with maximal effort for approximately 5 s, and the peak torque was recorded. measurement was done twice in both legs, and the higher of the two measurements for each leg was selected for analysis. the wbi was calculated as a subject s maximum isometric muscle strength divided by the body weight. measurements were performed at the start of the intervention and after 12 months of rehabilitation. the rehabilitation program used in this study was designed for patients with severe muscle weakness and low exercise tolerance, and is expected to be continued for a long period. when patients visited the hospital, they performed knee extension exercises in the sitting position and repetitive standing to increase lower - extremity muscle strength, as well as walking, relaxation including breathing exercises, guidance on the correct breathing method, and adl practice under a physiotherapist s supervision. the patients were asked to perform the same program every day at home, except on the day of an outpatient visit. the rehabilitation program on each day the exercises and the loading amount of muscle strength were decided from the results of the first evaluation. subjects for whom standing was difficult did the knee extension exercises in the sitting position, and those who could stand up easily did repetitive standing. the walking training involved continuous walking for 10 min ; however, it was stopped if symptoms of desaturation and dyspnea became aggravated in the subjects. the frequency of performing rehabilitation ranged from twice a week to once every 2 months, depending on the subject. the findings obtained before and after the intervention were compared by using the wilcoxon signed - rank test. spearman s correlation coefficient analysis was employed to examine the relation among the changes in weight, bmi, and wbi. all statistical analyses were carried out by using spss 16.0 software for windows (spss inc., chicago, il, usa), and significance was accepted if the p - value was 12 weeks is higher than that of a short - term program according to the american college of chest physicians / american association of cardiovascular and pulmonary rehabilitation guideline3. however, there have been no reports about pulmonary rehabilitation programs used by physiotherapists lasting for > 612 weeks. chaincharn.12 reported that unsupervised training for 12 months after direct intervention by a physiotherapist for 3 months led to improvement in muscle strength, endurance, the 6-min walking distance, dyspnea, and qol, and that the effects were still maintained at 24 months ; however, the effect of direct intervention by physiotherapists for > 3 months has not been investigated before. the present study revealed that the isometric knee extension strength and wbi were both increased at 12 months compared with those at baseline. weight and bmi decreased ; however, there was no significant correlation between the changes in wbi and weight. therefore, the increase in wbi was due to an increase in the muscle strength and was not secondary to weight loss. the rehabilitation program used in this study was designed for patients with severe muscle weakness and low exercise tolerance, and is expected to be continued for a long period. when patients visited the hospital, they performed repetitive standing and breathing exercises and underwent continuous walking training, under the physiotherapist s supervision. moreover, they were asked to perform the same program every day at home, except on the day of an outpatient visit. the increase in muscle strength in the legs after this intervention is thought to be related to repetitive standing and continuous walking training. exercise tolerance was lower with this program than that achieved with treadmill and ergometer exercises in previous reports13 ; however, the exercise tolerance achieved was suitable for patients who had severe muscle weakness at the baseline evaluation, and the program resulted in an adequate improvement in muscle strength. according a meta - analysis of the prolonged effect of rehabilitation, exercise tolerance is maintained after the end of the rehabilitation program14. in the present study, many patients also showed improvement in or maintenance of the 6-min walking distance 12 months later. the results of the nradl, sgrq, and cat showed the same trend. therefore, our long - term intervention program can be considered effective for maintaining exercise tolerance, adl, and qol in patients with chronic respiratory disease. beckerman.15 reported that during inspiratory muscle training for 1 year in patients with significant copd, there was an increase in exercise capacity, improvement in the qol, and decrease in dyspnea. however, the contents of the program in that study were different from those in our study. the results of the study by beckerman.15 showed that improvement in the breathing function contributes to the improvement in movement ability. furthermore, the results of this research showed that an intervention for increasing leg muscle strength and exercise tolerance directly leads to those improvements. the program used in this study included whole - body movements such as standing and walking, which are among the usual movements in daily living. patients with chronic respiratory disease often have dyspnea and inability to perform these movements. therefore, a program including daily - life movements is easy to implement in the rehabilitation of elderly patients with chronic respiratory disease, and can become a motivation for treatment. an easy - to - implement program that does not require a tool can also be continued easily at home, which may be a factor for improvement. after the initial period of rehabilitation, only a few patients continued to perform rehabilitation by themselves at home because many of them had no exercise habit or motivation to perform exercise. to deal with this problem, patients should be directly involved in long - term ongoing respiratory rehabilitation, even if the frequency of intervention is decreased. in this study, we evaluated the effect of rehabilitation through a comparative evaluation of patients before intervention and after 12 months of intervention. considering the chronic course of copd, a long - term clinical trial should be designed to assess the protective effect of ongoing pulmonary rehabilitation on the adl and qol covering several years. | [purpose ] the purpose of this study was to examine the effect of 12-month rehabilitation with low loading program on chronic respiratory disease. [subjects and methods ] twelve patients with chronic respiratory disease participated in this study, in which the effect of long - term rehabilitation for 12 months was assessed. nine patients had chronic obstructive pulmonary disease, two had asthma, and one had interstitial pneumonia. in all patients, symptoms, lower - extremity strength, walking distance, activities of daily living, and quality of life were investigated to examine the effect of respiratory rehabilitation. [results ] after 12 months, the isometric knee extension strength and weight - bearing index both showed a significant increase. [conclusion ] the findings of this study suggested that improvement in lower - limb muscle strength can be achieved through long - term intervention, and indicated the validity of repetitive standing and walking exercises. |
dental implants have improved the quality of life for many patients. currently titanium and titanium alloys are most widely used as dental implants due to their excellent biocompatibility, good mechanical properties, and long term follow - up in clinical success. even though titanium is a popular material, it has certain disadvantages such as greyish color, which is unaesthetic, especially in the anterior region where the gingival tissue is considerably thin. some studies have also reported of galvanic reaction that occurs after it comes in contact with saliva and fluoride. inflammatory response and bone resorption were also found to be induced due to titanium particles. in the last few years, zirconia dental implant has emerged as an alternative for titanium implant due to its potential to osseointegrate and having other beneficial properties like its translucency and white color which mimics the natural teeth. it is radiopaque similar to titanium and can be easily visualized on the radiograph. bacterial colonization around zirconia some studies have reported that zirconia has more biocompatibility as compared to titanium, as the latter produces corrosion products at the implant site. this review of literature aims to discuss various properties of zirconia like osseointegration, biocompatibility, and less bacterial colonization, which make it a biomaterial suitable to be used as dental implant, and tries to find out whether the researches done till date authenticate its use. literature search was done from 1975 to 2015 in pubmed database regarding mechanical properties, osseointegration, biocompatibility, soft tissue response, and antibacterial adhesion properties of zirconia. keywords used in literature search were zirconia dental implant, zirconia and osseointegration, zirconia and soft tissue response, zirconia and biocompatibility. abstract were screened and full texts of potentially eligible articles were obtained. all articles on surface coating of zirconia on implant surfaces, are excluded from the review. all of these papers included in - vitro studies, in - vivo studies and case reports. yttria - stabilized tetragonal zirconia polycrystalline (y - tzp) materials exhibit superior corrosion and wear resistance, as well as a high flexural strength (8001000 mpa) compared to other dental ceramics [table 1 ]. it was also found that flexural strength of zirconia increases by mechanical modification of its surface. when the compressive strength of blade type of zirconia implants was tested, it was found to be adequate in occlusion. fracture strength (512.9 n) of unloaded zirconia was found to be more than the fracture strength (401.7 n) of loaded zirconia [table 1 ]. a study performed by kohal. found low fracture strength of two - piece zirconia implants in both loaded and unloaded conditions, it was also found that the implant preparation and cyclic loading decrease the fracture strength of one - piece zirconia implants, but these values were still within clinically acceptable limits to withstand average occlusal forces even after an extended interval of artificial loading. whereas silva. reported in their study that crown preparation had no influence on the reliability of one - piece ceramic implant [table 1 ]. mechanical properties of zirconia zro2 is a polymorphic material and occurs in three forms : monoclinic, tetragonal, and cubic. the monoclinic phase is stable at room temperatures up to 1170c, the tetragonal at temperatures of 11702370c, and the cubic form at over 2370c. alloying pure zirconia with stabilizing oxides, such as cao, mgo, y2o3, or ceo2, allows the retention of the metastable tetragonal structure at room temperature. dental procedures, such as grinding or sandblasting, can trigger a tetragonal to monoclinic transformation in the surface region. this phase transformation results in compression of cracks, thereby retarding its growth and enhancing fracture toughness. this martensitic - like mechanism is known as transformation toughening. due to severe environmental conditions of moisture and stress, this mechanical property degradation in zirconia is known as aging of the material. the transformation is enhanced in water or in vapor, while the most critical enhancing effects of temperature occur in the range of 200300c. the transformation from tetragonal to monoclinic starts from surface and progresses to the core of the material. when the monoclinic phase dominates, it leads to reduction in strength, toughness, and density, which in turn leads to microcracking on the surface. low temperature degradation of the material involves roughening, increased wear and microcracking, grain pull - out, generation of particle debris, and premature failure. the aging process depends on various factors like porosity, residual stresses, grain size, and the content of stabilizer. it was found that decrease in grain size and increase in stabilizing oxide content reduce the transformation rate. aging is accelerated due to changes in processing technique and can be avoided by more accurate processing. some in vitro studies have found that the aging reduces the mechanical properties of zirconia, even though within clinical acceptable limits, in simulated dental treatment conditions. bone apposition takes place on different types of implant surfaces and depends on surface roughness of the implant. studies have shown that zirconia coating on the surface of titanium implants favours bone apposition, which was found to be more than that of titanium implants with no coating. akagawa., in their study, found no significant difference in bone implant contact (bic) between the loaded and unloaded zirconia implants. the bic was 81.9% for the unloaded group and 69.8% for the loaded group [table 2 ]. osseointegration of zirconia another study which examined the role of osseointegration around one - stage zirconia screw implant under various conditions for loading showed no difference in bone contact ratio among the single freestanding, connected freestanding, and implant - tooth supports of partially stabilized zirconia implants. these findings were in agreement with another study which compared the bic of submerged zirconia and non - submerged zirconia implants with submerged titanium as the control [table 2 ]. when bic of zirconia implants was compared with that of titanium and alumina, there was no statistical difference between the bic of all three types of implants. relatively bone healing around zirconia implants was found to be more than around titanium implants. some studies indicated that the zirconia implants might withstand occlusal loads over a longer period of time. similar rate of bone apposition on zirconia and surface - modified titanium implant surfaces during early healing was found when a histological examination of early bone apposition around zirconia dental implants at 2 and 4 weeks after insertion was compared to that of surface - modified titanium implants. there was no difference in osseointegration between acid - etched zirconia implants and acid - etched titanium implants. this was true even when the implant surfaces were pharmacologically and chemically modified [table 2 ]. while direct bone apposition can occur on different types of surfaces, it has been demonstrated that a certain degree of surface roughness is beneficial in accelerating bone apposition to the implant surface. since reduced treatment time is practiced more commonly in implant dentistry, the smooth surface of zirconia implants appears to be a disadvantage. a study performed to investigate the osteoblastic response to y - tzp with different surface topographies to increase the surface roughness by airborne particle abrasion and additionally acid etching showed cell proliferation with statistically significant higher values on day 3 for surface - treated zirconia as compared with machined zirconia. but no differences were found between the zirconia groups and sandblasted / acid - etched (sla) titanium at 6 and 12 days. it also found that roughening the zirconia implants enhances bone apposition and has a beneficial effect on the interfacial shear strength, which was later contradicted by hoffmann. so, recently, laser has been used to engrave a pattern on the zirconia surface. a scanning electron microscopic (sem) study done to find the influence of erbium - doped yttrium aluminium garnet (er : yag), carbon dioxide (co2), and diode laser irradiation on the surface properties of polished zirconia implants demonstrated that diode and er : yag lasers did not cause any visible surface alterations. torque removal forces have been used as a biomechanical measure of anchorage or osseointegration in which the greater forces required to remove implants may be interpreted as an increase in the strength of osseointegration. in the study of sennerby., it was found that coated zirconia implants and titanium implants showed higher removal torque value than the machined zirconia implants. the findings suggested that surface - modified zirconia implants can reach firm stability in bone. in another study wherein the removal torque values of machined zirconia implants, sandblasted zirconia implants, and acid - etched titanium implant were evaluated, machined zirconia had the least removal torque value. acid - etched titanium implants had the highest removal torque value, followed by sandblasted zirconia implants. the findings suggest that sandblasted zirconia implants can achieve a higher stability in bone than machined zirconia implants. even when the zirconia was coated on titanium implants, it increased the removal torque value. but in one of the studies that compared the biomechanical properties of six types of implant surfaces, it was found that removal torque value of zirconia implants was the least. it can be concluded that the removal of torque value of zirconia implants was improved after surface modification, but was not more than that of titanium implants. various in vitro tests were conducted on osteoblasts, fibroblasts, lymphocytes, monocytes, and macrophages to test the biocompatibility of zirconia. it was observed that zirconia had no cytotoxic effect on osteoblasts and made the cells capable of elaborating the extracellular matrix by synthesizing various essential and structural proteins. laser - modified zirconia showed better adhesion to osteoblasts due to the better wettability characteristics. wear products of zirconia could be cytotoxic as compared to titanium and other ceramics, when tested with fibroblasts. both powder and particles of zirconia tested in vitro on different cell lines (human and murine) of lymphocytes, monocytes, or macrophages did not induce high cytotoxicity or inflammation. biocompatibility tests were also conducted in vivo for zirconia, and it was found that when it was implanted in the soft tissue, it became encapsulated by a thin layer of fibrous tissue similar to that seen in the case of alumina. also, there was no cytotoxicity in the soft tissue in relation to wear products of zirconia. zirconia was also found to be biocompatible to hard tissue when tested in vivo according to the findings of a study which inserted pellets of stabilized zirconia with 6% y2o3 into the femur of monkeys. when compared with alumina, zirconia showed no difference in bone reaction. in the study by kohal., it was found that cell proliferation around zirconia was comparable to titanium, but surface modification of zirconia did not show improvement in osseointegration. biocompatibility of zirconia was also found to be good in another study conducted by gredes., in which they tested a newly created zirconia implant. studies conducted on the soft tissue response of zirconia implants [table 3 ] have reported comparable findings for both zirconia and titanium. found that the collagen fiber orientation around zirconia implants was parallel to the implant surface, similar to that of titanium. regarding the healing of soft tissue around the zirconia abutment and titanium abutment, it was reported by wellander. the distance from the peri - implant mucosa to the apical termination of the barrier epithelium for zirconia was found to be less than that of titanium. the same study also found that zirconia had less mucosal color change as compared to titanium, which was contradicted by zembic. brakel. found no significant difference in the soft tissue response around zirconia and titanium abutments., wherein zirconia and titanium implants were inserted in the extraction sites of monkeys and both implants showed same peri - implant soft tissue dimensions. soft tissue response to zirconia implants bacterial colonisation is commonly found around the natural tooth due to humid environment and constant temperature inside the oral cavity. since the microflora around implants is similar to that of natural teeth, microbial pathogens (i.e. actinobacillus actinomycetemcomitans, porphyromonas gingivalis, or prevotella intermedia) associated with periodontitis may contribute to implant failure. when zirconia was introduced in orthopedics, some studies evaluated the adhesion of oral bacteria in vitro. in a study which compared the inhibition of growth and adhesion of selected oral bacteria on titanium and zirconia, difference was found in the adhesion of some selected oral bacteria [table 4 ]. but in an in vivo study, zirconia showed significantly lesser adhesion of bacteria than titanium, which was contradicted by brakel. and egawa., who reported that the bacterial adhesion of zirconia was similar to that of titanium. bacterial colonization around zirconia implants with fewer studies on bacterial adhesion on zirconia surface, it can be concluded that plaque formation on this surface might be less. yttria - stabilized tetragonal zirconia polycrystalline (y - tzp) materials exhibit superior corrosion and wear resistance, as well as a high flexural strength (8001000 mpa) compared to other dental ceramics [table 1 ]. it was also found that flexural strength of zirconia increases by mechanical modification of its surface. when the compressive strength of blade type of zirconia implants was tested, it was found to be adequate in occlusion. fracture strength (512.9 n) of unloaded zirconia was found to be more than the fracture strength (401.7 n) of loaded zirconia [table 1 ]. a study performed by kohal. found low fracture strength of two - piece zirconia implants in both loaded and unloaded conditions, due to which it was also found that the implant preparation and cyclic loading decrease the fracture strength of one - piece zirconia implants, but these values were still within clinically acceptable limits to withstand average occlusal forces even after an extended interval of artificial loading. whereas silva. reported in their study that crown preparation had no influence on the reliability of one - piece ceramic implant [table 1 ]. mechanical properties of zirconia zro2 is a polymorphic material and occurs in three forms : monoclinic, tetragonal, and cubic. the monoclinic phase is stable at room temperatures up to 1170c, the tetragonal at temperatures of 11702370c, and the cubic form at over 2370c. alloying pure zirconia with stabilizing oxides, such as cao, mgo, y2o3, or ceo2, allows the retention of the metastable tetragonal structure at room temperature. dental procedures, such as grinding or sandblasting, can trigger a tetragonal to monoclinic transformation in the surface region. this phase transformation results in compression of cracks, thereby retarding its growth and enhancing fracture toughness. this martensitic - like mechanism is known as transformation toughening. due to severe environmental conditions of moisture and stress, this mechanical property degradation in zirconia is known as aging of the material. the transformation is enhanced in water or in vapor, while the most critical enhancing effects of temperature occur in the range of 200300c. the transformation from tetragonal to monoclinic starts from surface and progresses to the core of the material. when the monoclinic phase dominates, it leads to reduction in strength, toughness, and density, which in turn leads to microcracking on the surface. low temperature degradation of the material involves roughening, increased wear and microcracking, grain pull - out, generation of particle debris, and premature failure. the aging process depends on various factors like porosity, residual stresses, grain size, and the content of stabilizer. it was found that decrease in grain size and increase in stabilizing oxide content reduce the transformation rate. aging is accelerated due to changes in processing technique and can be avoided by more accurate processing. some in vitro studies have found that the aging reduces the mechanical properties of zirconia, even though within clinical acceptable limits, in simulated dental treatment conditions. bone apposition takes place on different types of implant surfaces and depends on surface roughness of the implant. studies have shown that zirconia coating on the surface of titanium implants favours bone apposition, which was found to be more than that of titanium implants with no coating. akagawa., in their study, found no significant difference in bone implant contact (bic) between the loaded and unloaded zirconia implants. the bic was 81.9% for the unloaded group and 69.8% for the loaded group [table 2 ]. osseointegration of zirconia another study which examined the role of osseointegration around one - stage zirconia screw implant under various conditions for loading showed no difference in bone contact ratio among the single freestanding, connected freestanding, and implant - tooth supports of partially stabilized zirconia implants. these findings were in agreement with another study which compared the bic of submerged zirconia and non - submerged zirconia implants with submerged titanium as the control [table 2 ]. when bic of zirconia implants was compared with that of titanium and alumina, there was no statistical difference between the bic of all three types of implants. relatively bone healing around zirconia implants was found to be more than around titanium implants. some studies indicated that the zirconia implants might withstand occlusal loads over a longer period of time. similar rate of bone apposition on zirconia and surface - modified titanium implant surfaces during early healing was found when a histological examination of early bone apposition around zirconia dental implants at 2 and 4 weeks after insertion was compared to that of surface - modified titanium implants. there was no difference in osseointegration between acid - etched zirconia implants and acid - etched titanium implants. this was true even when the implant surfaces were pharmacologically and chemically modified [table 2 ]. while direct bone apposition can occur on different types of surfaces, it has been demonstrated that a certain degree of surface roughness is beneficial in accelerating bone apposition to the implant surface. since reduced treatment time is practiced more commonly in implant dentistry, the smooth surface of zirconia implants appears to be a disadvantage. a study performed to investigate the osteoblastic response to y - tzp with different surface topographies to increase the surface roughness by airborne particle abrasion and additionally acid etching showed cell proliferation with statistically significant higher values on day 3 for surface - treated zirconia as compared with machined zirconia. but no differences were found between the zirconia groups and sandblasted / acid - etched (sla) titanium at 6 and 12 days. it also found that roughening the zirconia implants enhances bone apposition and has a beneficial effect on the interfacial shear strength, which was later contradicted by hoffmann. so, recently, laser has been used to engrave a pattern on the zirconia surface. a scanning electron microscopic (sem) study done to find the influence of erbium - doped yttrium aluminium garnet (er : yag), carbon dioxide (co2), and diode laser irradiation on the surface properties of polished zirconia implants demonstrated that diode and er : yag lasers did not cause any visible surface alterations. torque removal forces have been used as a biomechanical measure of anchorage or osseointegration in which the greater forces required to remove implants may be interpreted as an increase in the strength of osseointegration. in the study of sennerby., it was found that coated zirconia implants and titanium implants showed higher removal torque value than the machined zirconia implants. the findings suggested that surface - modified zirconia implants can reach firm stability in bone. in another study wherein the removal torque values of machined zirconia implants, sandblasted zirconia implants, and acid - etched titanium implant were evaluated, machined zirconia had the least removal torque value. acid - etched titanium implants had the highest removal torque value, followed by sandblasted zirconia implants. the findings suggest that sandblasted zirconia implants can achieve a higher stability in bone than machined zirconia implants. even when the zirconia was coated on titanium implants, it increased the removal torque value. but in one of the studies that compared the biomechanical properties of six types of implant surfaces, it was found that removal torque value of zirconia implants was the least. it can be concluded that the removal of torque value of zirconia implants was improved after surface modification, but was not more than that of titanium implants. various in vitro tests were conducted on osteoblasts, fibroblasts, lymphocytes, monocytes, and macrophages to test the biocompatibility of zirconia. it was observed that zirconia had no cytotoxic effect on osteoblasts and made the cells capable of elaborating the extracellular matrix by synthesizing various essential and structural proteins. laser - modified zirconia showed better adhesion to osteoblasts due to the better wettability characteristics. wear products of zirconia could be cytotoxic as compared to titanium and other ceramics, when tested with fibroblasts. both powder and particles of zirconia tested in vitro on different cell lines (human and murine) of lymphocytes, monocytes, or macrophages did not induce high cytotoxicity or inflammation. biocompatibility tests were also conducted in vivo for zirconia, and it was found that when it was implanted in the soft tissue, it became encapsulated by a thin layer of fibrous tissue similar to that seen in the case of alumina. also, there was no cytotoxicity in the soft tissue in relation to wear products of zirconia. zirconia was also found to be biocompatible to hard tissue when tested in vivo according to the findings of a study which inserted pellets of stabilized zirconia with 6% y2o3 into the femur of monkeys. when compared with alumina, zirconia showed no difference in bone reaction. in the study by kohal., it was found that cell proliferation around zirconia was comparable to titanium, but surface modification of zirconia did not show improvement in osseointegration. biocompatibility of zirconia was also found to be good in another study conducted by gredes., studies conducted on the soft tissue response of zirconia implants [table 3 ] have reported comparable findings for both zirconia and titanium. found that the collagen fiber orientation around zirconia implants was parallel to the implant surface, similar to that of titanium. reported that zirconia had similar probing depth as titanium. regarding the healing of soft tissue around the zirconia abutment and titanium abutment, it was reported by wellander. the distance from the peri - implant mucosa to the apical termination of the barrier epithelium for zirconia was found to be less than that of titanium. the same study also found that zirconia had less mucosal color change as compared to titanium, which was contradicted by zembic. brakel. found no significant difference in the soft tissue response around zirconia and titanium abutments., wherein zirconia and titanium implants were inserted in the extraction sites of monkeys and both implants showed same peri - implant soft tissue dimensions. bacterial colonisation is commonly found around the natural tooth due to humid environment and constant temperature inside the oral cavity. since the microflora around implants is similar to that of natural teeth, microbial pathogens (i.e. actinobacillus actinomycetemcomitans, porphyromonas gingivalis, or prevotella intermedia) associated with periodontitis may contribute to implant failure. when zirconia was introduced in orthopedics, some studies evaluated the adhesion of oral bacteria in vitro. in a study which compared the inhibition of growth and adhesion of selected oral bacteria on titanium and zirconia, difference was found in the adhesion of some selected oral bacteria [table 4 ]. but in an in vivo study, zirconia showed significantly lesser adhesion of bacteria than titanium, which was contradicted by brakel. and egawa., who reported that the bacterial adhesion of zirconia was similar to that of titanium. bacterial colonization around zirconia implants with fewer studies on bacterial adhesion on zirconia surface, it can be concluded that plaque formation on this surface might be less. limited amount of research on zirconia proves that zirconia is biocompatible with the surrounding tissues. zirconia is osseoconductive as reported in some studies and has also shown favourable interaction with the soft tissue. it has been found that zirconia reduces plaque formation on the implant surface, which leads to good healing and successful implant treatment. most of the studies on zirconia implants are short - term studies and evidence of success in long - term clinical trials is lacking. more research is needed on zirconia dental implants before we could use it for frequent treatment needs, as compared to titanium implants. | background : titanium has been the most popular material of choice for dental implantology over the past few decades. its properties have been found to be most suitable for the success of implant treatment. but recently, zirconia is slowly emerging as one of the materials which might replace the gold standard of dental implant, i.e., titanium.materials and methods : literature was searched to retrieve information about zirconia dental implant and studies were critically analyzed. pubmed database was searched for information about zirconia dental implant regarding mechanical properties, osseointegration, surface roughness, biocompatibility, and soft tissue health around it. the literature search was limited to english language articles published from 1975 to 2015.results:a total of 45 papers met the inclusion criteria for this review, among the relevant search in the database.conclusion:literature search showed that some of the properties of zirconia seem to be suitable for making it an ideal dental implant, such as biocompatibility, osseointegration, favourable soft tissue response and aesthetics due to light transmission and its color. at the same time, some studies also point out its drawbacks. it was also found that most of the studies on zirconia dental implants are short - term studies and there is a need for more long - term clinical trials to prove that zirconia is worth enough to replace titanium as a biomaterial in dental implantology. |
subjective tinnitus is characterized by the conscious perception of a sound in the absence of a corresponding physical source. this auditory phantom sound is usually described as a pure tone, a hissing, or a roaring noise. most of the people suffering from tinnitus report that the tinnitus sound is an ongoing and continuous perception which is typically more prominent in silent environments. resting state measures in a silent environment should therefore be a useful tool to investigate the aberrant brain activity associated with the tinnitus. comparison of resting brain activity of tinnitus patients and healthy controls under silent conditions should reveal the abnormal brain activity that is related to both tinnitus perception and tinnitus - associated distress. tinnitus - related alterations of resting state activity have indeed been demonstrated by several studies using electroencephalographic (eeg) and magnetoencephalographic (meg) recordings. changes in auditory areas of tinnitus sufferers comprise enhanced gamma activity [14 ], enhanced slow wave activity [1, 46 ], and reduced alpha activity. the relation between these changes of spontaneous brain activity and the tinnitus has been further strengthened by longitudinal studies providing evidence that a temporary or long - lasting reduction of tinnitus symptoms is associated with a normalization of this abnormal brain activity. for instance, adamchic and colleagues showed that reduced tinnitus severity after coordinated reset treatment relates to reduced delta and gamma power in temporal areas. mller and colleagues demonstrated increased auditory alpha power following successful tinnitus reduction with repetitive transcranial magnetic stimulation (rtms) and normalization of the delta and alpha power by means of neurofeedback has been shown to reduce tinnitus symptoms [10, 11 ]. in the current study, we sought to further investigate alpha activity in auditory areas of subjects with chronic tinnitus perception. it has been hypothesized that the oscillatory activity of the alpha frequency range (812 hz) in sensory regions of the human brain might represent a gating mechanism for incoming information that is not relevant to the subject and is therefore actively suppressed [12, 13 ]. it has been proposed that this reduced alpha power reflects a state of desynchronized neuronal networks, which is associated with auditory attention [14, 15 ]. in a normal hearing participant, this desynchronized state would be temporary and would enable neuronal couplings driven by correlated stimulus attributes. in tinnitus, a study on visual attention demonstrated an increase of alpha power in parietooccipital regions ipsilateral to the attended side together with a decrease of alpha power at the contralateral side. furthermore, thut and colleagues showed that the amount of this prestimulus hemispheric alpha lateralization is correlated with the reaction time of the participants. in a somatosensory discrimination task, haegens. were able to demonstrate that prestimulus alpha lateralization is associated with the cued target location and the reliability of the cue. in a cross - modal paradigm in which subjects had to switch between visual and auditory targets, mazaheri and colleagues recently showed that the prestimulus alpha activity switches between the respective sensory brain areas depending on the stimulus modality. taken together, all these results indicate an involvement of alpha oscillations in the active suppression of irrelevant and potentially distracting sensory information. this view on alpha activity is complemented by another line of research investigating spontaneous fluctuation of cortical alpha activity. romei and colleagues measured the spontaneous alpha fluctuation over visual areas and applied transcranial magnetic stimulation to evoke phosphenes. trials with a conscious perception of the phosphene were associated with reduced visual alpha activity in the visual cortex which suggests an enhanced excitability of the visual cortex during moments with low alpha power. similarly, another study showed that the detection of visual stimuli near the perceptual threshold was more reliable during episodes of alpha desynchronisation than during episodes of alpha synchronisation. our knowledge about the underlying neuronal mechanisms for these spontaneous fluctuations in the resting state is currently still relatively limited. nevertheless, evidence is accumulating that the variability of brain activity from one moment to the other is of functional importance for the central nervous system. measuring the variability of the blood oxygen level - dependent (bold) signal, garrett and colleagues demonstrated that the moment - to - moment variability increases when participants are engaged in a task rather than being in the resting state. furthermore, participants that are performing the task faster than average usually show larger variability of their bold signals. in another study investigating the variability of eeg phase synchronization in children with acute traumatic brain injury (tbi), nenadovic. reported that children with greater variability have higher chances for recovery from tbi. measurements of brain signal variability in other pathologies like alzheimer 's disease and autism also show large reductions, when patients are compared with healthy control groups. a conceptual framework for these findings is provided by the notion that variability in cortical processing is an essential feature of learning. according to this theory cortical map expansion occurs during learning processes for increasing processing capacities in order to enable replication with variation. analogous to a darwinian mechanism the behaviourally most useful circuit is then selected and consolidated. taken together, it can be hypothesized that disease - related alterations of neuronal plasticity should be reflected by alterations in the variability of neuronal activity. reduced variability may indicate a reduction of the dynamic range of brain response and impaired neuroplastic capacity. here we aimed to investigate whether the moment - to - moment variability of auditory alpha activity in tinnitus is altered as compared to controls. for this purpose, we used magnetoencephalographic recordings in the resting state and compared signal variability between subjects with chronic perception of tinnitus and healthy controls. our hypothesis was that the variability of auditory alpha activity is reduced in individuals with tinnitus. data of 42 subjects who participated in different studies [2831 ] were analysed retrospectively. twenty - one of the participants reported an ongoing perception of tinnitus for more than 6 months (mean duration : 4 years ; standard deviation : 3.3 years ; range : 512 years). tinnitus - related distress was measured with the german version of tinnitus questionnaire average distress of 21.5 points (standard deviation : 16.8 ; range : 359). the mean age of the tinnitus group was 44.4 years (standard deviation : 14.8 years ; range : 2269 years ; 6 female). the data of the tinnitus group was compared to an age- and gender - matched healthy control group (mean age : 43.7 years ; standard deviation : 15.3 years ; range : 2269 years ; 6 female). all participants gave informed written consent and the study was approved by the ethical review board of the university of konstanz. spontaneous brain activity was recorded using a whole - head meg system with 148 magnetometer (magnes tm 2500 wh, 4d neuroimaging, san diego, usa) at a sampling rate of 678.17 hz or 2034.51 hz and a hard - wired high pass filter of 0.1 hz. for data analysis, all data were downsampled to 600 hz. participants were instructed to relax in supine position with eyes open and fixating a point at the ceiling of the measuring chamber and not to engage in any deliberate mental activity. prior to data analysis, the meg channel positions were realigned towards standard magnetometer positions for each individual subject. a discrete fourier transform (dft) filter the continuous data set was cut into epochs of two seconds and those epochs containing artefacts were manually excluded from data analysis. following the artefact correction, we selected randomly a number of 90 epochs (i.e., 180 seconds of resting state data) from the remaining trials in order to make sure that the same amount of meg data was used for all subjects. we calculated the time - frequency representation of the spontaneous recordings from 1 to 100 hz with an increment of 0.5 hz. for each of the 90 trials, the meg data was multiplied with a hanning window before applying the fast fourier transform. in order to measure the moment - to - moment variability of alpha power, we calculated the coefficient of variation (cv) across the 90 trials for each individual data set and each sensor. the coefficient of variation for the frequency f and the trial t is defined as the ratio of the standard deviation f, t to mean f, t : (1)cvf, t=f, tf, t. thereby, the coefficient of variation expresses an unbound measure of the variability which is independent from the magnitude of the mean. statistical analyses including the mixed models analysis of variance (anova) were carried out using the open source r statistical software package available at http://www.r-project.org/ including the nlme library. comparison between the tinnitus and the control group was performed with a mixed models anova allowing a random intercept for each participant. in the first step of the analysis, we intended to reproduce the finding of reduced alpha activity in chronic tinnitus as proposed by previous studies. while the study by weisz. only investigated the reduction in the source space, figure 1(a) shows the normalized power spectrum for the tinnitus and the control group averaged over all sensors. in figures 1(b)1(d) we illustrate the topographical map for each group and the group difference. for the analysis of the spectral power over auditory regions, regions of interest (roi) were defined in order to cover the brain regions showing the strongest difference in alpha desynchronization between the tinnitus and the control group. therefore, the roi selection was based on the group difference in the 810 hz frequency range (figure 1(b)). spectral power of the left and the right temporal areas were averaged for the analysis. the analysis was done for the lower alpha band (810 hz) and the upper alpha band (1012 hz) separately. a linear mixed models analysis of variance (anova) for the lower alpha band revealed a significant group difference with f(1,40) = 9.58 and a p value of p = 0.0036. the anova for the upper alpha band revealed with f(1,40) = 3.86 and p = 0.056 a trend towards significance. the group differences for the lower and upper alpha power are depicted in figures 2(a) and 2(b). in order to analyse the variability of alpha power, we calculated a spectral analysis for each single trial. to illustrate the alpha variability we selected an example control subject and plotted the temporal alpha power for all 90 trials in figure 3. please note that this graph does not necessarily show a continuous timeline of temporal alpha activity since some trials in between might have been rejected during artifact correction and only 90 trials have been randomly selected. it rather demonstrates the relatively large variability of alpha activity from one trial to the other. in order to measure this variability, coefficients of variation (cv) statistical comparison of the cv in both groups indicated a strong reduction of temporal alpha variability in the tinnitus group. the analysis was done for the lower alpha band (810 hz) and the upper alpha band (1012 hz) separately. a linear mixed models analysis of variance (anova) for the lower alpha band revealed a significant group effect with f(1,40) = 9.04 and a p value of p = 0.004. for the upper alpha band, the group difference was not significant with f(1,40) = 1.90 and p = 0.18. further analysis of the alpha variability in the tinnitus group suggested a nonlinear relationship between the variability and the duration of the tinnitus. in subjects with a shorter duration of tinnitus we measured larger alpha variability than in subjects with longer tinnitus duration (see figure 5). a nonlinear function was fitted to the data explaining the auditory alpha variability by 1/tinnitus duration + a, with an estimate for a of 2.38 (t = 6.28, p 0.7) or the age of the subjects (p > 0.2). in the current study, we were able to repeat previous results by demonstrating reduced alpha activity in temporal regions in people with tinnitus as compared to healthy controls. in addition to former studies we differentiated in the current analysis between lower and higher alpha power and found evidence that this power reduction is more pronounced in the lower alpha frequency range (810 hz) than in the upper alpha frequency range (1012 hz). furthermore, we were able to show that the moment - to - moment variability of auditory alpha activity is significantly decreased in chronic tinnitus subjects. again, this effect was more prominent in the lower than in the upper alpha frequency range. moreover, alpha variability was more reduced in patients with a longer history of tinnitus. oscillatory activity in the alpha frequency range can be detected in all sensory areas and is by far the strongest oscillation that can be observed in the human brain [5, 15, 1719 ]. it has been shown that episodes with enhanced alpha activity in sensory areas are characterized by reduced excitability in the respective sensory modality, while episodes with low alpha activity (i.e., alpha desynchronization) are associated with enhanced neuronal excitability of this area [20, 21, 36, 37 ]. in this context, electro- und magnetoencephalographic recordings of auditory activity over sensory areas can be interpreted as a measurement of a neuronal mechanism gating sensory information processing. increases in alpha power recordings can therefore indicate suppression of sensory input that is currently not needed or even distracting, while reductions of alpha power suggest increased excitability of the sensory area for a more precise perception of potentially important sensory input. the link between enhanced neuronal excitability and reduced alpha power is currently not well established. recently, it has been shown that the locally enhanced neuronal excitability can be also characterized by increased functional coupling with remote brain areas (; see also weisz and obleser for a theoretical framework), meaning that the respective sensory area is ready to receive information from distant brain regions via already established functional connections. therefore, it might be that the alpha desynchronization is just one indicator of the enhanced neuronal excitability ; the integration in a distributed brain network might be another. how this state of enhanced excitability in the auditory areas is triggered in tinnitus remains a debate. several explanations are possible and might also depend on individual patient characteristics : (1) top - down attention to the auditory stream might trigger this state (e.g., in patients that routinely check if their tinnitus is still there), (2) a mismatch between the auditory phantom perception and the environment without a physical source for it might enhance the excitability in order to dissolve this mismatch, or (3) bottom - up mechanisms might also trigger regularly and/or constantly the excitability state. here, we used the reduced temporal alpha activity as a marker for the enhanced neuronal excitability. the recordings were done during resting state in a quite environment with no relevant auditory stimulation. during this resting state, we recorded strong alpha activity over temporal areas in healthy control subjects indicating reduced excitability of the auditory cortex. in tinnitus patients, however, we recorded reduced alpha activity over auditory areas indicating enhanced neuronal excitability. with this study we showed that auditory alpha activity is variable and fluctuates from one moment to the other. this result is in line with other research showing dynamic changes of brain activity under rest [22, 40, 41 ]. the dynamic change of alpha activity in sensory areas reflects thereby a variability of states with enhanced and reduced excitability. it has been hypothesized that this variability is beneficial to the system insofar as that it increases the dynamic range permitting more different responses to a broader range of incoming stimuli which finally leads to greater adaptability. the current results show that the variability of auditory alpha activity is reduced for the tinnitus group. furthermore, in recordings of tinnitus subjects with longer tinnitus duration even less variability was detected. due to the cross - sectional design of the study we can not distinguish whether the reduced variability reflects the predisposition to develop tinnitus or the consequence of tinnitus. thus, if the reduced moment - to - moment variability of brain activity represents a trait - marker reflecting reduced adaptability of the nervous system, one could conclude that only subjects with reduced alpha variability can enter the state of chronic tinnitus perceiving the phantom sound for many years. tinnitus subjects with greater variability might be able to adapt to the tinnitus, which results in a spontaneous remission of the symptoms. therefore, we do not see tinnitus subjects with great signal variability and a long history of tinnitus. if this hypothesis is true, the alpha variability should represent an indicator for spontaneous tinnitus remission. the second explanation favours neuroplastic changes of the auditory cortex as a consequence of the chronic tinnitus perception over the years. the continuous auditory phantom perception might attract the attention to the auditory stream leading to an ongoing enhancement of excitability in the auditory cortex. this long - term potentiation might lead to plastic changes in auditory areas that finally reduce the variability of auditory alpha activity in the long run. whether reduced alpha variability in tinnitus represents the predisposition or the consequence of tinnitus or in other words a trait or state marker should be addressed by longitudinal studies. this pilot study which investigates for the first time the moment - to - moment variability of oscillatory brain activity in tinnitus patients has several limitations. first, the analysis focussed on alpha activity in temporal brain areas, because the reduced alpha activity in the auditory cortex is the most robust neuroimaging finding in tinnitus. however, alterations in other frequency bands [14 ] and in other brain areas [4244 ] have been documented as well. the variability measure that we used in this study was normalized to the mean power. since we observed both reduced mean alpha power and reduced variability in the tinnitus group we can exclude that our finding of reduced variability is an artefact resulting from an increase in the mean alpha. nevertheless, the used procedure is just one possibility to quantify the variability of neuronal oscillations. in this study the control group was age- and gender - matched, but the groups were not matched for comorbidities of tinnitus like hearing loss, depression, or hyperacusis, which were not assessed in the whole study population. therefore, we can not rule out hearing loss, depression, or hyperacusis as alternative explanations for the reduced alpha variability. our study has also revealed that both the reduction of alpha power and alpha variability was mainly driven by low alpha activity (810 hz). this finding is new and somewhat unexpected and requires confirmation by investigations in independent samples. the current study supports the idea of reduced auditory alpha activity in chronic tinnitus patients. based on the concept that alpha activity reflects the level of inhibitory influence on sensory regions this finding can be interpreted as enhanced excitability of the auditory cortex in tinnitus. furthermore, we showed that the auditory alpha activity in healthy controls is dynamic and varies within the range of seconds. the moment - to - moment variability of auditory alpha in tinnitus subjects is significantly reduced with a tendency that subjects with a longer tinnitus duration show less variability. this might be an indicator for reduced adaptive potential of the auditory cortex in tinnitus patients and if confirmed by further studies has important implications for understanding the pathophysiological underpinnings of tinnitus. moreover, the reduced variability might represent a potential therapeutic target for neuromodulatory treatment approaches, for example, by auditory or brain stimulation. | subjective tinnitus is characterized by the conscious perception of a phantom sound which is usually more prominent under silence. resting state recordings without any auditory stimulation demonstrated a decrease of cortical alpha activity in temporal areas of subjects with an ongoing tinnitus perception. this is often interpreted as an indicator for enhanced excitability of the auditory cortex in tinnitus. in this study we want to further investigate this effect by analysing the moment - to - moment variability of the alpha activity in temporal areas. magnetoencephalographic resting state recordings of 21 tinnitus subjects and 21 healthy controls were analysed with respect to the mean and the variability of spectral power in the alpha frequency band over temporal areas. a significant decrease of auditory alpha activity was detected for the low alpha frequency band (810 hz) but not for the upper alpha band (1012 hz). furthermore, we found a significant decrease of alpha variability for the tinnitus group. this result was significant for the lower alpha frequency range and not significant for the upper alpha frequencies. tinnitus subjects with a longer history of tinnitus showed less variability of their auditory alpha activity which might be an indicator for reduced adaptability of the auditory cortex in chronic tinnitus. |
painful nonunion of the first metatarsophalangeal joint (mtpj) is one of the main severe complications of this surgery. the prevalence of nonunion is described between 5% and 10% across different operative techniques (e.g. plates, crossed screws, plate and compression screw). the implantation of hemicup - prosthesis has been successfully used for the hallux rigidus treatment with very promising results. to our best knowledge, we present the first case report using a hemicup - prosthesis as a salvage procedure for treatment of pseudoarthrosis of the first mtpj. we report a case about a 65-year - old male patient with pseudoarthrosis of the first mtpj, who suffered from a nonunion 12 months after a fusion made of two crossing screws (figure 1). postoperative x - rays showed a correct position of the two crossing screws and a correct position of the dorsiflexion (figure 2). the x - ray revealed a nonfusion during follow ups ; we recommended the possibility of an extracorporeal shockwave therapy but the patient refused. the blood samples which we had taken before the revision surgery did nt show any infection signs ; additionally, there was no fever or any clinical sign of infection. the patient is a high demand patient, who does sport (hiking and cycling up to 20 km) and gardening quite often. twelve months after the fusion he still had severe pain and was not satisfied at all with the result of the surgery. at this time we discussed the pros (definitive solution, a greater load - bearing capacity for our active high demand patient) and cons (a long follow - up treatment) of a revision fusion. on the other hand we informed him about the stability, function, the faster healing and mobilization of the prosthesis. our patient refused a revision because of the previous failed mtpj fusion ; for this reason we decided to use this new device. we took care that enough resection of the bone and a capsular release of the metatarsal head was made in order to achieve a dorsiflexion of more than 90 degrees intraoperative. afterwards a hemicup biopro prosthesis (biopro, farmingdale, ny, usa) 18.5 mm was implanted. intraoperative the final check up showed a dorsiflexion of 80 degrees and a good stability of the joint. he learned how to do exercises for mobilization of the great toe, which the patient continued at home. it showed a correct fitting of the prosthesis. in the clinical examination an active range of motion of 40/0/30 degrees seventeen months postoperative the patient was evaluated using the american orthopaedic foot and ankle society (aofas) hallux metatarso - interphalangeal scale and a visual pain rating scale from 1 to 10, where 0 s indicates the absence of pain and 10 indicates the worst pain imaginable. the patient rated the pain with 2 and reached 95/100 points of the aofas score at the last follow - up. the range of motion was scored 5 points because of a range of motion of the first metatarso - phalangeal joint of 60 degrees (40/0/20 degrees). our patient was very satisfied with the postoperative result of the procedure and would undergo the surgery again (figure 3). the arthritis of the first metatarsophalangeal joint is the most common disease of patients with forefoot arthritis and has a prevalence of 2 - 10% in the adulthood. as main factors in the development of the hallux rigidus are trauma, repeated microtrauma, osteoarthritis, abnormal long first metatarsal and osteochondral fractures blamed. patients with a degenerative arthrosis commonly suffer from pain in the first mtpj, limited dorsiflexion and increasing stiffness in the first toe. it can be radiographically divided into 3 grades : grade 1 (mild), grade 2 (middle) and grade 3 (severe). commonly grade 1 and 2 are treated with cheilectomy and grade 3 with arthrodesis or keller resection arthroplasty. there are a variety of possibilities of the arthrodesis of the first mtpj, but most commonly screws or a plating system are used. as main complications of the arthrodesis pseudoarthrosis, incorrect positioning of the dorsiflexion and arthrosis of the interphalangeal joint are considered. unfortunately, little data are available about the results of revision surgery in hallux rigidus patients. in case of failed fusion, the literature reveals several revision surgeries with innovative devices (e.g. plates, crossed screws, plate and compression screw) and interposition of an iliac bone graft. although not documented in literature, these techniques also show a high prevalence of complications, e.g. nonunion, shortening of the great toe and donor side morbidity after interposition of an iliac bone graft. we already use biopro first mpj (biopro) prosthesis for hallux rigidus treatment for many years. the results of these studies on primary implantation are comparable to our regarding range of motion and aofas score. non - porous coated and porous - coated implants in 5 sizes (17 mm, 18.5 mm, 20 mm, 21.5 mm, 23 mm) are available. a small plane must be removed with the oscillating saw. an excessive joint tension should be avoided, so it is important to remove enough of the bone. osteophytes should be removed to avoid an impingement and allow a normal movement of the joint. a sizer guide is used as next step in order to select the size of the implant. after finding the correct size of the implant, the implant is inserted. finally, it is important to check if the patient has a normal range of motion. postoperatively, the patient should achieve early weight bearing and should get a prompt access to physiotherapy. we conclude that the hemicup prosthesis is a new technique and option of the management of the pseudoarthosis of the first mtpj after arthrodesis. this early result of this salvage procedure is very promising. however, there must be longer follow ups and more patients would be beneficial to review the stability and long - term range of motion. | we report a case of a 65-year - old man with a painful nonunion of the first metatarsophalangeal joint (mtpj). it is one of the main severe complications of this surgery. its prevalence is described between 5% and 10% across different operative techniques. the implantation of hemicup - prosthesis has been successfully used for the hallux rigidus treatment with very promising results. in our case report, we introduce a treatment method of converting a pseudoarthrosis of the first mtpj, made of two crossing screws into a hemicup - prosthesis as a salvage procedure. this is to our best knowledge the first report using this device for treatment of pseudoarthrosis of the first mtpj. |
we and others have shown that it is possible to transfer phenotypes via microbial transplantation in antibiotic - treated animals, superseding the need for gf recipients in these types of studies, though potential problems with reproducibility and concerns of spreading antibiotic resistance genes should be acknowledged. nevertheless, gf animals still seem to be the best controlled model systems for microbial transplantation and thus, given the lack of reliable phenotype transfers, the antibiotic - treated models can not be regarded to serve as good models for discovering novel roles of gut microbiota in disease states where gut microbiota has not previously been implicated. the antibiotic - treated recipients should rather be considered when studying phenotype transfers in conditions already known to be associated with alterations in gut microbiota, such as obesity (table 1). when it is necessary to use gf animals, an approach where gf parents receive the microbial transplant and the subsequent offspring generations are used as study subjects is advisable to overcome the problems associated with an early gf life. when antibiotic treatment is used as an alternative to the gf state, e.g., because of limited access to certain gf mouse and rat strains, it should thoroughly be evaluated if the approach is truly applicable in the given situation. with this addendum, we identify a need for systematic experiments investigating the stability of microbial transplantations by addressing 1) the recipient status as either gf, antibiotic - treated or spf, and 2) different levels of protected housing systems. in addition, the developmental effect on host functions, in particular the immune system should be evaluated in the different recipient types. different research aims within translational microbiome research and the recommended use of either antibiotic - treated or germ - free rodent hosts for the purpose. status of hostresearch aimantibiotic - treatedgerm - freeinvestigate microbial phenotype transfer of manifestations known to be microbiota dependentxxinvestigate microbial phenotype transfer of manifestations not known to be microbiota dependent xinvestigate effect of disrupting the microbiome in certain life stages of the hostx investigate effect of targeting certain groups of bacteriax investigate effect of monocolonization xinvestigate effect of colonization with a few, defined organisms x applicability of antibiotic - treated or germ - free rodents. different research aims within translational microbiome research and the recommended use of either antibiotic - treated or germ - free rodent hosts for the purpose. randi lundberg is partly funded by innovation fund denmark and collaborates with the strategic research center 3 g (gut, grain & greens). | abstractwe recently investigated the applicability of antibiotic - treated recipient mice for transfer of different gut microbiota profiles. with this addendum we elaborate on perspectives and limitations of using antibiotics as an alternative to germ - free (gf) technology in microbial transplantation studies, and we speculate on the housing effect. it is possible to transfer host phenotypes via fecal transplantation to antibiotic - treated animals, but problems with reproducibility, baseline values, and antibiotic resistance genes should be considered. gf animals maintained in isolators still seem to be the best controlled models for long - term microbial transplantation, but antibiotic - treated recipients are also commonly utilized. we identify a need for systematic experiments investigating the stability of microbial transplantations by addressing 1) the recipient status as either gf, antibiotic - treated or specific pathogen free and 2) different levels of protected housing systems. in addition, the developmental effect of microbes on host physiological functions should be evaluated in the different scenarios. |
a 63-year - old male patient was re - admitted in our hospital for persistent atrial fibrillation (af) with left ventricular (lv) dysfunction and suspected left atrial (la) thrombus resistant to oral anticoagulation with warfarin. his first admission was 1 year before for af of recent onset with high ventricular response (ehra iii - iv) complicated by ventricular arrhythmias. during the hospitalization, coronary angiography demonstrated widespread coronary atheromasia, without hemodynamically significant lesions, the heart rate was reduced from 150 to 90/m with betablocker (carvedilol) and digoxin obtaining an improvement of ejection fraction (ef) (from 38% to 45%). four weeks later, he was checked and because of inadequate control of prothrombin time international normalized ratio (pt - inr) (2 values below 2), he underwent a three - dimensional (3d) transesophageal echocardiography (tee), which showed the presence of a spontaneous echo - contrast (sec) with a sludge effect in the la appendage (laa) that appeared enlarged and dysfunctional. in correspondence of both laa walls (more on the lateral wall) in the 80 projection, there was a thin wall thickening hardly to differentiate between a thrombus or pectinate muscles [figure 1 ]. therefore, cardioversion was again delayed with the indication to continue oral anticoagulation therapy with warfarin and repeat tee after sufficient time with pt - inr in range. transesophageal echocardiography 80 section showing the presence of spontaneous echo - contrast with sludge effect in the enlarged left atrial appendage, visible also in the left atrium (arrow). due to this echo - contrast la = left atrium, lv = left ventricle for 4 months, the next tee was postponed because of the inability to have 3 weeks of pt - inr in range. finally, when the pt - inr was in range for 3 consecutive weeks, a second tee showed again persistence of sec with sludge and suspect of thrombosis in laa. because of the difficult pt - inr control, warfarin was replaced with dabigatran 150 mg 2 cp / die and because of the persistent lv dysfunction, a third tee was planned to be eventually followed by af cardioversion. the third 2d/3d tee after 1 month of regularly intake of dabigatran therapy showed an important reduction of the sludge in the laa compared to the previous ones, with the only persistence of moderate sec [figure 2 ]. with this clear view, it was possible to exclude the presence of thrombotic formations and thanks to the 3d - analysis better identify the small immobile linear formations in the laa as pectinate muscles of the mid - distal portion [figure 3 ]. moreover, a reduced laa function was showed (max emptying rate : 22 cm / s and poor lateral wall motion). transesophageal echocardiography after 1 month of dabigatran ; 80 section showing disappearance of spontaneous echo - contrast in the enlarged left atrial appendage ; in correspondence of the lateral appendage wall, a thin wall thickening is visible hardly to differentiate between thrombus or pectinate muscles. la = left atrium, lv = left ventricle three - dimensional transesophageal echocardiography 80 section after 1 month of dabigatran without echo - contrast in the atrial appendage ; thanks to the three - dimensional analysis, it is possible to identify the small immobile linear formations in the lateral appendage wall (arrows) as pectinate muscles of the mid - distal portion. la = left atrium, laa = left atrial appendage, lupv = left upper pulmonary vein hence, electric cardioversion was performed with 100 j synchronized direct current shock, with restoration of sinus rhythm. the day after the patient was discharged with the improvement of clinical conditions and indication to continue therapy with dabigatran 150 mg. after 4 weeks, the patient was asymptomatic also for heavy exercise (nyha i), and the lvef recovered almost completely to a value of 57% at biplane simpson. in this case, we demonstrate for the first time, (1) the capability of dabigatran and not of warfarin in reducing the intense spontaneous echocontrast in laa following 6 months of therapy and (2) the ability of 3d tee in analyzing accurately the laa, after sec disappearance, and excluding laa thrombi to safely perform external cephalic version (ecv) in a patient with lv dysfunction secondary to af. the clinical importance of sec is its association with la thrombus, increased thromboembolic complications, and death. this link is stronger for dense sec as demonstrated by bernhardt in 128 patients with af and dense sec, performing serial, prospective tee, and cranial magnetic resonance imaging, and concluded that patients with dense sec have a high likelihood of cerebral embolism (28 of 128, 22%) and/or death, despite oral anticoagulation. despite effective anticoagulation (both with warfarin or direct new oral anticoagulants) while warfarin therapy is efficacious in treating la thrombus formation in patients with nonvalvular af, it does not affect red cell aggregation in vitro or la sec in patients. ito. using integrated backscatter concluded that quantitative value of sec was not changed by warfarin, whereas both studies demonstrated that patients with sec had larger la dimension and lower laa velocity. therefore, a controversial effect of warfarin is observed in reducing the risk of thromboembolism in af, without reducing la sec. the explanation for this paradox is that smoke - like echoes are influenced by various hematologic factors, such as the hematocrit and the fibrinogen concentration, but they are not affected by warfarin therapy. this is also confirmed by a case report with attenuation of la sec during antiplatelet therapy, considered to be due to the disaggregatory effect of platelets. concerning the thrombolytic effect of dabigatran in laa in several clinical cases, nagamoto. demonstrated the disappearance of thrombus in laa with dabigatran 220 mg / day in three patients > 80 years with paroxysmal and persistent af without a previous story of anticoagulation therapy ; morita. with dabigatran 300 mg / day in a 72-year - patient with permanent af (cha2ds2-vasc 5), after a little reduction of the thrombus with warfarin ; lee. with dabigatran 220 mg / day in a 72-year - patient with permanent af (cha2ds2-vasc 3), after 1 year of inadequate use of warfarin ; vidal and vanerio in a 59-year - patient with uncertain duration af, after a period with warfarin at low time in therapeutic range. our case is the first one in humans to demonstrate the disappearance of dense sec in the laa after therapy with dabigatran 150 mg twice daily and not with well - controlled warfarin, adding a supplementary effect previously demonstrated with antiaggregation therapy to the thrombolytic effect. 3d echo adds information to the analysis of cardiac structures and can depict more accurately laa thrombi ; in our patient, the real time 3d tee echo allowed an additional diagnostic capability in the differential diagnosis of suspected laa thrombi. in this patient with lv dysfunction secondary to af, we observed the disappearance of dense sec in the laa after therapy with dabigatran and not with well - controlled warfarin. this allowed us to analyze accurately the laa with 3d tee echo excluding thrombi and to perform ecv to obtain a statistically significant improvement of lv function. | while warfarin therapy is efficacious in treating left atrial (la) thrombus formation in patients with nonvalvular atrial fibrillation (af), it does not affect red cell aggregation in vitro or la spontaneous echo - contrast in patients. in this patient with left ventricular (lv) dysfunction secondary to af, we observed the disappearance of dense echo - contrast in the atrial appendage after therapy with dabigatran and not with well - controlled warfarin. this allowed us to analyze accurately the appendage with three - dimensional (3d) transesophageal echocardiography (tee) excluding thrombi and to perform electrical cardioversion to obtain a significant improvement of lv function. this case demonstrates the capability of dabigatran and not of warfarin in reducing the intense spontaneous echocontrast in atrial appendage and the ability of 3d tee in analyzing accurately the appendage, excluding thrombi to safely perform cardioversion. |
recent progress in genome sequencing has significantly widened a division within the major model organisms, some of which are genetically tractable whereas some of which are not but are valuable in other spheres of biology. in particular, the availability of the complete genome sequence in several species has allowed researchers to monitor gene transcription on a global scale for the first time, making possible an impressive leap from the study of individual genes or proteins to an integrated understanding of how gene networks enable complex functions to be carried out. the promise of such methods for systems biologists, behavioralists and neuroethologists is enormous, as they help to integrate the different biological levels from genotype to phenotype. the honeybee (apis mellifera) has an exceptional track record as a behavioral model. it has contributed greatly to our understanding of insect navigation, social behavior and learning under natural conditions, and is now poised to become a valuable model in drug and pesticide evaluation because of the ease with which behavioral changes in this species can be monitored. in contrast, the recombinant - dna - based genetic information available for the honeybee and other hymenoptera is rudimentary and, consequently, little is known about the molecular and cellular mechanisms underlying the functioning of the brain and other vital systems in this social insect. we have examined the practicability of using spotted microarrays representing several thousand cdnas amplified from a standard unannotated library to generate a catalog of genes differentially expressed during behavioral development in the honeybee. the adult life of the honeybee worker is divided into two easily accessible but behaviorally quite different stages, which offer a unique opportunity to compare molecular processes in ' naive ' individuals with those in experienced foragers. young adult bees (nurses) perform ' simple ' in - hive duties and largely depend on olfactory stimuli and colony context, whereas older bees (> 2 - 3 weeks) are engaged in complex, far - ranging foraging tasks and have strongly developed visual and olfactory perception. in contrast to experienced foragers, younger workers show no daily rhythms and, somewhat surprisingly, can not be trained in laboratory olfactory tasks until they are 6 - 7 days old. interestingly, both colony status and artificial treatments can either accelerate or delay the rate of adult development. the arrays were co - hybridized with rnas extracted from naive, newborn individuals, experienced foragers, and drug - treated and untreated 3-day - old bees. we used caffeine treatment following our finding that it accelerates the development of associative olfactory learning in newborn workers (r.m., unpublished work). the candidate clones revealed by microarray analysis were verified by northern blot hybridization, sequenced and scored against the protein and dna databases. this approach yielded a set of differentially expressed transcripts encoding conserved proteins with putative functions. additionally, several genes of unknown function that may be involved in the control and execution of adult maturation were also identified. our study shows that an analysis of a partial transcriptome can be successful in gaining insights into the dynamics of gene expression in a valuable model organism where conventional genetics is difficult to implement. the expression pattern of approximately 2,500 unique honeybee transcripts, equivalent to approximately 20% of the estimated number of genes in the honeybee genome, was analyzed using cdna probes reverse transcribed from mrnas extracted from naive and experienced honeybees. head rna represents transcripts from the brain, cephalic glands and mandibular parts, whereas abdomen rna comes largely from the digestive system, undeveloped reproductive system, the lymph - propelling organ, the sting and certain glands. data from independent co - hybridizations were analyzed to identify spots (array elements) whose gene - expression ratio differed between the two conditions. selected microarray data are shown in table 1. by comparing transcript expression levels in the heads of naive and experienced individuals we selected 24 clones showing a more than twofold difference. an additional 11 clones were selected by comparing gene expression in the abdomens (table 1). whereas honeybee behavioral development is largely controlled by genetic factors, both colony status and artificial treatments have been shown to either accelerate or delay the maturation of adult workers. we therefore examined gene expression in caffeine - treated bees following our finding that this drug accelerates the development of memory of olfactory associative learning in young workers (r.m. we used cdna probes reverse transcribed from mrnas extracted only from the heads of caffeine - treated and untreated 3-day - old bees. we selected 11 clones showing a greater than twofold change in the level of expression in treated versus untreated individuals (table 1). some of the differentially expressed cdnas from the head and abdomen detected in these two experiments were found to represent the same transcripts ; therefore, only 37 unique genes were revealed by this approach, both upregulated and downregulated, and are listed in table 1. although spotted cdna arrays can rapidly generate massive datasets, they are prone to methodological problems, such as the variability in absolute hybridization intensity owing to differences in the amount of dna deposited on the chip for various genes, the existence of alternatively spliced variants and repetitive elements, and the lack of linearity between the quantified signals and the expression levels of the corresponding genes. to ensure that the candidate transcripts identified by the microarray approach were indeed differentially expressed in naive and experienced bees, we conducted northern blot verification for 36 selected array data points. we found good correlation between the array and northern blot data, and the differential expression patterns detected by microarray were reproduced in the northern blot analyses of the selected transcripts. one notable exception is clone best129, which microarrays showed to be upregulated in heads of caffeine - treated bees, but northern blots showed to be downregulated (figure 1). to determine whether a change in the level of a particular transcript in one compartment correlates with a similar change in other organs or tissues, we examined the expression patterns of 15 confirmed candidates in all three major body compartments (head, thorax and abdomen). by adding mrna from the thorax, we expanded the range of tissues examined to include the wing and leg muscles. as shown in figure 2, this experiment gave additional information on the spatio - temporal expression of our candidate genes. for example, genes for the chaperonin hsp20 and the transcriptional regulator stck were both upregulated in the head but downregulated in the abdomen of foragers. the relative levels of mrnas in different compartments were uneven, indicating an additional level of complexity of the regulatory mechanisms controlling the expression of these genes in time and space. transcripts associated with ' functional ' glands, including those for the royal - jelly proteins (rjps) and certain enzymes, were restricted to the head, whereas -glucosidase-2 was predominantly expressed in the abdomen. however, despite the fact that the cdna library used in this study had been prepared from brain mrnas, we found that the majority of the transcripts were expressed in other body compartments. this is consistent with previous studies in other species showing that the majority of genes are expressed with little spatial specificity. in - depth analysis of the 37 clones listed in table 1 that were detected as differentially expressed by microarray analysis revealed both evolutionarily conserved and hitherto unknown genes. details of the genes, including putative identifications and functional attributes, are shown in table 1. the clones fall into three main groups. the first comprises genes encoding proteins whose functional attributes are based on large data sources from many organisms : ribosomal proteins (best : 99), metabolic enzymes (best : 22, 105, 138), developmentally regulated proteins and mitochondrial proteins (best : 54, 56), all sufficiently conserved in sequence and domain composition throughout many evolutionary lineages to be predicted with some confidence. this category also includes heat - shock cognate proteins (best : 5, 30, 31), which belong to a large, highly conserved family that includes molecular chaperones, stress proteins, signal transducers and developmental regulators. the second group comprises transcripts encoding proteins that have at least one recognizable motif or domain, but whose functional attributes are generally unclear. for example, cell adhesion molecules are particularly heterogeneous between different organisms in terms of their domain combinations, making in silico comparisons particularly problematical (best : 97). the third group comprises ' orphan ' genes that show no significant similarity to sequences deposited in genbank and are likely to represent either honeybee - specific genes or genes that have not yet been sequenced in other species. alternatively, these sequences might represent genes that encode highly divergent proteins that can not be assigned to a known functional class without a three - dimensional structure. one example of a novel gene that is significantly downregulated in experienced foragers, especially in the abdomen, is clone best123. the expression pattern of approximately 2,500 unique honeybee transcripts, equivalent to approximately 20% of the estimated number of genes in the honeybee genome, was analyzed using cdna probes reverse transcribed from mrnas extracted from naive and experienced honeybees. head rna represents transcripts from the brain, cephalic glands and mandibular parts, whereas abdomen rna comes largely from the digestive system, undeveloped reproductive system, the lymph - propelling organ, the sting and certain glands. data from independent co - hybridizations were analyzed to identify spots (array elements) whose gene - expression ratio differed between the two conditions. selected microarray data are shown in table 1. by comparing transcript expression levels in the heads of naive and experienced individuals we selected 24 clones showing a more than twofold difference. an additional 11 clones were selected by comparing gene expression in the abdomens (table 1). whereas honeybee behavioral development is largely controlled by genetic factors, both colony status and artificial treatments have been shown to either accelerate or delay the maturation of adult workers. we therefore examined gene expression in caffeine - treated bees following our finding that this drug accelerates the development of memory of olfactory associative learning in young workers (r.m. we used cdna probes reverse transcribed from mrnas extracted only from the heads of caffeine - treated and untreated 3-day - old bees. we selected 11 clones showing a greater than twofold change in the level of expression in treated versus untreated individuals (table 1). some of the differentially expressed cdnas from the head and abdomen detected in these two experiments were found to represent the same transcripts ; therefore, only 37 unique genes were revealed by this approach, both upregulated and downregulated, and are listed in table 1. although spotted cdna arrays can rapidly generate massive datasets, they are prone to methodological problems, such as the variability in absolute hybridization intensity owing to differences in the amount of dna deposited on the chip for various genes, the existence of alternatively spliced variants and repetitive elements, and the lack of linearity between the quantified signals and the expression levels of the corresponding genes. to ensure that the candidate transcripts identified by the microarray approach were indeed differentially expressed in naive and experienced bees, we conducted northern blot verification for 36 selected array data points. we found good correlation between the array and northern blot data, and the differential expression patterns detected by microarray were reproduced in the northern blot analyses of the selected transcripts. one notable exception is clone best129, which microarrays showed to be upregulated in heads of caffeine - treated bees, but northern blots showed to be downregulated (figure 1). to determine whether a change in the level of a particular transcript in one compartment correlates with a similar change in other organs or tissues, we examined the expression patterns of 15 confirmed candidates in all three major body compartments (head, thorax and abdomen). by adding mrna from the thorax, we expanded the range of tissues examined to include the wing and leg muscles. as shown in figure 2, this experiment gave additional information on the spatio - temporal expression of our candidate genes. for example, genes for the chaperonin hsp20 and the transcriptional regulator stck were both upregulated in the head but downregulated in the abdomen of foragers. the relative levels of mrnas in different compartments were uneven, indicating an additional level of complexity of the regulatory mechanisms controlling the expression of these genes in time and space. transcripts associated with ' functional ' glands, including those for the royal - jelly proteins (rjps) and certain enzymes, were restricted to the head, whereas -glucosidase-2 was predominantly expressed in the abdomen. however, despite the fact that the cdna library used in this study had been prepared from brain mrnas, we found that the majority of the transcripts were expressed in other body compartments. this is consistent with previous studies in other species showing that the majority of genes are expressed with little spatial specificity. in - depth analysis of the 37 clones listed in table 1 that were detected as differentially expressed by microarray analysis revealed both evolutionarily conserved and hitherto unknown genes. details of the genes, including putative identifications and functional attributes, are shown in table 1. the first comprises genes encoding proteins whose functional attributes are based on large data sources from many organisms : ribosomal proteins (best : 99), metabolic enzymes (best : 22, 105, 138), developmentally regulated proteins and mitochondrial proteins (best : 54, 56), all sufficiently conserved in sequence and domain composition throughout many evolutionary lineages to be predicted with some confidence. this category also includes heat - shock cognate proteins (best : 5, 30, 31), which belong to a large, highly conserved family that includes molecular chaperones, stress proteins, signal transducers and developmental regulators. the second group comprises transcripts encoding proteins that have at least one recognizable motif or domain, but whose functional attributes are generally unclear. for example, cell adhesion molecules are particularly heterogeneous between different organisms in terms of their domain combinations, making in silico comparisons particularly problematical (best : 97). the third group comprises ' orphan ' genes that show no significant similarity to sequences deposited in genbank and are likely to represent either honeybee - specific genes or genes that have not yet been sequenced in other species. alternatively, these sequences might represent genes that encode highly divergent proteins that can not be assigned to a known functional class without a three - dimensional structure. one example of a novel gene that is significantly downregulated in experienced foragers, especially in the abdomen, is clone best123. our rationale for comparing gene expression in naive, newly born bees and experienced foragers was based on previous findings suggesting that adult development in this insect is associated not only with behavioral maturation and division of labor, but also with significant molecular and cellular changes in the brain and other tissues. these changes include alterations in cholinergic and glutamatergic systems, increases in the levels of hormones and biogenic amines and volumetric changes in some brain neuropils. by comparing patterns of gene expression in naive versus experienced bees, we expected to identify transcripts that might control maturation of the brain and the innate immune system, activation of metabolic pathways, learning ability and other physiological functions. furthermore, we were curious to determine if caffeine - induced improvements in memory consolidation and associative learning lead to changes in gene expression that can be analyzed by microarrays. in this study we have not detected any differences in the level of expression of genes involved specifically in neuro - transmission, most probably as the result of experimental restrictions imposed by the use of heterogeneous body parts. it has been acknowledged that the marked cellular heterogeneity and low expression levels of many genes in the nervous system pose a serious challenge for microarray technologies. one recent study of two different mouse strains has shown that of more than 7,000 expressed mouse genes detected on the array, only 24 were identified that were differentially expressed in all six brain regions in the two different strains. in spite of its relatively small size (around 1 mm), the honeybee brain contains 1 million neurons organized in functionally specialized and anatomically divergent neuropils. differences in the levels of mrnas expressed in such a small amount of tissue are probably at the detection limit of current microarray experiments. judging from their level of expression, the genes revealed in our present study are either moderately or highly expressed (figure 2), and might therefore be more suitable for parallel comparisons than less abundant transcripts. nonetheless, the identification of a number of differentially expressed transcripts in the honeybee encoding molecules from functionally defined gene - activity cascades, including some in the nervous system, underscores the utility of this approach as a screen for detecting changes that occur during complex biological processes. as expected, a number of upregulated genes encoding metabolic enzymes and proteins expressed in ' functional ' glands have been detected. the rjps are related at the sequence level to the yellow protein family in drosophila, but have diverse, poorly understood functions in the honeybee. they are highly abundant in the hypopharyngeal gland of worker bees and are components of the so - called bee - milk, used to feed the queen larva. however, at least one member of the rjp family is also expressed in the brain. one of the -glucosidases described in this work (best122) seems to be a novel protein expressed predominantly in the abdomen (figures 1,2), in contrast to the previously identified -glucosidase-1 (best22), which is head - specific. the expression levels of mitochondrial genes (best54, 56, 109, 134) were significantly reduced in foragers, suggesting that electron - transport system activity declines in older bees. it has been proposed that decreased levels of mitochondrial transcripts reflect changes in the electron - transport system and oxidative phosphorylation and contribute to the etiology of aging. similarly, the decreased levels of mrnas encoding ribosomal proteins (best99) in foragers are likely to indicate the loss of homeostatic function in older individuals, as reported for other species. other genes known to be involved in cellular differentiation showed changes in the level of expression in newly born bees and experienced foragers. for example, sparc (best36), which encodes a calcium - binding glycoprotein expressed in extracellular matrix of various cell types undergoing morphogenesis, development and remodeling, is highly expressed in young bees, whereas a crystallin - like chaperonin, hsp20, encoded by l(2)efl (lethal 2 essential for life, best30) is upregulated in the heads of foragers. other genes upregulated in foragers encode proteins such as lim (best57), which affects muscle adherens junction integrity and mechanosensory function, imaginal disc growth factor (idgf ; best121), and a novel protein (best112). among transcripts induced by caffeine treatment, we have identified those for a putative member of the lim family (with a protein - protein interacting domain lim ; best57), idgf (best121) and a glutamine synthetase (best138). the increased levels of mrnas encoding two proteins implicated in immune responses, namely transferrin (best92) and hymenopteacin (best125), may indicate a reaction to physical injury and/or bacterial infection during injections. on the other hand, transferrin is also a key component in the control of iron homeostasis and it is conceivable that cellular changes induced by caffeine treatment, unrelated to injury, lead to the induction of its transcript. such multifunctional roles for proteins are increasingly being found to be a rule rather than an exception, and even the best characterized of all proteins are increasingly being found to have bioinformatically unpredicted multifunctional characteristics. for example, many ribosomal proteins have been found to be involved in extra - ribosomal activities such as iron binding, chromosomal condensation, dna replication, transcription, rna processing and dna repair, and in protein - protein interactions in the p53 and mdm2 pathways involved in malignant transformation. the identification of caffeine - inducible transcripts suggests that the honeybee might be a convenient organism in which the efficacy of drug treatment could be rapidly tested before more costly and time - consuming experiments with vertebrates. a number of the honeybee cdnas, such as that for the putative pdz - domain - containing protein (best61), show higher similarity to vertebrate sequences than to invertebrate sequences. this observation may indicate an under - representation of genbank entries from insect species more closely related to the honeybee than is drosophila melanogaster. one example of an unexpected evolutionary divergence of dipteran and hymenopteran sequences is the ligand - binding domain of the honeybee nuclear receptor ultraspiracle (usp) (r.m., unpublished work ; genbank accession number af263459), which clearly falls in the vertebrate - crab - tick - locust group rather than the dipteran - lepi - dopteran group. the reason for the divergence of usp sequences is unknown, but could result from a change in the type of ligand bound or the loss of ligand altogether. our study has revealed a collection of predicted proteins common to the honeybee and drosophila as well as proteins that may be unique to the honeybee. together with another study in the honeybee that compared the expression pattern of 288 ' caste - related ' genes, this dataset will be valuable for future comparative studies of gene - expression patterns in both insects. these comparisons will help to determine whether the behavioral differences between the ' simple ' fly and the ' sophisticated ' honeybee result from the invention of novel proteins, or are the output of changing patterns of expression of a basic set of genes common to both species. the haploid genome in the honeybee is similar in size to that in drosophila (approximately 180 million base pairs) and is expected to contain a comparable number of genes, approximately 13,600. however, these two genomes produce radically different living systems, both structurally and functionally, supporting the view that there is essentially no correlation between the gene number and the phenotype of a given species. we show here that array - based transcriptome analysis can be successfully used for basic cataloguing of gene expression in an organism for which genome and/or transcriptome information is virtually unavailable. furthermore, in contrast to the most widely studied invertebrate model organisms - the fruit fly d. melanogaster and the nematode caenorhabditis elegans - the honeybee has a range of complex individual and social behaviors, including a change from doing simpler activities when young to more complex activities when older. thus, the availability of gene - expression data in the honeybee, combined with the ease with which honeybee brain chemistry and behavior can be perturbed, further strengthens the value of this insect in behavioral and pharmacological studies. foraging honeybee workers were captured near the hive entrance and snap - frozen in liquid nitrogen (ln). to ensure that fully matured workers were collected, only those that carried pollen or nectar were selected. we estimate their age to vary from 20 - 35 days. to obtain newly emerged honeybees, a single brood frame was removed from the hive and incubated at 32c (80% humidity). individual insects were collected within 30 - 60 min after emergence and snap - frozen in ln. all dissections were done under permanent cooling (dry ice or ln). to obtain caffeine - treated bees, a colony of 30 newly born individuals in a small cage was fed for 3 days with honey containing 10 mm caffeine. we used a standard, unannotated and non - normalized honeybee brain cdna library made in lambda zap, kindly provided by g. robinson, university of illinois, urbana - champaign. mass excision of cdna - containing plasmids was carried out as recommended by the stratagene uni - zap xr manual. single colonies were picked manually and inoculated into 96-well microtiter plates containing 100 l of lb medium per well. following an overnight incubation, 5-l bacterial aliquots from each well were taken for direct pcr amplification using 2 units of taq dna polymerase from promega (plus 0.025 units of stratagene pfu) and 50 pmol of each primer (reverse and m13 - 20) per 100 l reaction. twenty - four pcr reactions from each of the 48 microtiter master plates were checked for the presence of amplified inserts. the pcr products were cleaned up by precipitation with iso - propanol and after several washes with 70% ethanol were resuspended in 3 ssc, 0.01% sarcosyl. array construction, labeling and hybridization were carried out according to protocols established by the brown lab in stanford with minor modifications. arrays were prepared by printing 4,608 samples in triplicate on polylysine - coated glass slides (menzel) using a robotic device from genetic microsystems (model 418). this estimate is based on random sequencing of 106 clones (aproximately 72% unique), the frequency of ' empty ' clones resulting from abortive bacterial growth or failed plasmid purification (10%) or the success rate of insert amplification (85%). total rna was extracted from frozen tissues by homogenization in a guanidine - thiocyanate buffer and sedimented overnight at 100,000 g through a cscl cushion (1.2 ml of 5.7 m cscl overlaid with 4 ml rna extract). we found that incorporation of fluorochromes was greatly improved in samples purified by cscl sedimentation. labeled probes were prepared by incorporating cy3 and cy5 dutp (amersham) during reverse transcription of total rna (superscriopt ii, life technologies). hybridization was carried out for 2 - 5 h at 62c in a small volume (60 - 120 l) of expresshyb buffer (clontech) under a plastic coverslip. slides were scanned with the affymetrix 428 scanner and analyzed using affymetrix pathways software v.1.0 and microsoft excel spreadsheets. high - throughput automated sequencing was done by the australian genome research facility (agrf) in brisbane using double - stranded plasmid dna templates. database searches were carried out at the national center for biotechnology information (ncbi) using the blast server. additional searches were conducted at the keck center for comparative and functional genomics, university of illinois at urbana - champaign to determine if our cdnas are included in the recently established honeybee est database. the following nine clones have not previously been found : best : 28, 54, 121, 123, 132, 134, 136, 143 and 147 (see table 1 for genbank accession numbers). total rna was isolated using trizol reagent from gibco brl followed by mrna purification on oligo(dt)25 magnetic beads from dynal (oslo). rna samples were denatured by mixing with an equal volume of formamide, containing 0.05% bromophenol blue and 0.01% sybrgreen ii, incubated at 90c for 5 - 7 min and immediately chilled on ice. electrophoresis was carried out in small horizontal tanks (hoeffer he33) using 1.5% agarose gels submerged in tbe buffer (50 mm tris - borate, 1 mm edta ph 8.2) at 20 v / cm. alternatively, the glyoxal - based system (ambion) was used for rna separation, in particular to analyze larger (> 2 kb) transcripts (see the manufacturer 's instruction manual (ambion, catalogue number 1946) for details). following electrophoretic resolution the gels were quantified with the vistra fluoroimager and then soaked in 1 m ammonium acetate, 0.02 m naoh and blotted onto hybond n+ nylon membranes (amersham) by capillary transfer. rna was crosslinked to the membrane by uv irradiation, and after a brief wash in 2 ssc the blots were prehybridized for 5 - 30 min. hybridization was carried out either at 68c (expresshyb solution, clontech), or at 42c (ultrahyb buffer, ambion) for 16 h using p - labeled probes (rediprime kit, amersham). blots were washed 3 - 4 times in 2 ssc, 0.1% sds at 50c and exposed to a phosphorstorage screen (molecular dynamics) without drying. computer generated images (md phosphor - imager 400s) of individual gels were analyzed using imagequant software. foraging honeybee workers were captured near the hive entrance and snap - frozen in liquid nitrogen (ln). to ensure that fully matured workers were collected, only those that carried pollen or nectar were selected. we estimate their age to vary from 20 - 35 days. to obtain newly emerged honeybees, a single brood frame was removed from the hive and incubated at 32c (80% humidity). individual insects were collected within 30 - 60 min after emergence and snap - frozen in ln. all dissections were done under permanent cooling (dry ice or ln). to obtain caffeine - treated bees, a colony of 30 newly born individuals in a small cage was fed for 3 days with honey containing 10 mm caffeine. we used a standard, unannotated and non - normalized honeybee brain cdna library made in lambda zap, kindly provided by g. robinson, university of illinois, urbana - champaign. mass excision of cdna - containing plasmids was carried out as recommended by the stratagene uni - zap xr manual. single colonies were picked manually and inoculated into 96-well microtiter plates containing 100 l of lb medium per well. following an overnight incubation, 5-l bacterial aliquots from each well were taken for direct pcr amplification using 2 units of taq dna polymerase from promega (plus 0.025 units of stratagene pfu) and 50 pmol of each primer (reverse and m13 - 20) per 100 l reaction. twenty - four pcr reactions from each of the 48 microtiter master plates were checked for the presence of amplified inserts. the pcr products were cleaned up by precipitation with iso - propanol and after several washes with 70% ethanol were resuspended in 3 ssc, 0.01% sarcosyl. array construction, labeling and hybridization were carried out according to protocols established by the brown lab in stanford with minor modifications. arrays were prepared by printing 4,608 samples in triplicate on polylysine - coated glass slides (menzel) using a robotic device from genetic microsystems (model 418). this estimate is based on random sequencing of 106 clones (aproximately 72% unique), the frequency of ' empty ' clones resulting from abortive bacterial growth or failed plasmid purification (10%) or the success rate of insert amplification (85%). total rna was extracted from frozen tissues by homogenization in a guanidine - thiocyanate buffer and sedimented overnight at 100,000 g through a cscl cushion (1.2 ml of 5.7 m cscl overlaid with 4 ml rna extract). we found that incorporation of fluorochromes was greatly improved in samples purified by cscl sedimentation. labeled probes were prepared by incorporating cy3 and cy5 dutp (amersham) during reverse transcription of total rna (superscriopt ii, life technologies). hybridization was carried out for 2 - 5 h at 62c in a small volume (60 - 120 l) of expresshyb buffer (clontech) under a plastic coverslip. slides were scanned with the affymetrix 428 scanner and analyzed using affymetrix pathways software v.1.0 and microsoft excel spreadsheets. high - throughput automated sequencing was done by the australian genome research facility (agrf) in brisbane using double - stranded plasmid dna templates. database searches were carried out at the national center for biotechnology information (ncbi) using the blast server. additional searches were conducted at the keck center for comparative and functional genomics, university of illinois at urbana - champaign to determine if our cdnas are included in the recently established honeybee est database. the following nine clones have not previously been found : best : 28, 54, 121, 123, 132, 134, 136, 143 and 147 (see table 1 for genbank accession numbers). total rna was isolated using trizol reagent from gibco brl followed by mrna purification on oligo(dt)25 magnetic beads from dynal (oslo). rna samples were denatured by mixing with an equal volume of formamide, containing 0.05% bromophenol blue and 0.01% sybrgreen ii, incubated at 90c for 5 - 7 min and immediately chilled on ice. electrophoresis was carried out in small horizontal tanks (hoeffer he33) using 1.5% agarose gels submerged in tbe buffer (50 mm tris - borate, 1 mm edta ph 8.2) at 20 v / cm. alternatively, the glyoxal - based system (ambion) was used for rna separation, in particular to analyze larger (> 2 kb) transcripts (see the manufacturer 's instruction manual (ambion, catalogue number 1946) for details). following electrophoretic resolution the gels were quantified with the vistra fluoroimager and then soaked in 1 m ammonium acetate, 0.02 m naoh and blotted onto hybond n+ nylon membranes (amersham) by capillary transfer. rna was crosslinked to the membrane by uv irradiation, and after a brief wash in 2 ssc the blots were prehybridized for 5 - 30 min. hybridization was carried out either at 68c (expresshyb solution, clontech), or at 42c (ultrahyb buffer, ambion) for 16 h using p - labeled probes (rediprime kit, amersham). blots were washed 3 - 4 times in 2 ssc, 0.1% sds at 50c and exposed to a phosphorstorage screen (molecular dynamics) without drying. computer generated images (md phosphor - imager 400s) of individual gels were analyzed using imagequant software. differential expression of 36 ests (indicated by red boxes) detected by microarrays and confirmed by northern blotting (lower panels for each est). the three columns correspond to transcripts identified as differentially expressed in the heads and abdomens of newly born bees and experienced foragers, and in the heads of 3-day - old bees after caffeine treatment. northern blot hybridization showing the pattern of expression of selected genes in the head, thorax and abdomen. (a) genes downregulated in foragers ; (b) genes upregulated in foragers ; (c) genes predominantly expressed in only one compartment. identity of genes differentially expressed between naive and experienced honeybees and in caffeine - treated honeybees best number, cdna number ; gene, gene identifier ; array data, fold change calculated from array hybridization data using pairwise comparisons for heads, abdomens and caffeine - treated bees ; predicted function, putative function inferred from sequence similarity ; percentage similarity to the closest relative in genbank. accession numbers of bests reported in this paper : 5, bi946410 ; 22, bi946425 ; 28, bi946431 ; 30, bi946433 ; 31, bi946435 ; 36, bi946440 ; 54, bi946454 ; 56, bi946456 ; 57, bi946458 ; 61, bi946461 ; 82, bi946480 ; 92, bi946487 ; 97, bi946490 ; 99, bi946493 ; mrjp2 (102), af000632 ; gld (105), ab022907 ; 108, bi946499 ; 109, bi946500 ; 112, bi946503 ; 121, bi946511 ; 122, bi946512 ; 123, bi946513 ; 124, bi946514 ; hymenoptaecin (125), amu15956 ; 127, bi946517 ; 128, bi946519 ; 129, bi946520 ; 130, bi946522 ; 131, bi946524 ; 132, bi946525 ; 133, bi946526 ; 134, bi946527 ; 136, bi946528 ; 138, bi946532 ; 143, bi946537 ; 147, bi946542 ; 148, bi946543. | backgroundthe honeybee (apis mellifera) has been used with great success in a variety of behavioral studies. the lack of genomic tools in this species has, however, hampered efforts to provide genome - based explanations for behavioral data. we have combined the power of dna arrays and the availability of distinct behavioral stages in honeybees to explore the dynamics of gene expression during adult development in this insect. in addition, we used caffeine treatment, a procedure that accelerates learning abilities in honeybees, to examine changes in gene expression underlying drug - induced behavioral modifications.resultsspotted microarrays containing several thousand cdnas were interrogated with rnas extracted from newly emerged worker bees, experienced foragers and caffeine - treated bees. thirty - six differentially expressed cdnas were verified by northern blot hybridization and characterized in silico by sequencing and database searches. experienced foragers overexpressed royal jelly proteins, a putative imaginal disc growth factor, a transcriptional regulator (stck) and several enzymes, including -glucosidases, aminopeptidases and glucose dehydrogenase. naive workers showed increased expression of members of the sparc and lectin families, heat - shock cognate proteins and several proteins related to rna translation and mitochondrial function. a number of novel genes overexpressed in both naive and experienced bees, and genes induced by caffeine, have also been identified.conclusionswe have shown the usefulness of this transcriptome - based approach for gene discovery, in particular in the context of the efficacy of drug treatment, in a model organism in which routine genetic techniques can not be applied easily. |
hypertension is known to be a direct or indirect cause of death globally [1, 2 ]. findings from previous longitudinal studies of blood pressure (bp) suggests that essential hypertension in adults can be detected in early life [3, 4 ]. because of the public health value of prevention of hypertension, a lot of work has been done to find out the prevalence of childhood hypertension [35 ]. part of the findings of these studies is that prevalence of hypertension in children has racial and rural - urban variations [3, 4 ]. in african children, higher risks of cardiovascular disease have been found among urbanites than rural dwellers, however, no consistent trend was found along socioeconomic and ethnic lines [46 ]. these inconsistencies in the trend of essential hypertension in africa have made it difficult to extrapolate adult risk for high bp from a child 's bp. however, studies have been conducted in the past to identify anthropometric indices that can be used as markers of child bp and heart rate (hr) in africa and other continents [4, 7 ]. these studies have shown that bp in children is closely related to height (ht), weight (wt), body mass index (bmi), ponderosity index (pi), skin fold thickness, and mid - upper arm circumference. however, the few studies that have examined this relationship among nigerian children [4, 6 ] have not explored the relationship of abdominal circumference (ac) to their bp and hr whereas previous studies have highlighted its important relationship with obesity and abdominal adiposity [811 ]. in the year 2000, savva. reported that abdominal circumference and waist - to - hip ratio were better predictors of cardiovascular disease risk factors in children than bmi. one of the comprehensive studies in the area of nigerian child anthropometry and cardiovascular risk factors was the study of balogun.. they observed significant correlation between anthropometric (wt, ht, bmi, and upper - arm circumference) and cardiovascular (systolic blood pressure (sbp), diastolic blood pressure (dbp), hr, rate pressure product (rpp), and pulse pressure (pp)) parameters of nigerian children. this study found a 4% prevalence of hypertension among these children and noted that weight was the most viable predictor of bp and resting myocardial oxygen consumption. they also recommended that wt norms rather than age be used in evaluating abnormal bp levels of nigerian children. it is important to know the bp relationship with ac of nigerian children and if there are changes in the reported relationship between anthropometric and cardiovascular parameters among children in nigeria. this is because the country 's economy have been changing and population increasing over the past two decades. information derived will help health professionals to adjust to the current trend and policy makers to evolve preventive measures to cope with changes in cardiovascular risk factors. this study therefore examined (1) the relationship of wt, ht, bmi, and ac with bp and hr of school age children in nigeria ; (2) which of these anthropometric parameters is the most viable predictor of bp ? one thousand and twenty - six (512 males and 514 females) apparently healthy primary school pupils whose ages ranged from 6 to 14 years (mean age = 10.12 2.46 years) in ten public (8) and private (2) primary schools in ile - ife, nigeria were recruited for the study. ile - ife is an urban city in osun state of nigeria with 10 wards (city areas) and a population of 355,341 people (male = 176,707 ; female 178,634) based on 2006 population census. to select the participating primary schools, the list of primary schools was obtained from the local schools authority and separated according to the city areas where they are located without minding whether they were public or private schools. schools in the same ward were written out on small sheets of paper, which were folded and a neutral person was asked to pick one from each ward. before the commencement of the study, informed consent of the parents of the pupils was sought and ethical clearance obtained from the ethics and research committee of obafemi awolowo university teaching hospitals complex, ile - ife. the participants were screened to eliminate any disability such as infantile poliomyelitis or limb length discrepancy that may make measurement of anthropometric and cardiovascular parameters difficult. on arrival into a class, the names of the pupils were written out alphabetically on a sheet of paper, 18 names were then selected from the list by selecting every 3rd name. height was measured using a validated height metre. the subjects stood erect, barefoot on a flat surface, with the occiput, upper back, buttocks, and heels, touching the height metre. in line with the view of steele and spurgeon, the upper margin of the external auditory canal opening was in the frankfurt horizontal plane, and the point of greatest ht to the nearest 0.1 cm weight was measured to the nearest 0.1 kg with a weighing scale (hanson company, ireland). abdominal circumference was measured to the nearest 0.1 cm below the rib cage and above the umbilicus at the end of normal expiration using an inelastic tape (butterfly, china). all measurements were taken by the same examiner within the school premises with subjects on minimum clothing. blood pressure was measured once after ten minutes of quiet sitting with a standardized aneroid sphygmomanometer and a stethoscope (u - mec, china). heart rate was measured by auscultation for 30 seconds and later multiplied by two to get the hr per minute. the subject was comfortably seated in a chair with the arm well supported with a pillow. the pp and pi were calculated using the formula pp = sbp dbp and pi the bmi was obtained by dividing the wt by the square of the ht, that is, bmi = wt / ht, while rpp was evaluated using the formula : rpp = sbp hr. mean arterial pressure (map) was estimated using the formula map = dbp + 1/3 pp. data was analyzed using the statistical package for social sciences (spss) version 10.0. chicago, iii, usa). both descriptive (range, mean, median, mode, and standard deviation), and inferential statistics were used for data analysis. pearson product moment correlation coefficient was computed to determine the correlation between the anthropometric characteristics of the pupils and their cardiovascular parameters. student 's t - test was used to determine the significant difference between the physical characteristics and physiological parameters of boys and girls. stepwise regression analysis was employed to determine the combined and individual contribution of the anthropometric parameters towards the prediction of the criteria variables (cardiovascular parameters). a total of 1032 pupils were initially selected, however, 6 children were not measured because when examined, 4 of them were febrile, while 2 had lower limb deformity. therefore, this report is based on the data collected from 1026 apparently healthy school - age pupils (mean age of 10.12 2.46 years) who participated in this study. the physical characteristics and the resting physiological parameters of all the subjects are presented in table 1. on the average, the sbp and dbp of the pupils were 98.63 12.38 mmhg and 62.85 10.18 mmhg with a 95th centile of 121.00 and 80, respectively. the mean rpp was 8725.54 1828.1 mmhg beats per minute, while the mean pp was 35.79 8.64 mmhg. the correlation between the anthropometric characteristic and the cardiovascular measures are presented in table 2. age was positively related to sbp, dbp, rpp, and pp but has inverse relationship with hr. the correlation between some anthropometric (age, ht, wt, bmi, and ac) and cardiovascular (bp, pp) parameters were positive and significant (p <.01). however, hr had significant negative correlation with age, ht, bmi, and ac (p <.01), and wt (p <.05). the correlation between pi and sbp was significant (p <.01) but was not significantly correlated with hr and rpp. as shown in table 3 however, females were significantly (p <.05) heavier and had significantly (p <.05) higher ac and bmi than their male counterparts. weight (24.8%), ac (2.0%) and bmi (1.6%) when combined contributed significantly (p <.05) towards predicting (28.4%) the levels of sbp. addition of bmi to wt caused a variation of 1.6% in predicting sbp while addition of ac to wt and bmi explained 2.0% of the total variation in sbp. age (9.7%) and weight (1.5%) accounted for 11.2% of the total variance in dbp (table 5) while ac (4.6%) and age (0.7) were two viable predictors (5.3%) of hr. the only determinant of rpp was wt which accounted for 1.5% in its variance. this cross - sectional study investigated the relationship of some anthropometric and cardiovascular indices of school age nigerian children. the main purpose of this research was to find out if some of the previously established anthropometric determinants of hypertension in nigerian children are still viable while exploring the usefulness of other parameters. it was observed that significant correlations still exist between anthropometric parameters (age, ht, wt, bmi, and ac) and bp. weight (24.8%) was found to be the most viable predictor of sbp while age was the best determinant of dbp (9.7%). although weight contributed little to the prediction (1.5%) of dbp age did not contribute to the prediction of sbp. this finding is consistent with the report of andy. but not with that of balogun. andy. reported that age and wt were the two most important determinants of bp levels of nigerian children, but the influence of wt was greater., age was not a viable predictor of bp in the study of balogun. they attributed this inconsistency to age restriction (120 years and 820 years, resp.). the reason why weight should preferentially predict systolic and why age preferentially predicts diastolic bp in children is open to speculation. however, it is known that bp rises with age but sbp increases more than dbp. also, increases in cardiac output increase the sbp, whereas increases in peripheral resistance increase dbp. our finding suggests that increasing wt may be associated with increase cardiac output while ageing may be associated more with increasing peripheral resistance thus explaining their respective correlations to each other. furthermore, age range of subjects seems an important factor in determining anthropometric marker for bp and hypertension. the age range of the current cohorts was 614 years, and the 95th centile bp was found to be 121/80 mmhg. by using this centile as the cutoff diagnostic criteria for hypertension [15, 16 ], 8% (n = 81) prevalence of hypertension was found. in contrast balogun. found the 95th centile bp to be 133/92 mmhg and a prevalence of 4%. therefore, the higher bp range may be attributed to the influence of the older (1520 years) and most likely heavier subjects in their study. apart from wt and age, ac (2.0%), and bmi (1.6%) were the other predictors of sbp, while ac (4.6%) and age (0.7) were predictors of hr. our findings are consistent with reports from other populations [9, 11, 17 ].. found pi to be one of the most important contributors to the associated variation in bp levels of american children. the important contributions of ac and bmi to the prediction of bp and hr are to be expected because of their known correlation to adiposity. previous studies have shown that abdominal circumference was the most practical anthropometric measurement for assessing a patient 's abdominal fat content and that ac correlates with the amount of fat in the abdomen [7, 9, 12 ]. the present study found that ac correlated more (r = 0.48, p <.01) than bmi (r = 0.39, p <.01) with sbp in nigerian children, a finding that agrees with the other reports [7, 9 ]. these two studies reported that ac correlated more than bmi with cardiovascular risk factors and most strongly with insulin and sbp. this finding agrees with the report of balogun. who reported no significant difference in resting bp of nigerian boys and girls. this is in contrast with the report of oviasu. who observed significant difference in the bp of male and female children in their study of subjects with age range similar to the present cohort. these differences in observation may reflect ageing effect, ethnic, or regional variations in the relationship between anthropometric and cardiovascular indices of children [6, 9, 14 ]. this is more so because gender differences in growth pattern have long been known and ethnic and rural - urban differences in body somatotypes and sensory characteristics had been reported [18, 19 ]. further work will be needed to clarify this trend. this study has revealed that wt is a more viable predictor of sbp, while age predicts dbp better and ac predicts cardiovascular risk. based on these findings, it is recommended that body wt norms be used in evaluating abnormal sbp while using age to evaluate abnormal dbp levels in nigerian school - aged children. ac should be included as an anthropometric predictor of resting bp and hr in this population with a call for more action towards the prevention of childhood hypertension in nigeria. significant correlations exist between age, ht, wt, bmi, ac, and bp with wt being a more viable predictor of sbp and age a more viable predictor of dbp. body weight norms should be used in evaluating abnormal sbp levels, while age should be used to evaluate abnormal dbp levels in nigerian children. | purpose. this study investigated the anthropometric indices associated with variations in cardiovascular parameters among primary school pupils in ile - ife. method. one thousand and twenty - six pupils (age range 614 years, mean age 10.12 years) from ten schools were recruited with parents ' informed consent. anthropometric (height (ht), weight (wt), abdominal circumference (ac)) and cardiovascular (systolic blood pressure (sbp), diastolic blood pressure (dbp), heart rate (hr)) parameters were measured using standard instruments and procedures. blood pressure (bp) was measured after ten minutes of quiet sitting. body mass index (bmi), rate pressure product (rpp) and pulse pressure (pp) were estimated. results. age, ht, wt, bmi, and ac correlated significantly (p <.01) with bp and pp. ac and bmi were predictors of bp, hr, rpp, and pp. conclusion. significant correlations exist between age, ht, wt, bmi, ac, and bp with weight being a more viable predictor of sbp and age a more viable predictor of dbp. |
nearly one third of the patients develop perivalvular abscesses leading to an increased rate of systemic embolization and death. we report a rare case of infective endocarditis in a patient with a biological aortic valve prosthesis with paravalvular abscess that acutely penetrated into the right atrium. acute left - to - right shunting caused severe hemodynamic depression and multiple organ failure. a 62-year - old, hiv positive, male patient, who underwent bioprosthetic aortic valve replacement five years previously, was admitted to an external hospital with sepsis. no specific focus was found by an initial ct - scan of the chest and abdomen. a transesophageal echocardiography (tee), performed upon admission, showed no endocarditis. exactly one week after admission the patient developed symptoms of a stroke and ct detected a thromboembolism in the region of the posterior cerebral artery. subsequent tee, revealed a vegetation on the right coronary cusp of the aortic valve bioprothesis and an aortic root abscess. his neurological symptoms declined during the following days. on day 12 after admission, however, acute deterioration of the patient 's hemodynamic conditions required urgent transfer to our hospital. on admission to the intensive care unit the patient required high dose vasopressor therapy. transthoracic echocardiography (tte) showed hyperdynamic biventricular function and shunting from the aortic root to the right atrium (fig. 1). position, dimension and exact anatomic conditions of the fistula were obtained by contrast enhanced computed tomography (fig. after removal of the bioprosthesis, a large periannular abscess opening into the right atrium was uncovered (fig. 3). a pericardial patch plasty was performed to exclude the abscess and another to seal the right atrium interiorly. postoperatively, his clinical status improved quickly and the patient left the icu nineteen days after surgical treatment in stable physical condition and without any neurological deficit. a high in - hospital mortality rate of up to 40% has been documented in prosthetic valve endocarditis. in this case, several risk factors such as immunodeficiency, presence of an intracardiac abscess, transient cerebral ischemia and in particular staphylococcal sepsis were present. the european society of cardiology guidelines for the treatment of infective endocarditis recommends cardiac surgery in these cases within a few days.. recently showed that cardiac surgery performed within 48 h after diagnosis of infective endocarditis with severe valve disease and large vegetations results in a significantly improved outcome. to our best knowledge this work is the first randomized trial confirming the superiority of early surgical intervention. in a linear regression model with the data from 24 non - randomized studies thuny. also presented a significant enhanced correlation between early cardiac surgery and survival. they occur in about one third of all patients with infective endocarditis. in the absence of cerebral hemorrhage, cardiac surgery could be performed with a moderate rate of 36% for neurological detoriation. less invasive interventions, such as atrial or ventricular septal defect occluder, the amplatzer duct occluder or coils, described feasible in patients with paravalvualar leaks. however, they seem not to be beneficial under time - critical conditions and implantation of non - biologic material in an infected field can not be recommended. finally, detection of a fistula in a cardiac chamber or the pericardium resulting in shock remains an inescapable indication for emergent cardiac surgery. in our case the patient was not yet intubated, so we did not perform tee to prevent further aggravation of the hemodynamic conditions due to sedation. two - dimensional tte reliably detects left - to - right shunting but only the ct - scan allowed exact description of the lesion and planning of cardiac surgery. to reduce further risk of deterioration in - hospital transfer of the critically ill patient it is described as effective point of care imaging method with high sensitivity and specificity to verify diagnosis in several case reports. therefore, in the intensive care setting this new technique should be taken into account in future. our case report supports the early surgery strategies and shows that maximum therapeutic efforts can achieve good outcomes even in complex medical situations associated with poor prognosis. r. schramm added the surgical parts of the text and took the picture of the intraoperative view of the heart. | introductionheart failure is the most common cause of death due to infective endocarditis. we report a case of a patient presenting with severe shock due to an infection - associated left - to - right cardiac shunt.presentation of casea 62-year - old man, who underwent aortic valve replacement five years previously, was admitted to icu due to acute hemodynamic deterioration. a few days earlier, he had a septic episode with blood cultures positive for staphylococcus aureus and clinical features of infective endocarditis. in icu, transthoracic echocardiography revealed shunting from the aortic root to the right atrium resulting in severe cardiogenic shock.discussionthis case report describes a near fatal complication of infective endocarditis, detected by routine use of transthoracic echocardiography.conclusionour case outlines the relevance of early cardiac surgery strategies in patients with infective endocarditis and we briefly discuss the current literature. |
herpes zoster caused by the neurodermotropic virus called varicella zoster virus is distributed worldwide. this benign localized viral disease has been recognized as a distinct entity since ancient times. it manifests as a result of reactivation of the virus laid dormant in the sensory ganglion[13 ] following a clinical or sub clinical varicella (chicken pox) infection early in life or occasionally in utero. the reactivation of the virus may be due to immunosuppression (inherited, acquired or iatrogenic) or spontaneous. in hiv disease it presents in diverse manner such as multidermatomal involvement, crusted, nodular or vesiculopustular, ulcerative, and ecthymatous[46 ] lesions that may be widely disseminated[479 ] or localized. although in most cases the manifestations are classical, there are occasions, particularly of visceral involvement that can challenge the knowledge of even an experienced physician. another important and most troublesome complication is post herpetic neuralgia (phn), which is very tiring to the patient as well as to the physician and to date there is no single effective drug to cure this distressing problem. two hundred five patients, in the age group 385 years with herpes zoster attending the various departments of the calicut medical college, kerala, india, were included in the study for a period of 2 years. preliminary information such as age, sex, address, and occupation were noted. a detailed history regarding the prodromal and presenting symptoms, day of occurrence of skin lesions after prodrome, nature of pain, its intensity and duration, and other symptoms if any, were elicited and recorded. history suggestive of provocative factors such as drugs, recent trauma, surgery, irradiation, immunosuppressive and cytotoxic chemotherapy, diabetes mellitus, pulmonary tb, hiv infections were inquired. a thorough dermatological examination regarding the segment of involvement, morphology, and pattern of the lesions, regional lymph node enlargement, motor complications, dissemination of the lesions in other areas of the body etc. whenever necessary, opinion from other specialists such as the ophthalmologist, chest physician, diabetologist, and the general physician were sought. at first tzanck smear (in leishman 's stain) examination for ballooned epithelial cells and multinucleated giant cells, complete haemogram, blood sugar, urine routine exam, and enzyme - linked immunosorbent assay test for hiv antibodies were conducted. all the patients were reviewed weekly for 1 month and monthly for two more months. the age and sex distribution is given in table 1.the maximum incidence was in the age group of 3140 years (24%), which is followed by that of 2130 years (19%), and 4150 years (15.6%). minimum incidence was observed in the age group 110 years and 7180 years (1.9% and 3.9%). out of 205 cases, 130 (63.4%) had definite history of chicken pox, most of them within 20 years of age. the remaining 75 cases (36.6%) were neither aware of nor had chicken pox at all. age and sex distribution of zoster sixty - two patients had one or more suspected provoking factors. twenty patients were on steroids for ailments such as bronchial asthma, contact dermatitis, exfoliative dermatitis, pemphigus, lepra reactions, systemic lupus erythematosis, idiopathic thrombocytopenic purpura, and erythema multiforme. nineteen patients had various types of malignancies and were on chemotherapy / radiotherapy or on both, 11 patients were diabetic, ten patients had hiv infection [figures 1 and 2 ], and two patients had pulmonary tuberculosis. extensive herpes zoster in an hiv patient disseminated herpes zoster in an hiv patient twenty - two cases had significant constitutional symptoms such as fever, headache and arthralgia, 2 - 3 days prior to or concurrently with the onset of vesicles. 94.6% cases had segmental neuralgia some times during the course of the disease varying in intensity from mild to very severe producing insomnia. pain preceded the vesicles in 123 (60%) cases. concurrently started with the vesicles in 65 and followed by 23 days in six patients. the segment wise distribution of zoster is given in table 2 thoracic dermatome was commonly (87) affected [figure 3 ] and among thoracic, t4 segment was common, followed by the trigeminal nerve (56) [ophthalmic branch (33), maxillary (15), and mandibular (8) ]. twenty - five patients had cervical, 16 lumbar, and 10 had sacral nerve involvement. segmental distribution of zoster herpes zoster of the thoracic segment one hundred five had lesions on the right side and 100 cases on the left side. sixty percent had tender regional adenopathy at the time of presentation, in the rest, 25% developed lymphadenopathy by 1 week. the period of resolution ranged from 718 days with an average of 912 days. out of 205 patients 71 (34.6%) developed complications. the complications observed were secondary bacterial infection (28 cases, 13.6%), severe ulceration (four cases, 1.95%), scarring (eight cases, 3.9%), phn (21 cases, 10.2%), motor weakness (three cases in a 25-year old female right ulnar weakness, 71-year old man and 42- year old woman ramsay - hunt syndrome with facial weakness), depigmentation (five cases, 2.4%) and post herpetic itching (two cases). it was a continuous deep nagging pain in seven and intermittent lancinating or radicular pain in the rest. the study of 205 patients of herpes zoster revealed that the majority of the patients affected were adults, (55%) below the age of 40 years and (45%) above 40 years. but in contrast to other reports in the literature.[1315 ] males outnumbered females in the ratio of 1.75:1.3 in this study which is similar to a study from south india but in contrast to the western studies where both males and females were equally affected. trauma and stress as a result of their occupation and outdoor activity may be the predisposing factor for the male preponderance in indian setup. in 70% of the patients the commonest provocative factor was steroid intake for various ailments (20 cases) followed by malignancies. with or without irradiation (19 cases) diabetes mellitus (11 cases) and hiv infection (10 cases).[1724 ] depressed cmi associated with the above conditions[182527 ] could be the possible factor responsible for the development of zoster and for its extensiveness. the prevalence of prodromal or concurrent constitutional symptoms were found to be lower in this study (10.7%), particularly in older patients comparing to older indian studies. in accordance to the previous literature reports,[2931 ] in most of the patients, the presenting symptom was pain (94.6%) then vesiculations (94%) and pain coinciding with the onset of the eruptions of vesicles were in more than two thirds of the cases. 87.5% of the patients below the age of 20 years had neuralgic pain at the onset of the lesions contrary to the previous studies. 85% of the patients had strict dermatomal distribution, but 15% had vesicles beyond the dermatome. even with this slightly altered morphology in the latter group, the course of the illness was not different from the former excepting for the slightly prolonged resolution time. thoracic segment was common, then cranial nerve involvement, unlike the previous studies where thoracic segment was followed by cervical and lumbar segments. even though the reports claim that the clinical picture as well as the course of the disease is severe in fifth decade and above, in this study, severe rash with hemorrhagic and extensive grouped vesicles were encountered only in less than 15% of the older population. phn was observed mainly in older age group (above 60) and was more in thoracic segments in contrast to reports in ophthalmic zoster. since more than 70% of the cases were of less than 50 years of age, the incidence of phn is less in this study. out of ten hiv patients, nine had localized lesions as in normal patients, but one had dissemination, bulla formation, and necrosis. out of ten hiv cases, zoster was the presenting symptom in three patients.[18313436 ] but unusual morphologies as reported in some studies were not encountered in this study except in one. | background : even though herpes zoster is a common condition its incidence and pattern of occurrence in the era of hiv disease is significant.aim:to analyze the incidence, pattern of occurrence and evolution of herpes zoster with special attention to provocative factors if any.materials and method : this was an analytical study conducted for 2 years based on a preformed proforma containing preliminary information, a detailed clinical evaluation regarding the segment of involvement, morphology, pattern of lesions, complications, disseminations etc. and investigations to establish provocative factors if any.results:incidence of herpes zoster was mainly in the fourth and third decades of life. a definite history of chicken pox was present in only 63.4% cases. in the majority (70%) herpes zoster occurred spontaneously. in 30% cases, immunosuppression due to chemotherapy, malignancy, hiv infection, diabetes mellitus were observed. the commonest segment affected was thoracic (42.4%) followed by cranial (28.2%) and cervical (12.1%). majority resolved in 714 days except immunosuppressed. 34.6% of the patients had complications such as secondary bacterial infection, post herpetic neuralgia, and motor weakness. ten patients had hiv infection as a provocative factor.conclusion:the results of incidence and clinical pattern of herpes zoster is almost parallel to the previous studies. any factors of immunosuppression should be checked, especially hiv, particularly in disseminated and long - lasting cases. |
neonicotinoids are a relatively new class of insecticides that share a common mode of action that affect the central nervous system of insects, resulting in paralysis and death. studies suggested that neonicotinoids residues can accumulate in pollen and nectar of treated plants and represent a potential risk to pollinators. the honey italian observatory stated that in 2008 more than half of italian hives, and that 600,000 of a total of 1,100,000 have been put out of production for the depopulation of entire apiaries. one result might be expected given that the previous year, the european food safety authority (efsa) stated that the bee die - off had hit the 50% bee population, compared to the annual average of 15%. neonicotinoids can also be persistent in the environment and, when used as seed treatments, translocate to residues in pollen and nectar of treated plants. the potential for these residues to affect bees and other pollinators remains uncertain. despite these uncertainties, neonicotinoids are beginning to dominate the market place because of their high systemicity, the broad spectrum of action, and the reduced dose. in light of these findings, the italian ministry of agriculture has asked the ministry of health to suspend action. the ministry of health, after consultation with the pesticides committee, issued the ministerial decree of september 17, 2008 that stated the precautionary suspension of the authorized use for the seeds tanning of plant protection products containing the active substances clothianidin, thiamethoxam, imidacloprid, and fipronil. on june 25, 2012, a decree of the ministry of health extended to january 31, 2013 stating the neonicotinoids suspension for seeds treatment. similar measures have already been taken by other european states. recently, many researchers detected these insecticides in honey bees, honey, soil, pollen, and treated seeds for agriculture [612 ]. measurement of pesticide residues in different matrices involves two basic steps, namely, sample preparation (extraction and clean up) and instrumental analysis. ideally, a sample preparation should be rapid, simple, cheap, and environment friendly and provide clean extracts.. quechers (quick easy cheap effective rugged safe) technique, which was developed between 2000 and 2002 and first reported in 2003, is a fast and complete extraction and clean up procedure and also employs the use of dispersive - solid phase extraction (d - spe) for sample clean up. in this paper, we report a rapid modified quechers method for multiresidue analysis for 6 neonicotinoids (acetamiprid, clothianidin, imidacloprid, nitenpyram, thiacloprid, and thiamethoxam) in honey with good selectivity, sensitivity, and cost effectiveness. in order to demonstrate the suitability of the method for routine regulatory purposes, the certified analytical standards of all the 6 pesticides (acetamiprid, clothianidin, imidacloprid, nitenpyram, thiacloprid, and thiamethoxam) and internal standard tris(1-chloro-2-propyl)phosphate (tcpp) were purchased from ultra scientific (bologna, italy) (100.0 0.5 g / ml each) in acetonitrile. all the solvents and chemicals used in the study were of analytical reagent (ar) grade, ethanol was supplied by romil (milan, italy), and formic acid, ammonium formiate, and acetonitrile were by carlo erba (milan, italy). distilled water was purified at 18.2 m with a milliq ultra (millipore, vimodrone (mi), italy) purification system. a mixture of dispersive spe citrate extraction tube supelco (4 g magnesium sulphate, 1 g sodium chloride, 0.5 g sodium citrate dibasic sesquihydrate, and 1 g sodium citrate tribasic dihydrate) was used, supplied by sigma - aldrich (milan, italy). ultra high - performance liquid chromatography uhplc - ms / ms (thermo scientific, tsq quantum access max) equipped with thermo hypersilgold column (50 mm 2.1 mm, 1.9 m) was used for quantification of neonicotinoids. the flow rate was 400 l / min, the column temperature 30c, and the injection loop volume 5 l. a binary gradient of 0.05% hcooh and hcoonh4 2 mm in water (a) and 0.05% hcooh and hcoonh4 2 mm in ch3oh (b) was employed. the mobile - phase gradient was programmed as follows : 0 min, 10% b ; 7 min, 95% b ; 8 min, 95% b ; 9 min, 10% b ; and 10 min, 10% b. mass spectral analyses were performed using an lc - tsq quantum access max operating in the positive ion mode using a h - esi interface. the neonicotinoids and the internal standard tcpp were detected in ms / ms conditions, programming the chromatographic run in srm mode (selected reaction monitoring) as reported in table 1. preliminary tunings were carried out with continuous introduction of a dilute solution of certified standards. flow rate of syringe pump infusion of 5 l / min and the voltages on the lenses were optimized in tsq tune master (excalibur software). the standard mix solution at 5 g / ml of standard pesticides was diluted by transferring 500 l (100.0 0.5 g / ml) into a volumetric flask (10 ml, class a certified). the standard mix solution at 1 g / ml of standard pesticides was diluted by transferring 100 l (100.0 0.5 g / ml) into a volumetric flask (10 ml, class a certified). the standard mix solution at 0.1 g / ml of standard pesticides was diluted by transferring 200 l of solution at 5 g / ml into a volumetric flask (10 ml, class a certified). the specificity of the analytical method for neonicotinoids detection was confirmed by obtaining positive results from honey containing the analyte, coupled with negative results from samples which do not contain it (negative controls). the matrix effect was assessed by preparing pesticide standards in blank matrix extracted from untreated honey. the matrix extracts were analyzed before spiking to confirm the absence of the test pesticides in them. the quantification of pesticide was based on a six - point matrix - matched calibration graph by plotting the detector response (srm area ratio with respect to internal standard tcpp) against concentration of the calibration standards within the range 150 g / a linear regression of six calibration points for each component was used to determine the relationship with the analyte concentrations calculated for each component on the basis of their occurrence in the reference material. the regression equations with slope, y - intercept, and coefficient of correlation (r) were evaluated for acetamiprid, clothianidin, imidacloprid, nitenpyram, thiacloprid, and thiamethoxam. statistical test (mandel and residual analysis with normal distribution of the calibration points) loq was estimated by the response of method noise level by approximately ten and lod is, therefore, 3.3-fold lower. method recovery studies were performed at two spiking concentration levels (10 g / kg and 40 g / kg). the sample matrix was prepared by homogenizing a series of different honeys in order to develop a highly specific method. the samples were prepared by weighing 5.0 0.5 g of honey spiked in 50 ml tube (meus srl, piove di sacco (pd), italy). these sample tubes were vortexed (velp, usmate (mb), italy) for 30 seconds after adding 10 ml of water and 10 ml of acetonitrile, in order to homogenize and fluidize the sample, and 50 l of tris(1-chloro-2-propyl)phosphate (tcpp) at 50 mg / l. in each tube was added a mixture of salts (4 g magnesium sulphate, 1 g sodium chloride, 0.5 g sodium citrate dibasic sesquihydrate, and 1 g sodium citrate tribasic dihydrate). the extract was stirred for 1 minute in vortex, in order to maximize the distribution of the analytes in the organic phase. the samples were centrifuged at 3000 rpm for 5 minutes and the supernatant was filtered at 0.45 m ptfe filters (vwr, milan, italy). the extract was analyzed by uhplc - ms / ms, making 6 replicates for each concentration. the average percentage of recovery and the relative standard deviation (rsd, repeatability) were evaluated. combined uncertainty in estimation was determined for all the neonicotinoids at the two fortification levels studied (10 and 40 g / kg) as the statistical procedure of the eurachem / citac guide cg 4. as the uncertainty of standard concentration declared in the supplier 's certificate was given without any confidence level, rectangular distribution was assumed for calculating standard uncertainty (1)u1= u(x)/c(x)3, where u(x) represents the uncertainty value given in the certificate and c(x) the concentration of the standard solution. the relative uncertainty due to honey weighing was calculated using normal distribution given by (2)u2=(0.00005)wi, where wi is the weight of the sample, and 0.00005 is the value of uncertainty of the balance at 95% confidence level as reported in the certificate. uncertainty associated with the calibration curve, was calculated according to (3)u3= (sb1) ({ 1p}+{1n}+ { (c0c)2sxx})1/2, where s is the standard deviation of the residuals of the calibration curve, b1 is the slope of the calibration curve, p is the number of measurements of the unknown, n is the number of points used to form the calibration curve, c0 is the calculated concentration of the analyte from the calibration curve, c is the arithmetic mean of the concentrations of the standards used to make the calibration curve, and sxx is calculated as given in (4)sxx=(cj c)2, where j = 1, 2,, n. cj is the concentration of each calibration standard used to build up the calibration curve. the random errors of extraction, clean up, and uhplc analyses steps were approximated by standard deviations which were calculated from repeated determinations of analytes expressed as repeatability. the precision was calculated according to (5)u4 = s(nx), where s is the standard deviation of the results obtained from the recovery study, n is the number of assays and x is the mean value of the concentration recovered. the volume of the solution is subject to 3 sources of uncertainty : calibration, repeatability, and temperature effects. (a) calibration : the uncertainty in the certified internal volume of the flask and of the pipettes. for example, the manufacturer gives a volume of 10.00 0.02 ml (v a) for the flask, when measured at a temperature of 20c. because the value of the uncertainty is given without a confidence level or distribution information, an assumption is necessary. in this work, the standard uncertainty is calculated by assuming a triangular distribution according to (6)u5 = (a/3)v. in the same way, the volumes of the pipettes used to prepare the solutions at different levels are calculated by assuming a triangular distribution. the contributions due to the dilution operations performed for each concentration level are calculated separately and combined to give the standard uncertainty of the volume. (b) repeatability : the uncertainty due to variations in filling is considered in the repeatability experiments. (c) temperature : the temperatures of the flask and solution differ from the temperature at which the volume of the flask was calibrated. according to the manufacturer, the flask was calibrated at a temperature of 20c, whereas the laboratory temperature varies by 2c. the uncertainty from this effect can be calculated from the estimate of the temperature range and the coefficient of the volume expansion. in the case of acetonitrile as a solvent, this effect is negligible. the combined uncertainty (u) was calculated as = x[(u1 + u2 + u3 + u4) ], where cx is the mean neonicotinoids concentration, and reported as expanded uncertainty (2u) which is twice the value of the combined uncertainty at 95% confidence level. in order to identify the major species produced in collisional experimental fragmentation of ms / ms analysis, a mass characterization study was firstly performed for direct infusion of each investigated neonicotinoids. mass scans in positive ions mode were performed with h - esi source ionization ; all investigated molecules showed a good fragmentation. the collision energy was modulated from 5 to 50 of instrumental maximum to obtain the better fragmentation pattern. the esi spectrum is characterized by the parent ion [m + h ] for all molecules. the neutral losses of no2 and/or hcl were observed for clothianidin, imidacloprid, nitenpyram, and thiamethoxam. the fragment at m / z 126, corresponding to [c6h5-ocl ] was a characteristic for acetamiprid, nitenpyram, and thiacloprid (table 1). the discussed srm data were in agreement with what reported by sabatino. and ferrer. the chromatographic method has been developed on the results of preliminary studies carried out on matrix - fortified standards. the best results were obtained using an elution gradient starting with a binary gradient of 0.05% hcooh and hcoonh4 2 mm in water and 0.05% hcooh and hcoonh4 2 mm in ch3oh combined with the thermo hypersil gold 50 2.1 mm (1.9 m i.d.) column. under the described chromatographic conditions, the studied molecules were resolved in less than 5 minutes (figure 1) and well recognizable on the basis of m / z signals, and good sensitivities were obtained ; each analyte showed a typical mass spectrum profile previously identified by direct infusion. the concept of a single extraction and dilution of the extracts was chosen in this study to achieve good results in the shortest time. in 2011, tanner and czerwenka applied two steps of purification with d - spe applying the quechers methodology to the honey. our protocol eliminated the second purification step, limiting the extraction to the use of d - spe citrate extraction tube and reducing times and costs of analyses. nevertheless, results were satisfactory in terms of statistical parameters, the selectivity for the analytes of interest, and reduction of the matrix effect (see paragraph below). this protocol permitted to analyze a high number of samples per day and is, therefore, suitable for a routine application in control laboratories. the proposed analytical protocol is currently applied in icqrf catania laboratory in the frame of italian ministry quality control investigation. analytical parameters of the proposed method were evaluated according to the criteria given in section 2. the linearity of each pesticide was established by plotting uhplc response area ratio versus concentration. the analytes showed linear behavior in the studied concentration range of 150 g / l. the correlation coefficient (r) was found to be 0.995 for all pesticides. lod and loq were estimated as the lowest concentrations of pesticide injected that yielded a signal / noise ratio of 3 and 10, respectively. loqs evaluation showed the lowest value 0.10 g / kg for thiacloprid to the higher value of 4.00 g / kg for nitenpyram. the loqs attained in the proposed method fit with maximum residue limits (mrls) of 10 g / kg for nonallowed pesticides. the single - step extraction method adopted for honey samples provided satisfactory recovery which ranged from 75% (nitepyram) to 114% (imidacloprid) for the fortification level of 10 g / kg and from 92 (thiacloprid) to 109% (imidacloprid) for the fortification level of 40 g / kg. the precision of the method was good, not exceeding a coefficient of variation of 12%, with the exception of nitenpyram at the lowest fortification level. therefore, the method could be considered sufficiently accurate and precise for the purpose. the study of uncertainty was performed at 2 concentration levels (10 and 40 g / kg), identifying and studying the most important parameters that determined the uncertainty of the analytical method. the parameters selected were point calibration, standard solution, weigh, volume, and precision ; their contributions to method uncertainty were calculated as indicated in the experimental section. the different contributions of uncertainty for each concentration level, together with the relative combined standard uncertainty, are shown in tables 3 and 4 for each neonicotinoid. results showed that the contribution to uncertainty due to the dilution operations and the standard purities was constant for each concentration level and for each analyte. the same value of uncertainty concerning the amount of weighed sample was used for each level and for all pesticides because the quantity of analyzed sample did not change among the experiments ; moreover, this contribution could be considered negligible. the uncertainty associated with repeatability has a moderate contribution to the expanded uncertainties, showing the higher value for nitenpyram, thiamethoxam, and clothianidin. the 10 g / kg level showed the uncertainty of calibration point as the main constituent of total uncertainty, followed by the volume contribution. on the contrary, the volume uncertainty was the major source to total uncertainty at the 40 g / kg level, while the uncertainty of repeatability and calibration point had approximately similar values. when the uncertainty of the result is reported, the combined standard uncertainty is multiplied with a so - called coverage factor, yielding an expanded uncertainty. a factor k = 2 was used because of the resemblance of the expanded uncertainty to a 95% confidence interval. sanco/12495/2011 recommended a default expanded uncertainty of 50% to be used by regulatory authorities in cases of enforcement decisions (mrl exceedances). our results showed a relative uncertainty (u%) ranging from 21 (thiamethoxam) to 49% (acetamiprid) at levels of 10 g / kg. lower values were obtained for the 40 g / kg level. at this level, | rapid and reliable multiresidue analytical methods were developed and validated for the determination of 6 neonicotinoids pesticides (acetamiprid, clothianidin, imidacloprid, nitenpyram, thiacloprid, and thiamethoxam) in honey. a modified quechers method has allowed a very rapid and efficient single - step extraction, while the detection was performed by uhplc / ms - ms. the recovery studies were carried out by spiking the samples at two concentration levels (10 and 40 g / kg). the methods were subjected to a thorough validation procedure. the mean recovery was in the range of 75 to 114% with repeatability below 20%. the limits of detection were below 2.5 g / kg, while the limits of quantification did not exceed 4.0 g / kg. the total uncertainty was evaluated taking the main independent uncertainty sources under consideration. the expanded uncertainty did not exceed 49% for the 10 g / kg concentration level and was in the range of 1619% for the 40 g / kg fortification level. |
the variety of biochemical functions that are being carried out by rna molecules is mesmerizing. many rnas such as ribosomal rna, rnaase p or trna attain a defined secondary and tertiary structure that is vital to their function. experimentally determined structures are only available for a small fraction of rnas that are of interest. this makes the computational prediction of the base - pairing pattern (the secondary structure) of rna an important problem. one major breakthrough was the development of dynamic programming algorithms that could predict the minimum free energy secondary structure of rna sequence assuming that the structures are non - nested (15). subsequently, dynamic programming algorithms have been extended to allow certain classes of pseudoknots (6,7). many rna secondary structure prediction algorithms (including the one presented here) are based on the idea of iteratively adding substructures to an initially unfolded sequence (8,9). genetic algorithms are an example of such algorithms and have proven very useful for exploring pseudoknotted structures and sub - optimal rna structures (1014). allowing pseudoknots is desirable simply because rna structures determined by x - ray crystallography or nmr revealed that many rnas contain non - nested base pairing interactions. allowing all possible base pairing interactions leads to the potential problem for structure prediction approaches that not only are there many more conformations to consider, but also many conformations are not sterically feasible. here, we describe a computational approach for rna secondary structure prediction that has no restriction in terms of pseudoknot complexity, but additionally checks the steric feasibility of the considered conformations. the described approach of rna secondary structure prediction is based on the idea of maximizing matching helices in a secondary structure (10). a flow chart of the algorithm is shown in figure 1. briefly, the method works as follows : initially, a list (called a stem - list) of all possible helices with more than 3 bp is generated. the secondary structure prediction is performed by picking the best - scoring structure obtained after 50 folding simulation runs. the score is set to be the sum of the free energy contribution of the already placed helices. each folding simulation run is performed by picking helices from the stem list with a boltzmann - weighted probability. estimating the free energy contribution of an rna double - helix is accomplished using the rna vienna package (2). each chosen helix is represented by a very coarse - grained 3d representation in a virtual 3d workspace. an rna double helix is represented by a cylinder (using a radius of 6.5 and a length of 2.7 times the number of base pairs) that is capped with a half - sphere on both ends. this shape is called a capsule. a schematic diagram of the mapping of an rna secondary structure into a highly coarse - grained 3d representation is shown in figure 2. the main reason for choosing capped cylinders over regular cylinders is the computational efficiency of collision detection. single stranded regions between helices are represented as constraints for the maximum distance between the ends of the capped cylinders. a newly chosen capped cylinder is placed into the 3d simulation space at a random position such that the distance - constraints are fulfilled. the distance constraints are a function of the single - stranded sequence lengths between connected helices. the maximum distance between helix ends is 2.0 + n8.0 with n being the sequence separation. figure 2.scheme for mapping between an rna secondary structure (a) and the used 3d coarse - grained representation (b). only those rna secondary structures can be a (partial) solution of a secondary structure prediction, for which the algorithm succeeds in placing the corresponding capped cylinders such that they do not collide and do not violate distance constraints. a flow - chart depicting the algorithm for predicting rna secondary structures. scheme for mapping between an rna secondary structure (a) and the used 3d coarse - grained representation (b). only those rna secondary structures can be a (partial) solution of a secondary structure prediction, for which the algorithm succeeds in placing the corresponding capped cylinders such that they do not collide and do not violate distance constraints. if cylinders collide, the newly placed capped cylinder is placed at a different random position. if after 20 attempts the newly placed capped cylinder is still colliding with previously placed capped cylinders, the positions of all capped cylinders are optimized in order to minimize collisions and constraint violations. if no collision - free position can be found, the newly chosen helix and its capped cylinder representation is discarded. helices that are part of the stem - list and that share bases with the newly placed helix are removed from the stem - list. in the next iteration the next helix is chosen until no more helices can be placed. once no more fifty simulation runs are performed and the overall best - scoring structure is returned to the user. the web server has been implemented using the grails framework (18), which is based on the groovy programming language. for a secondary structure prediction request, the web server launches the cylofold binary on a linux compute cluster. after the prediction result has been generated, the program varna (19) a user of the cylofold prediction web server can start a secondary structure prediction request by entering (pasting) a nucleotide sequence (as raw characters or in fasta form, both acgu and acgt alphabets are accepted) into the web form and pressing submit. the maximum sequence length that is currently accepted by the web server is 300 nt. the initial return of the web server is a unique i d, which is needed if one wants to access results at a later time. due to the compute - intensive approach for the prediction, it can take several minutes for the server to finalize a secondary structure prediction. the user can access the results by one of three methods : a simple reload of the initial result page will update the status of the prediction and will eventually contain the prediction results. alternatively, the user can bookmark the initial result page in the web browser and return to it at a later time. lastly, the unique i d provided after submitting the secondary structure prediction compute request can be used to access the results using another web form available on the server home page. a typical output from a completed rna structure prediction the prediction result is presented to the user in three different formats : (i) an image of the predicted rna secondary structure created by varna (19) ; (ii) an extended bracket notation in which nested base pairs are denoted as pairs of nested parentheses and helices corresponding to pseudoknot interactions are denoted as letters ; (iii) the ct file format that is also generated by other programs such as mfold (5). this format contains a list of the indices of the bases and their predicted base - pairing partners. the shown sequence corresponds to the bacteriophage t2 gene 32 mrna pseudoknot (pdb 2tpk). the shown sequence corresponds to the bacteriophage t2 gene 32 mrna pseudoknot (pdb 2tpk). a user of the cylofold prediction web server can start a secondary structure prediction request by entering (pasting) a nucleotide sequence (as raw characters or in fasta form, both acgu and acgt alphabets are accepted) into the web form and pressing submit. the maximum sequence length that is currently accepted by the web server is 300 nt. the initial return of the web server is a unique i d, which is needed if one wants to access results at a later time. due to the compute - intensive approach for the prediction, it can take several minutes for the server to finalize a secondary structure prediction. the user can access the results by one of three methods : a simple reload of the initial result page will update the status of the prediction and will eventually contain the prediction results. alternatively, the user can bookmark the initial result page in the web browser and return to it at a later time. lastly, the unique i d provided after submitting the secondary structure prediction compute request can be used to access the results using another web form available on the server home page. a typical output from a completed rna structure prediction the prediction result is presented to the user in three different formats : (i) an image of the predicted rna secondary structure created by varna (19) ; (ii) an extended bracket notation in which nested base pairs are denoted as pairs of nested parentheses and helices corresponding to pseudoknot interactions are denoted as letters ; (iii) the ct file format that is also generated by other programs such as mfold (5). this format contains a list of the indices of the bases and their predicted base - pairing partners. the shown sequence corresponds to the bacteriophage t2 gene 32 mrna pseudoknot (pdb 2tpk). the shown sequence corresponds to the bacteriophage t2 gene 32 mrna pseudoknot (pdb 2tpk). the performance of the new rna secondary structure prediction method was evaluated using two different data sets. data set 1 (corresponding to the results shown in table 1) consists of 26 rna sequences, whose tertiary structure is available in the protein data bank (pdb). the reference secondary structure was obtained by extracting the base pair information from the pdb coordinate file using the program rnaview (20). data set 2 consists of 241 rna sequences and secondary structures originating from pseudobase (21,22). table 1.prediction results corresponding to 26 rna structures that are available in the protein dank bankpdbdescriptionlpkfcfpkhkufmccsnsppvmccsnsppvmccsnsppvmccsnsppv1a60tymv trna - like structure4413.60.740.770.710.961.000.930.830.770.910.830.770.911cx0hdv ribozyme7222.20.010.000.000.010.000.000.010.000.000.010.000.001e95srv-1 pseudoknot3633.31.001.001.001.001.001.001.001.001.000.700.501.001hvuhiv rt bind. intron700.00.910.870.950.810.830.790.810.830.790.960.960.961l2xviral rna pseudoknot2722.20.941.000.890.941.000.890.790.631.000.790.631.001q9a23s rrna sarcin / ricin270.00.910.831.000.770.830.710.861.000.750.770.830.711u8dguanine riboswitch6711.90.870.870.870.880.781.000.880.781.000.880.781.002a43luteoviral pseudoknot2623.00.931.000.880.931.000.880.750.571.000.750.571.002g1wtmrna pseudoknot2218.10.811.000.670.861.000.750.810.671.000.810.671.002gissam- riboswitch948.50.800.760.850.800.760.850.860.860.860.550.550.552hoothi - box riboswitch830.00.700.670.740.580.620.540.580.620.540.580.620.542k95p2b - p3 telo - merase rna4837.50.890.801.000.890.801.000.750.80.710.540.400.752oiul1 ribozyme ligase adduct710.00.860.780.950.980.961.000.980.961.000.981.001.002qushammerhead ribozyme682.90.950.911.000.950.911.000.950.911.000.951.001.002qwysam - ii riboswitch5226.90.480.460.500.480.460.50.340.310.40.350.310.402rp0pemv1 mrna pseudoknot2615.30.881.000.780.881.000.780.840.711.000.840.711.002tpkt2 gene 32 mrna p.k.3627.71.001.001.001.001.001.000.710.580.880.630.580.70361ddomain e of 5s rrna190.00.861.000.750.861.000.750.830.830.830.830.830.833diglysine riboswitch17330.10.890.850.930.740.720.760.740.720.760.740.720.763fu2class - i preq1 riboswitch3218.80.790.631.000.790.631.000.790.631.000.790.631.003phptymv p.k. frame - shifting p.k.2722.20.941.000.890.941.000.890.790.631.000.790.631.00meanall0.830.850.830.820.840.810.750.710.830.730.650.83meanno pseudoknots5.00.810.840.800.820.840.820.730.650.840.660.550.80l, sequence length ; pkf, fraction of pseudoknot interactions ; for each of the four different prediction methods (cf, cylofold ; pk, pknotsrg ; hk, hotknots 2.0 ; uf, unafold) we report three different measures of prediction quality (sns, sensitivity ; ppv, positive predictive value). prediction results corresponding to 26 rna structures that are available in the protein dank bank l, sequence length ; pkf, fraction of pseudoknot interactions ; for each of the four different prediction methods (cf, cylofold ; pk, pknotsrg ; hk, hotknots 2.0 ; uf, unafold) we report three different measures of prediction quality (sns, sensitivity ; ppv, positive predictive value). in order to quantify the time - complexity of the folding method, we fitted a function of the form an (with n being the number of residues in the input sequence) to the execution time needed for the cases of the 241 sequence set. we found that the execution time (measured in seconds) of the structure prediction is well described by the function 2.74 10n. the timing evaluation was performed on a computer with 4 gb of ram and an intel 64-bit xeon processor (3.0mhz). we report in tables 1 and 2 prediction results for these two data sets together with the corresponding results obtained by running the rna secondary structure prediction programs hotknots 2.0 (8), pknotsrg (7) and unafold (23). table 2.prediction results for a set of 241 rna sequences that are part of pseudobase for the programs cylofold, pknotsrg (7), hotknots 2.0 (8) and unafold (23)mccsnsppvcylofold0.7520.7630.747pknotsrg0.7480.7530.756hotknots 2.00.6110.5650.684unafold0.5970.5320.692sns, sensitivity of predicted base pairs ; ppv, positive predictive value. prediction results for a set of 241 rna sequences that are part of pseudobase for the programs cylofold, pknotsrg (7), hotknots 2.0 (8) and unafold (23) sns, sensitivity of predicted base pairs ; ppv, positive predictive value. the average matthews correlation coefficient (mcc) obtained by comparing the base pairing pattern of the predicted secondary structures with their respective reference secondary structure is for data set 1 and cylofold 0.83 ; this can be compared to pknotsrg (0.82), hotknots 2.0 (0.75) and unafold (0.73) (see row of table 1 named we divided this data set into two subsets according to the fraction of pseudoknot base pairs in the respective structures. the eight pdb structures with 5% correspond to an average mcc of 0.81 for cylofold, 0.82 for pknotsrg, 0.73 for hotknots 2.0 and 0.66 for unafold. using the larger data set 2, one obtains an average mcc of 0.752 for cylofold and 0.748 for pknotsrg one can see the rna secondary structure predictions obtained by cylofold correspond to the highest mcc (compared to the programs pknotsrg, hotknots 2.0 and unafold). it also has the highest average base pair prediction sensitivity (0.763). for another measure, the positive predictive value (how often are predicted base pairs part of the reference secondary structure), all programs obtain averages between 0.68 and 0.76 for data set 2 with pknotsrg leading with a value of 0.756. it should be noted that the mcc is often used as an overall measure of prediction quality, while sensitivity, specificity and positive predictive value capture certain other aspects of the prediction quality. the key advantage of cylofold is that there is no restriction in terms of the classes of pseudoknots that are being considered. also, it should be noted that the employed model of simulated rna folding by placing helices with a probability according to their free energy contribution is in essence very simple (24). in that sense it is surprising how well the method performs, and it should be an encouragement to continue to develop rna folding algorithms that are substantially different from established approaches. we show using two different data sets that the prediction accuracy (mcc) is comparable to the rna secondary structure prediction program pknotsrg. another novel aspect is that at each step during the simulated folding process, the steric feasibility of the predicted structures is checked for steric feasibility using a highly coarse - grained 3d representation. the method is made available in the form of a user - friendly web server. this project has been funded in whole or in part with federal funds from the national cancer institute, national institutes of health, under contract hhsn26120080001e. this research was supported by the intramural research program of the nih, national cancer institute, center for cancer research. | computational rna secondary structure prediction approaches differ by the way rna pseudoknot interactions are handled. for reasons of computational efficiency, most approaches only allow a limited class of pseudoknot interactions or are not considering them at all. here we present a computational method for rna secondary structure prediction that is not restricted in terms of pseudoknot complexity. the approach is based on simulating a folding process in a coarse - grained manner by choosing helices based on established energy rules. the steric feasibility of the chosen set of helices is checked during the folding process using a highly coarse - grained 3d model of the rna structures. using two data sets of 26 and 241 rna sequences we find that this approach is competitive compared to the existing rna secondary structure prediction programs pknotsrg, hotknots and unafold. the key advantages of the new method are that there is no algorithmic restriction in terms of pseudoknot complexity and a test is made for steric feasibility. availability : the program is available as web server at the site : http://cylofold.abcc.ncifcrf.gov. |
glycogen storage disease type ii (gsdii), also known as pompe s disease or acid maltase deficiency, is a rare, autosomal recessive, progressive neuromuscular disease caused by the deficiency of lysosomal acid -glucosidase (gaa) or acid maltase. the enzyme catalyzes the hydrolysis of -1,4 and -1,6 links of glycogen and its deficiency leads to intra - lysosomal accumulation of glycogen. in patients with the classic infantile form, the deposition of glycogen in the heart, skeletal, and respiratory muscles causes severe cardiomyopathy, hypotonia, and respiratory failure, typically leading to death within the first year of age. in late - onset gsdii patients glycogen deposition is confined mainly to skeletal and respiratory muscles, causing progressive limb - girdle myopathy and respiratory insufficiency. a considerable number of patients become wheelchair dependent and may require assisted ventilation later in life. an inverse correlation is usually observed between the amount of residual gaa activity and disease severity, and in general the symptoms do not emerge until the gaa activity remains above 30% of average normal activity. until recently, no effective therapy for gsdii patients was available, even if physical activity alone or in parallel with an high - protein and low - carbohydrate dietary regime has been demonstrated to improve quality of life and motor function. enzyme replacement therapy (ert) with recombinant human -glucosidase became available in 2000, and currently a number of studies have been published on the efficacy and safety of ert in gsdii disease. clinical studies in infants have shown that ert led to improvement in skeletal and cardiac muscle function and to increased survival in many patients. few studies on the efficacy of this treatment in older children and adults have been published so far [69 ]. overall these studies demonstrated that ert is associated, over a 12- to 36-month period, with a stabilization of pulmonary function and with an improved exercise tolerance, as estimated by the 6-min walking test (6mwt), which has been demonstrated to be an appropriate outcome measure. however, besides being intrinsically imprecise, the 6mwt does not provide specific information on the function of the different organs and systems involved in exercise, or the mechanism of exercise limitation. insights into these issues, together with a more precise quantification of the exercise intolerance, could derive from standard cardiopulmonary exercise testing, associated with measurements of pulmonary o2 uptake, cardiac and skeletal muscle functions carried out during incremental exercise up to voluntary exhaustion. in the present study we report a 12-month follow - up of four late - onset gsdii patients who underwent ert. in particular, data related to cardiovascular and metabolic responses to exercise and respiratory function are provided. all measurements were non - invasive, so they could be easily repeated as a function of time. four late - onset gsdii patients (2 males and 2 females, 45 6 years) were investigated. ert with recombinant human -glucosidase (myozymegenzyme corporation, cambridge, mass) was administered intravenously at a dose of 20 mg / kg every 2 weeks. before treatment (before) and after 12 months of ert (after) the patients were evaluated in our laboratory. the subject were fully informed of any risk and discomfort associated with the experiments before giving their written informed consent to participate to the study, which was approved by the local institutional ethics committees. all procedures were in accordance with the recommendations found in declaration of helsinki (2000) of the world medical association. pulmonary function variables, forced vital capacity (fvc) and forced expiratory volume in 1 s (fev1), were measured with the patients in sitting position by a spirometer. exercise tolerance was assessed on an electronically braked cycle ergometer (corival, lode, the netherlands). an incremental exercise was performed : after sitting for a few minutes at rest the patient performed at 1530 w for 5 min, and thereafter the workload was increased by 510 w (according to the subject s estimated level of physical fitness) every minute until voluntary exhaustion was reached. for cardiovascular, gas exchange, and muscle oxygenation variables mean values were calculated during the last 30 s of each load. the highest values reached by the patient were taken as peak values.pulmonary ventilation (ve, expressed in btps), o2 uptake (vo2), and co2 output (vco2), both expressed in stpd, were determined breath - by - breath by a metabolic cart (sensormedics vmax29c ; the netherlands). expiratory flow measurements were performed by a mass flow sensor (hot wire anemometer), calibrated before each experiment by a 3-l syringe at three different flow rates. tidal volume and ve were calculated by integration of the flow tracings recorded at the mouth of the subject. vo2 and vco2 were determined by continuously monitoring partial pressures of oxygen (po2) and carbon dioxide (pco2) at the mouth throughout the respiratory cycle and from established mass balance equations, after alignment of the expiratory volume and expiratory gases tracings and a / d conversion. calibration of o2 and co2 analyzers was performed before each experiment by utilizing gas mixtures of known composition. gas exchange ratio (r) was calculated as vco2/vo2. heart rate (hr) was monitored from the electrocardiogram ; stroke volume (sv) was estimated beat - by - beat by impedance cardiography (physio flow, manatec, paris, france). the accuracy of this device has been previously evaluated during incremental exercise in healthy subjects against the direct fick method. oxygenation changes in a superficial portion of the vastus lateralis muscle were evaluated by near - infrared spectroscopy (nirs). a portable nir single - distance continuous - wave photometer (heo-100, omron, japan), the instrument provides separate measurements of changes in deoxygenated hb and mb concentrations, as well as of oxygenated hb and mb concentrations, expressed in arbitrary units. further details on the method can be found in a previous article by our group. for a detailed discussion regarding advantages and limitations of nirs measurements in skeletal muscle, concentration changes of deoxygenated hb + mb ([deoxy(hb + mb) ]), with respect to an initial value arbitrarily set equal to zero, were calculated and expressed in arbitrary units. [deoxy(hb + mb) ] was taken as an oxygenation index, since this variable is relatively insensitive to changes in blood volume. [deoxy(hb + mb) ] data were expressed as a percentage of the values determined after the exercise by obtaining a maximal deoxygenation of the muscle, by pressure cuff inflation (at about 300 mmhg) carried out at the inguinal crease of the thigh (subject in the sitting position on the cycloergometer), for a few minutes until [deoxy(hb + mb) ] increase reached a plateau. results were expressed as mean values standard deviation (x sd). the statistical significance of differences between means was checked by paired students t - test. no side effects of therapy were observed. we did not use validated scales to assess quality of life, however, the patients reported a progressive subjective improvement of daily performance, and particularly less fatigability both at work and during activities of daily living. after 9 months of treatment one of the patients spontaneously started to perform 23 times per week short sessions (about 15 min) of exercise on a stationary bicycle. 1. in before fvc and fev1 were, on the average, 73% and 74% of the predicted normal value, respectively. for both variables, either expressed in l or as a percentage of the predicted value, ert was associated with a slight improvement, which, however, did not reach statistical significance, presumably as a consequence of the low number of patients (see also below). the patients tolerated the exercise testing relatively well and none of them complained of significant discomfort, pain or delayed onset muscle soreness. peak values of work rate, vo2, co and ([deoxy(hb + mb) ] are shown in fig. 2 (also for [deoxy(hb + mb) ] peak the data were not available for one of the patients). vo2peak increased by 10% from before (1.01 0.42 l / min, or 17.2 4.4 ml / kg / min) to after (1.14 0.36 l / min, or 19.7 3.5 ml / kg / min). on the average vo2peak values obtained in after correspond to 5 mets, or 5 times the resting energy expenditure (1 met = 3. ml o2 kg min), and to 59% of the sex- and age - predicted peak value. the increase in vo2peak was associated with a clear tendency for an increase in copeak, from 12.3 5.3 to 14.8 4.5 l / min. both hrpeak (which increased from 134 16 beats / min to 145 25 beats / min) and peak stroke volume (from 91 29 to 101 21 ml) contributed to the increased copeak. for copeak, hrpeak and svpeak the increase, in after vs. before, the same can be said for [deoxy(hb + mb) ] peak, taken as an estimate of skeletal muscle fractional o2 extraction (for details see). the major finding of the present study is that in patients with glycogen storage disease type ii enzyme replacement therapy is associated, over a 12-month period, with a stabilization of pulmonary function and with a mild improvement of exercise tolerance. two widely accessible and simple parameters, fvc and fev1, were used to monitor respiratory function. a nonsignificant trend in fvc and fev1 improvement these findings are consistent with those recently reported in larger ert trials on late - onset gsdii patients treated for 12, 18 and 36 months. the stabilization or the slight improvement of pulmonary function observed in our study, as well as in the previously mentioned ones, is in contrast to the progressive deterioration that characterizes the natural course of the disease (annual decline of almost 23% in the percentage of predicted fvc). significantly higher values of vo2peak after were associated with (and presumably were responsible for) a significant improvement of exercise tolerance, as shown by the significantly higher peak work rate values. the increase in vo2peak was associated with an improvement of copeak and [deoxy(hb + mb) ] peak. in two of the patients [deoxy(hb + mb) ] peak values were markedly lower than the values observed by our group in healthy subjects, but higher than those obtained on patients with mitochondrial myopathies or myophosphorylase deficiency. + mb) ] peak values were substantially unchanged compared with those determined at rest ; in this patient, characterized by the lowest work rate and vo2peak values, the variable was unaffected by ert. as nicely discussed by poole., it would be an oversimplification to interpret skeletal muscle fractional o2 extraction simply as a result of muscle factors. the enhanced peak fractional o2 extraction described with training in two of our patients may indeed be the result of a combination of factors such as increased bulk blood flow and o2 delivery ; enhanced vasodilation and capillary recruitment ; improved intramuscular matching of o2 delivery and o2 utilization ; enhanced peripheral o2 diffusion ; improved endothelial function ; reduced levels of inflammatory, catabolic and pro - apoptotic mediators and oxidative stress ; increased mitochondrial volume density and activity of oxidative enzymes ; enhanced oxidative phosphorylation, etc. all these factors, which could be at least in part related to a decreased disease - induced damage in muscle tissue, could explain (and be a result of) the increased exercise tolerance. in late - onset gsdii patients skeletal muscle pathology is extremely heterogeneous, ranging from substantially unaffected fibers to a complete destruction of contractile machinery. the pathogenic mechanisms of muscle damage are still under debate, but autophagy is increasingly identified as a pivotal contributor to muscle destruction and mitochondrial abnormalities have been repeatedly found. regarding the response to ert, clinical trials have indicated that ert is effective in glycogen clearance in cardiac muscle, whereas a reversal of the damage in skeletal muscle has not always been achieved, and highly variable responses between patients should be expected. unfortunately, muscle biopsies were not performed in the present study, thus we have no data of the degree of muscle damage in the patients in before and in after. as discussed above, the subjective improvement observed by one of the patients during the first few months of ert induced her to spontaneously adopt a home - based light exercise training protocol. thus, it can not be excluded that the beneficial effects described in the present study after ert can be attributed, at least in part, to the increased level of physical activity. recently, evidence has been provided that ert and exercise training could have additive positive effects on these patients exercise tolerance and, ultimately, on their quality of life. for some of the variables determined in the present study (spirometry data, copeak, [deoxy(hb + mb) ] peak) the observed increases did not reach statistical significance. this could be attributed to the low number of patients, which does not allow us to exclude the possibility of a type 2 error. thus, the findings need to be validated on a larger group of patients and with a longer follow - up period. in conclusion, our findings showed that in late - onset gsdii patients ert is associated with a mild improvement of pulmonary function and exercise tolerance over a 12 month period. the improved exercise tolerance seems associated with improvements both in cardiovascular and in skeletal muscle functions. the findings need to be validated on a larger group of patients and with a longer follow - up period. in addition, the results highlight the role that cardiopulmonary exercise testing, with simultaneous non - invasive measurements of pulmonary o2 uptake, cardiac output and skeletal muscle oxygenation, can play in the assessment and follow - up of late onset gsdii patients. | enzyme replacement therapy (ert) has recently became available for patients with glycogen storage disease type ii. previous studies have demonstrated clinical efficacy of enzyme replacement therapy, however, data on physiological variables related to exercise tolerance are scarce. four glycogen storage disease type ii late - onset patients (45 6 years) performed an incremental exercise on a cycle ergometer, up to voluntary exhaustion, before (before) and after 12 months of ert (after). peak workload, oxygen uptake, heart rate, cardiac output (by impedance cardiography) and vastus lateralis oxygenation indices (by continuous - wave near - infrared spectroscopy, nirs) were determined. peak workload and oxygen uptake values significantly increased during ert (54 30 vs. 63 31 watt, and 17.2 4.4 vs. 19.7 3.5 ml / kg / min, respectively, in before vs. after). on the other hand, for both peak cardiac output (12.3 5.3 vs. 14.8 4.5 l / min) and the nirs - determined peak skeletal muscle fractional o2 extraction, expressed as a percentage of the maximal values during a transient limb ischemia (30 39% vs. 38 28%), the observed increases were not statistically significant. our findings suggest that in glycogen storage disease type ii patients enzyme replacement therapy is associated with a mild improvement of exercise tolerance. the findings need to be validated during a longer follow - up on a larger group of patients. |
systemic sclerosis (ssc) is a connective tissue disease characterized by fibrosis and vasculopathy involving multiple organ systems. classification into diffuse or limited cutaneous forms depends on the extent of skin thickening, with the former affecting areas proximal to the elbows or knees, and the latter limited to the face and distal extremities. many clinical complications of ssc are due to dysfunction of vascular beds throughout the body. involvement of the microvasculature leads to cutaneous and mucosal telangiectasias, digital ulcers, and tissue ischemia. if medium - sized blood vessels are involved, manifestations include gangrene, digital loss, renal crisis, and pulmonary arterial hypertension. while occlusive vasculopathy is a well - recognized feature of ssc, less is known about the occurrence and the consequences of frank vascular inflammation. albeit rare, typical vasculitis with inflammatory infiltrates damaging blood vessels has been reported in patients with ssc. distinguishing between noninflammatory vasculopathy and vasculitis can pose a significant diagnostic challenge in the absence of histologic examination. here, we review reported cases of large-, medium-, and small - vessel vasculitis in association with ssc. the distinction between ssc vasculopathy and vasculitis can be difficult to make based on clinical presentation alone, but knowledge of the underlying pathogenesis and histopathology can be very helpful. in the current pathogenic model of ssc, a vascular injury of unknown cause leads to endothelial apoptosis and initiates the process of ssc vasculopathy. autoantibodies, reperfusion injury, infection, and defects in vascular repair have all been implicated as possible instigators. increased levels of endothelial cells in the circulation have been cited as evidence that the intactness of the vascular lining is jeopardized [3, 4 ]. subsequent endothelial dysfunction results in the imbalance of vasoactive factors : decreased levels of vasodilators such as endothelial nitric oxide synthase and prostacyclin synthase, as well as increased levels of the vasoconstrictor endothelin-1 and vascular endothelial growth factor [5, 6 ]. continuous endothelial dysfunction likely contributes to activation of adventitial fibroblasts with resultant intimal proliferation, eventual luminal narrowing, and tissue hypoxia [4, 7 ]. histopathology of ssc vasculopathy reflects the underlying pathogenesis, with myofibroblast proliferation and matrix deposit in the subendothelial layer leading to obliterative thickening of vessel walls. inflammatory infiltrates are absent, and the internal elastic lamina remains intact. in contrast to vasculopathy, concurrent vasculitis in ssc shows histopathologic evidence of inflammation, with presence of mononuclear infiltrates and destruction of the vascular wall. notably, both vasculopathic and vasculitic changes were seen in five of nine (55%) digital amputation specimens from ssc patients, emphasizing that small vessel vasculitis and stenosing vasculopathy may coexist. further support has come from autopsy studies of ssc patients, where 24% of 58 cases showed noninflammatory intimal proliferation in two or more organs, but 9% had features of inflammatory polyarteritis. thus, vasculitis is known to occur even in the setting of a disease predisposing towards vasculopathy, and histology is required to distinguish the two pathogenic processes. giant cell arteritis is a common vasculitis of the elderly involving large- and medium - sized arteries, typically the temporal, ophthalmic, vertebral, and axillary arteries as well as the aorta. the american college of rheumatology (acr) criteria include at least three of the following : (1) onset at age > 50, (2) new headache, (3) claudication of the jaw or tongue, (4) temporal artery tenderness to palpation or decreased pulsation, (5) esr > 50 mm / h, and (6) temporal artery biopsy showing vasculitis with mononuclear inflammatory infiltrate or granulomatous inflammation with presence of giant cells. typical histomorphologic findings include disruption of the internal elastic lamina, thinning of the media, and occlusion of the lumen by hyperplastic intima. pathogenic studies have established that giant cell arteritis is a t - cell driven disease with participation of th1 and th17 lineages [11, 12 ]. steroids remain the mainstay of therapy, with many cases resolving after one to two years. while giant cell arteritis is relatively common among the vasculitides, it has only been reported in three cases of concurrent ssc, all of which were women in their sixth decade with limited skin involvement [1315 ] (table 1). two of the three had the classic presentation of headache, jaw claudication, and elevated sedimentation rate (esr), with evidence of vessel wall inflammation and giant cells on temporal artery biopsy. the case reported by sari - kouzel was unusual in that the presenting symptom was pain and discoloration of the right foot in the setting of normal esr. the lower extremity ischemia eventually progressed to gangrene necessitating a below - the - knee amputation. while ssc vasculopathy may have contributed to ischemic tissue damage, the histology from the amputation specimen yielded evidence for vasculitis with the presence of inflammatory infiltrates, giant cells in the vessel wall, and vascular lumen obliteration. all patients were started on corticosteroids (prednisolone or prednisone 30 to 80 mg daily) with slow taper over 56 months and resolution of symptoms. takayasu arteritis is a relatively rare large vessel vasculitis (incidence 0.42/million / year) affecting mostly young women of asian origin although the incidence among the middleaged with atherosclerosis has been rising [20, 21 ]. acr diagnostic criteria include at least three of the following : (1) onset before age 40, (2) claudication of an extremity, (3) decreased brachial artery pulse, (4) > 10 mmhg in systolic blood pressure between the arms, (5) bruit over the subclavian arteries or aorta, and (6) stenosis / occlusion of the aorta, its major branches, or large arteries in proximal upper or lower extremities. similar to giant cell arteritis, the histopathology in takayasu arteritis shows mononuclear infiltrates in the vessel wall, intimal thickening, destruction of elastic laminas, giant cell formation, and expansion of the adventitial layer. elastic lamina destruction can lead to aneurysm formation while transmural inflammation drives intimal proliferation, adventitial scarring, and vascular lumen narrowing. treatment with steroids leads to remission in 40% of patients while 40% may relapse or require addition of a second drug such as methotrexate or azathioprine [24, 25 ]. four cases of takayasu arteritis in the setting of ssc have been reported. as the overwhelming majority of patients with takaysu arteritis are female, all four of these cases were women, with ages ranging from 29 to 68 [1619 ]. the presenting symptoms included arm claudication and lightheadedness, and physical examination revealed pulselessness in upper extremities, blood pressure discrepancies > 10 mmhg as measured in both arms, and in one case bruits involving the neck and the back. computed tomographic angiography in all cases showed stenosis of various aortic branches, including the brachiocephalic trunk, common carotid, subclavian, celiac, and renal arteries. thoracic outlet syndrome was concurrently diagnosed in one case of arteritis, with imaging demonstrating compression of the brachial artery by the scalenus muscle. two of the four patients were older than 40 years of age at the time of takayasu arteritis diagnosis, raising the question whether they indeed had typical large vessel vasculitis or whether a component of vessel - obstructive and progressive atherosclerosis was part of the disease process. polyarteritis nodosa (pan) is a necrotizing vasculitis affecting medium - sized vessels, with a constellation of clinical findings that reflect multiorgan involvement. pan can be distinguished from the small - vessel vasculitides such as microscopic polyangiitis by the absence of antineutrophil cytoplasmic antibodies. the acr diagnostic criteria for pan include at least three of the following : (1) weight loss > 4 kg, (2) livedo reticularis, (3) testicular pain or tenderness, (4) myalgias, weakness, or leg tenderness, (5) mono- or polyneuropathy, (6) hypertension, (7) elevated blood creatinine or urea, (8) serum hepatitis b antigen or antibody, (9) aneurysms or occlusions of visceral arteries, or (10) granulocytes on biopsy of small- or medium - sized arteries. a recent retrospective study of 348 patients with pan found general symptoms in 93.1%, neurologic involvement in 79%, and skin involvement in about 50%. five - year relapse - free survival was 59.4% for nonhepatitis - b - associated pan and 67% for hbv - associated pan. predictors of mortality included age greater than 65 years, new onset hypertension, and gastrointestinal manifestations requiring surgery. treatment relies on glucocorticoids in mild disease and a combination of glucocorticoids and cyclophosphamide in moderate to severe disease. only one case of pan has been described in a 28-year - old woman with diffuse ssc, characterized by raynaud 's phenomenon and skin sclerosis over the hands, arms, and chest (table 2). biopsy of these lesions revealed necrotizing arteritis in the deep dermis. despite the histologic appearance of the nodules, the patient technically did not meet acr criteria for pan as she had normal blood pressure and renal function, negative hepatitis b serologies, and no other symptoms such as weakness or neuropathy. primary angiitis of the central nervous system (pacns) is a rare poorly characterized entity affecting small- and medium - sized vessels of the central nervous system (cns) but not organs or vessels outside the cns. in general, pacns is distinguished from secondary cns vasculitis with the exclusion of infections, malignancy, systemic vasculitis or connective tissue disease, or drug - induced vasculitis. clinical presentations of pacns include confusion, new onset headache, seizures, stroke or cerebral hemorrhage, and myelopathy. the duration from symptom onset to diagnosis can range from 3 days to 3 years. multiple laboratory data abnormalities can occur but none is specific for the diagnosis, with esr described to be normal in a number of cases. characteristic changes on cerebral angiography include multifocal segmental stenosis, dilatation, or occlusion of small- and medium - sized leptomeningial and intracranial vessels as well as formation of collateral vessels. further supportive evidence can be obtained from leptomeningeal or parenchymal biopsies, which are specific but not sensitive for vasculitis given the focal segmental nature of the disease ; therefore, a negative biopsy does not rule out the diagnosis. histology can show either granulomas in small vessel walls, lymphocytic infiltrates, or necrotizing vasculitis. calabrese and mallek have proposed the following diagnostic criteria for pacns : (1) recent onset of headache, confusion, or multifocal neurologic deficits, (2) cerebral angiographic changes suggestive of vasculitis, (3) exclusion of systemic disease or infection, and (4) leptomeningeal or parenchymal biopsy to confirm vasculitis and to exclude infection, malignancy, and noninflammatory vascular occlusive disease. once the diagnosis is made, aggressive treatment with high - dose steroids and cyclophosphamide has been suggested as the management of choice. one case of pacns has been described in ssc (table 2). a 45-year - old woman with limited ssc diagnosed at age 21 presented with new onset headache for 3 days and confusion, later developing hypertension to the 230s/190s and generalized seizure. computed tomography of the head and spinal fluid was unremarkable. cerebral angiogram showed an occluded medium - sized branch of the middle cerebral artery as well as narrowing of several distal medium - sized arteries in the anterior and middle cerebral artery distribution. the patient was empirically started on methylprednisolone 100 mg iv every 4 hours for suspicion of pacns, and her mental status was normalized within 14 hours. a repeat cerebral angiogram of the posterior circulation showed multiple 1.5 cm segments of smooth narrowing in medium - sized arteries. the steroid dosage could not be tapered below prednisone 50 mg daily, as each time the patient developed right facial and arm paresthesias and expressive aphasia. despite a negative leptomeningeal biopsy, cyclophosphamide was started at 100 mg daily and gradually increased to 200 mg daily with complete resolution of symptoms. mixed cryoglobulinemia is the presence of polyclonal immunoglobulins that precipitate in the serum with cold exposure, often secondary to a connective tissue disease such as systemic lupus erythematosus or sjogren 's syndrome. the presence of cryoglobulins (cgs) may be asymptomatic or may lead to manifestations of the cryoglobulinemic syndrome, including purpura, arthralgia, myalgia, glomerulonephritis, and peripheral neuropathy. the diagnosis of the latter entails a combination of clinical presentation, laboratory testing showing the presence of circulating cryoglobulins, and histopathologic appearance such as leukocytoclastic vasculitis (most common). similarly, cryofibrinogenemia is the presence of cold - induced precipitants in the plasma but not in the serum. connective tissue diseases, malignancy, and infection have been known to be associated with this condition, which can be asymptomatic or can manifest as painful ulcers, purpura, or perniosis, reflecting possible underlying cold - induced thromboses, increased blood viscosity, or vascular reactivity. the diagnosis of clinically significant cryofibrinogenemia requires not only circulating cryofibrinogen (cf) but also clinical features and histopathologic evidence of small - vessel thrombosis and perivascular infiltrate. for both mixed cryoglobulinemia and cryofibrinogenemia, treating the underlying disease (whether infection, connective tissue disease, or malignancy) can sometimes improve symptoms. plasmapheresis and immunosuppression with glucocorticoids and/or cytotoxic therapy have also been used in severe disease although with unclear efficacy. connective tissue diseases have been associated with the presence of both cg and cf, perhaps more so than cf alone. in the few studies and reports involving ssc, these cold - induced precipitants do not appear to trigger symptoms. in one study, one out of 19 patients with both cg and cf carried the diagnosis of ssc. in another study, 10 out of 20 ssc patients had the presence of polyclonal igg and igm cryoglobulins in the serum, but none exhibited clinical signs of cryoglobulinemic syndrome. in one report of long - standing ssc with the presence of cryoglobulins (both igg and igm), the patient presented with paresthesias, transient aphasia, vision changes, and delirium (table 2). cerebral angiogram was normal, and electroencephalogram revealed generalized slowing of action potentials, and computed tomography of the extremities revealed calcinosis. while peripheral neuropathy can be a manifestation of mixed cryoglobulinemia, central nervous system involvement would be highly unusual ; therefore it, is unclear whether the presence of cryoglobulins in this case is an incidental finding. another man with long - standing ssc presented with sudden onset gangrene in the fingers and toes after cold exposure and was found to have very elevated cryofibrinogen. he did not respond to prostaglandin e1 or subcutaneous heparin and died shortly after presentation. no biopsy was done to ascertain the etiology for the gangrene, therefore, either underlying ssc vasculopathy or thrombosis from the cryofibrinogenemia could have been possible causes. behcet 's disease is characterized by recurrent oral aphthous ulcers and other systemic manifestations believed to be due to systemic vasculitis, including genital aphthous ulcers, ocular disease, skin involvement, gastrointestinal ulcers, neurologic disease, and arthritis. it is more common along the ancient silk road, with 0.11% prevalence in turkey and 2.6% per 100,000 in southern china. diagnosis is made based on clinical features including presence of recurrent oral aphthae plus two of the following without other systemic disease : (1) recurrent genital aphthae, (2) eye lesions including uveitis or retinal vasculitis, (3) skin lesions including erythema nodosum, pseudovasculitis, papulopustular lesions, or acneiform nodules, or (4) positive pathergy test. treatment for mucocutaneous and joint disease includes colchicine (mixed results), glucocorticoids, and other immunosuppressives such as azathioprine. more serious disease with internal organ involvement has been treated with cyclophosphamide and high - dose steroids. two cases of behcet 's disease with concurrent ssc have been reported [32, 33 ] (table 2). recurrent oral and genital ulcers were the predominant symptoms in both cases, and neither had eye involvement. in the case of the 54-year - old woman with diffuse ssc, the diagnosis of behcet 's disease was made based on recurrent oral and genital ulcers and presence of pathergy ; skin involvement was nonspecific. her more bothersome symptoms were related to her ssc including restrictive lung disease, esophageal dysmotility, and polyarthritis, as well as secondary sjogren 's syndrome. the recurrent ulcers initially responded well to azathioprine 100 mg daily ; later she was switched to colchicine with unknown effect. in the case of the 62-year - old man with limited ssc based on biopsy - proven sclerodactyly, the presence of recurrent oral and genital ulcers as well as erythema nodosa led to the diagnosis of behcet 's disease. he later developed esophageal and gastric ulcers that were felt to be consistent with enteric behcet 's. relapsing polychondritis (rpc) is an inflammatory disease of unknown etiology involving cartilagenous tissues in multiple organs, typically the ears, nose, eyes, respiratory tract, and joints. association with systemic vasculitis, connective tissue disease, or myelodysplastic syndrome occurs in up to one - third of the cases. the original diagnostic criteria by mcadam required three of six clinical manifestations : (1) bilateral auricular chondritis, (2) nonerosive seronegative polyarthritis, (3) nasal chondritis, (4) respiratory tract chondritis, (5) ocular inflammation, or (6) cochlear and/or vestibular dysfunction. the criteria were later modified to include the presence of three or more of the above, one clinical manifestation with corroborating histology, or chondritis at more than two sites responsive to steroids or dapsone. given the rarity and relapsing nature of the disease, treatment has been empiric, with dapsone and glucocorticoids favored for mild nonvisceral disease, and high - dose steroids and possible adjunctive immunosuppressants for organ - threatening disease. only one case of rpc has been reported in association with ssc (table 2). a 35-year - old man with limited ssc presented with a known nasal septal perforation presented with left auricular swelling and polyarthralgia. the left auricle later was ulcerated with drainage of pus and was felt to be super infected with pseudomonas aeruginosa. however, the ulcer did not improve with antibiotics but rather with prednisolone 15 mg daily. the spectrum of necrotizing small - vessel vasculitis known as anca - associated vasculitis (aav) includes wegener 's granulomatosis (wg), microscopic polyangiitis (mpa), and churg - strauss syndrome (css). while formally classified as small - vessel vasculitis, aav can involve medium - sized vessels. cytoplasmic antineutrophil cytoplasmic antibodies (c - ancas) directed against proteinase 3 (pr3) are more commonly found in wgs whereas perinuclear ancas (p - anca) targeting myeloperoxidase (mpo) are more frequently seen in mpa and css. variable organ involvement makes diagnosis a challenge, with alveolar hemorrhage and crescentic glomerulonephritis frequently occurring in wg and mpa, while polyneuropathy can be seen in anca - associated css. disease stage can range from localized without end - organ damage to severe generalized with organ failure. treatment of generalized disease involves induction with cyclophosphamide (whether oral or intravenous) followed by maintenance with less toxic drugs such as azathioprine, methotrexate, or myclophenolate mofetil. standard therapy leads to remission in 90% to 94% of aav patients within six months although relapses frequently occur (18 to 40% in wg at 24 months). of all the small vessel vasculitides, aav is the most frequently reported in association with ssc, raising the question whether an overlap syndrome exists that combines features from both diseases. a study by rho. found 31 reports containing 63 cases of aav in ssc up to 1994. fifty of the 63 cases provided sufficient clinical and laboratory information and were included in the analysis. autoantibody profiling showed 72% with positive ana (titers unknown), 70% with anti - scl-70 antibody, and 72% with positive anti - mpo antibody. the most common end - organ involvement included kidneys (82%) and lungs (70% had pulmonary fibrosis). outcomes were not described in 17/50 (34%), but analysis of the remaining cases yielded a 7-year survival rate of 67.9%, with a high mortality rate within the first year. furthermore, anti - scl-70 antibody was found to be a significant predictor of developing aav in ssc (or 3.1, 95% confidence interval 1.11 - 8.55, p value.031). there was no difference in aav occurrence between ssc subtypes (p value.998), the presence of mpo versus. pr3 anca (p value.196), or prior use of d - penicillamine (p value.143). in our review of the literature, we found eleven additional cases of aav in ssc that were not cited as part of the rho study [4855 ] (tables 3 and 4). seven (64%) were female with a mean age of 53 14 years (range from 19 to 71 years). ssc disease duration was known for 9 of the 11 cases, with a mean of 4.49 3.37 years (range from 0.42 to 10 years). the time from ssc diagnosis to aav onset was known for 10 cases and reached a mean of 3.85 2.67 years (range from 0.42 to 8 yrs). seven patients (64%) had diffuse skin involvement, and four (36%) were previously treated with d - penicillamine. renal involvement in the form of crescentic glomerulonephritis on renal biopsy was seen in 9 cases (82%) while pulmonary fibrosis was seen in 3 cases (27%). eight cases (88%) had positive ana of at least 1 : 320, 5 cases (45%) with positive anti - scl-70 antibody, and only 1 case (9%) with anticentromere antibody. almost all cases (91%) had positive anti - mpo antibody while none had anti - pr3 antibody. these findings highlight the importance of considering crescentic glomerulonephritis related to aav as a potential cause of renal insufficiency in ssc patients. classically, scleroderma renal crisis occurs in up to 20% of patients with diffuse ssc, and renal involvement manifesting as hypertension, proteinuria, or azotemia can be found in 4560%. however, causes other than scleroderma renal crisis should be considered as a differential diagnosis, especially in settings of normotension or anca positivity. while lumen - occlusive vasculopathy is a prominent feature of ssc, frank vasculitis may also occur. coexistent ssc and vasculitis have been reported for vessels of all sizes, either before or after ssc diagnosis, and in either ssc subtype (limited or diffuse). we found in the literature 88 cases of vasculitis in ssc, with the vast majority being anca - associated vasculitides (74 cases : 63 cited by previous studies, 11 new). rare cases of takayasu 's arteritis (4 cases), giant cell arteritis (3 cases), and behcet 's disease (2 cases) have been reported in patients affected by ssc. only single - case reports have focused on other vasculitic syndromes, including mixed cryoglobulinemia / cryofibrinogenemia, polyarteritis nodosa, primary angiitis of the central nervous system, and relapsing polychondritis, suggesting that the association between ssc and these disorders may be a chance event. although most patients exhibited classic symptoms and signs for the respective vasculitides, confirmation of diagnoses and distinction from ssc rested on histology. prompt treatment with immunosuppression usually resulted in stabilization of symptoms. in ssc patients with renal insufficiency and anca positivity, crescentic glomerulonephritis related to aav | systemic sclerosis (ssc) is a multiorgan connective tissue disease characterized by autoantibody production and fibroproliferative stenosis of the microvasculature. the vascoluopathy associated with ssc is considered to be noninflammatory, yet frank vasculitis can complicate ssc, posing diagnostic and therapeutic challenges. here, we have reviewed the literature for reports of small-, medium-, and large - vessel vasculitis occurring in ssc. amongst 88 reported cases of vasculitis in ssc, patients with anca - associated vasculitis appear to present a unique subclass in that they combined typical features of ssc with the renal manifestation of anca - associated glomerulonephritis. other vasculitic syndromes, including large - vessel vasculitis, behcet 's disease, cryoglobulinemia, and polyarteritis nodosa, are rarely encountered in ssc patients. anca - associated vasculitis needs to be considered as a differential diagnosis in ssc patients presenting with renal insufficiency, as renal manifestations may result from distinct disease processes and require appropriate diagnostic testing and treatment. |
suboptimal blood pressure (> 115 mmhg sbp) is the number one attributable risk for death throughout the world. guidelines of advisory bodies (national heart, lung, and blood institute nhlbi, who) emphasize to increase awareness, prevention, and control of risk factors because awareness and early diagnosis of the vulnerability of hypertension and prehypertension can substantially reduce the risk. anthropometric measures can be used as predictor for cardiovascular risk factors and essentially aids in prevention and control. however, there seems to have considerable variability of sensitivity among the anthropometric measures such as bmi, waist circumference, waist to height ratio and so on to predict cardiovascular risks among populations across geographies, ethnicity, and demography. the debate over a more sensitive anthropometric predictor of cardiovascular risks is amplified on the basis of the reports demonstrating variability in the efficacy of anthropometric parameters in predicting cardiovascular risks. according to several workers in india and abroad bmi alone is less accurate as a predictor and waist circumference and/or waist to height ratio is advocated as more sensitive indicator / s of cardiovascular risks. on the contrary, some researchers argue that sensitivity of bmi is better and it sufficiently correlates with cardiovascular risk factors as hypertension. yet another group reports bmi and waist circumference both are equally good predictors of cardiovascular risks. in this background, a study to evaluate sensitivity of anthropometric measures on cardiovascular risk factor as high blood pressure seems imperative, more so when similar studies are not reported from this region (tripura). in the present work, the focus is on correlation and the degree of association of anthropometric measures with blood pressure indices in apparently healthy female adolescent and young adult students. there was a general perception that women to be less vulnerable to cardiovascular complications but it is acknowledged that women are more prone to several other impediments for their inherent physiology, which may have a negative synergistic effect if hypertension or prehypertension coexists. moreover, prognosis of cardiac complications in women may be less satisfactory. also in menopause the so - called female advantage is reversed due to rapid decrease in female steroid hormones, and thus, sex - associated differences must be considered in hypertension management of women. so identification of cardiovascular risk factors and its more sensitive anthropometric indicator even in apparently healthy female population is crucial for prevention and control of cardiovascular causalities in the long run. in this respect a cross - sectional study on anthropometric measures, blood pressure indices, and some hematologic parameters was conducted among the female students (women 's polytechnic) in tripura, as a part of the academic dissertation during july 2014 and february 2015. total 210 (n) female students of women 's polytechnic studying in various disciplines in the age group 1622 years participated in the study. written consent of the participants and guardians were taken based on recommendation of world medical association declaration of helsinki sixth revision guidelines. any participant with immediate family history of sever cardiac anomalies were excluded from the study. all these factors including age, demography (urban / rural), ethnicity (data not shown), and medical history were self - reported by the participants. participants on any medication and with any significant medical history were excluded from the study. trained female students of the institute carried out all measurements during college hours. body weight (kg), height (m), waist circumference (cm) of the participants were collected in college uniform and subsequently adjusted. weight was measured in a doctor 's weight measuring machine (krup s) and height was measured by a standard measuring tape against a wall. waist circumference (cm) was measured at the midpoint between the lower costal margin and the top of iliac crest, while the participant was in the standing position using a non - stretch tape (who). sbp and dbp (first and fifth korotkoff sounds, respectively, using stethoscope, microtone) were measured to the nearest even digit by auscultation with an appropriate - size cuff and an aneroid sphygmomanometer (diamond, isi 3390). heart rates (hr times / minute) were measured manually using stop watch (samsung). hemoglobin concentration (gm / dl) were detected by sahli 's method (marienfeld hemoglobinometer) using 0.1n hcl (merck). sahli 's method is a efficacious method of hemoglobin estimation in the field work, and is significantly economical in resource limited set up like in this case. tulip diagnostics) in the participants for database purpose and rh typing was not done. total 210 (n) female students of women 's polytechnic studying in various disciplines in the age group 1622 years participated in the study. written consent of the participants and guardians were taken based on recommendation of world medical association declaration of helsinki sixth revision guidelines. any participant with immediate family history of sever cardiac anomalies were excluded from the study. all these factors including age, demography (urban / rural), ethnicity (data not shown), and medical history were self - reported by the participants. participants on any medication and with any significant medical history were excluded from the study. trained female students of the institute carried out all measurements during college hours. body weight (kg), height (m), waist circumference (cm) of the participants were collected in college uniform and subsequently adjusted. weight was measured in a doctor 's weight measuring machine (krup s) and height was measured by a standard measuring tape against a wall. waist circumference (cm) was measured at the midpoint between the lower costal margin and the top of iliac crest, while the participant was in the standing position using a non - stretch tape (who). sbp and dbp (first and fifth korotkoff sounds, respectively, using stethoscope, microtone) were measured to the nearest even digit by auscultation with an appropriate - size cuff and an aneroid sphygmomanometer (diamond, isi 3390). heart rates (hr times / minute) were measured manually using stop watch (samsung). hemoglobin concentration (gm / dl) were detected by sahli 's method (marienfeld hemoglobinometer) using 0.1n hcl (merck). sahli 's method is a efficacious method of hemoglobin estimation in the field work, and is significantly economical in resource limited set up like in this case. tulip diagnostics) in the participants for database purpose and rh typing was not done. data are expressed as mean, standard deviation, and range (max - min). mean differences of parameters among the bmi classes are reported with statistical significance for dependent variables (anova). correlation (r) analysis was used to identify the better (p) indicator of elevated blood pressure (dependent variable) in the studied population and its impact was assessed by multiple regression analysis of parameters. bmi was calculated from weight (kg) and height (m) in kg / m. it was such that the participants could be divided in total seven groups depending upon their bmi class. normal (bmi 18.524.9 kg / m), overweight (2530 kg / m), obese class i (3035 kg / m), obese class ii (3540 kg / m), severe thinness (< 16 kg / m), moderate thinness (1617 kg / m) and mild thinness (1718.5 kg / m). mean pressure was calculated as dp + 1/3 pp, whereas rate pressure product (rpp) was calculated as sp hr 10. bmi was calculated from weight (kg) and height (m) in kg / m. it was such that the participants could be divided in total seven groups depending upon their bmi class. normal (bmi 18.524.9 kg / m), overweight (2530 kg / m), obese class i (3035 kg / m), obese class ii (3540 kg / m), severe thinness (< 16 kg / m), moderate thinness (1617 kg / m) and mild thinness (1718.5 kg / m). mean pressure was calculated as dp + 1/3 pp, whereas rate pressure product (rpp) was calculated as sp hr 10. the sample population (n = 210) could be categorized into seven bmi categories. anthropometric measures and hemoglobin (g / dl) of the population with sample size (n) is depicted in [table 1 ]. age and demography (urban / rural) is depicted in [table 3 ]. it is apparent that mean age of obese (class i / ii) and severe thin participants are higher compared with other bmi categories, as well as from the overall population. sbp, dbp, and mean pressure is comparatively higher in obese (i / ii) and overweight participants with statistically significant (95.5% confidence) mean differences. bmi is positively correlated to dbp [r (+) 0.252185854, p = 0.0001 ], mean pressure [r (+) 0.248430338, p = 0.0002 ] and sbp [r (+) 0.203482052, p = 0.001 ] [table 5 ]. bmi is also positively correlated to rpp and hemoglobin level but the correlation is not significant. waist circumference is positively correlated with sbp, dbp, mean pressure, rpp, and hemoglobin level ; however, significant correlation is found with dbp (r = (+) 0.227278779, p = 0.0006) and mean pressure (r = (+) 0.200640562, p = 0.001). wthr is also positively correlated with sbp, dbp, mean pressure, rpp, and hemoglobin level and is significantly correlated with dbp (r = (+) 0.217848832, p = 0.0007) and mean pressure (r = (+) 0.189695053, p = 0.002). hr and pp are negatively correlated to bmi, waist circumference, and wthr but the relationship is not statistically significant. direct impact of independent variables (bmi, waist circumference, and wthr) on the dependent variables (sbp, dbp, and mean pressure), which have significant correlation are depicted in [table 6 ]. impact of anthropometric measures with blood pressure indices is most significant for bmi (p 0.020) followed by wthr (p 0.500) and waist circumference (p 0.520) in the population. 74.88% of the population are from urban tripura and among rbc antigens o (30.80%) is the most common in the population followed by a mean (sd) of bmi, waist circumference, waist to height, and hemoglobin level in the population mean (sd) of sbp, dbp, pulse pressure, heart rate in the population. the present study was conducted among 210 female adolescent and young adult students of tripura to analyze the fidelity of using bmi as an indicator of suboptimal blood pressure in apparently healthy females. analysis of mean of parameters helped to initially identify significant independent (anthropometric measures) and dependent factors (blood pressure indices, hr, and so on). significance of correlation was used to pinpoint the most sensitive anthropometric index and the regression analysis fortified the argument. overweight and obese (i / ii) participants (according to bmi categories) have wthr more than 0.50, the cutoff value for all age groups. sbp, dbp, and mean pressure is comparatively higher in obese (i / ii) and overweight participants (95.5% confidence). when anthropometric parameters were correlated to blood pressure indices, hr, and so on, it was observed that dbp and mean pressure are positively correlated to anthropometric measures to a significant extant followed by sbp. therefore, dbp seems to be better responder in correlating anthropometric measures with blood pressure indices in the studied population. dbp is an important parameter that dictates cardiovascular outcome and is related to physiological stress and causality due to cardiac failure. its significance in prevention and management of cardiovascular complications is established by the fact that a small reduction of 2 mmhg in dbp in the mean of the population distribution could have a great public health impact on the number of chd and stroke events prevented. it is observed that pp, as well as, hr negatively correlates to anthropometric measures in the studied population. pp is a reliable indicator of vascular distensibility, whereas hr is an indicator of sympathovagal regulation. the negative correlation (anthropometric measures and pp / hr), although insignificant but can be explained on the basis of autonomic function and or energy metabolism in women with the aid of female steroid hormones. on the contrary, bmi among independent parameters is significantly correlated to most of the dependent factors (blood pressure indices and so on) than that of wthr and waist circumference and the direct impact of bmi on the blood pressure indices are also more significant statistically. schematic representation of the decision pathway (original figure) although it is observed that the blood pressure indices in the studied population is not alarming but normal bp in higher margin and hypertensive bp is regarded as a cause of concern in women. in the studied population 20% of the participants are either overweight or obese and have risk of developing cardiovascular complications. individuals with prehypertensive levels of blood pressure have an increased risk of developing cardiovascular disease relative to those with optimal levels and the association is pronounced among those with high bmi. also, high - normal blood pressure is associated with an increased risk of cardiovascular disease. therefore, this study is significant and aids to identify participants at risks. in the studied population, it is observed that bmi is significantly associated with blood pressure indices and, therefore, is a good indicator of cardiovascular risks. it has been observed that the long - term reproducibility of bmi is superior and it significantly correlates to hypertension and prehypertension in various age groups even in normal individuals. therefore, bmi is a superior predictor of cardiovascular risks in apparently healthy adolescent and young adult female students of tripura. the participants included in this academic study were the students of women 's polytechnic, govt. of tripura. bmi is a superior indicator of blood pressure indices and can identify participants at risk of cardiovascular complications even in apparently healthy adolescent and young adult females. screening on the basis of bmi may aid to awareness generation and prevention of complications. | background : anthropometric measures are used as indicators of elevated blood pressure, but reported to have variable sensitivity among populations. this study was undertaken to identify the better indicator of cardiac - risk factors by statistical comparison of bmi, waist circumference, and waist to height (wthr) ratio in apparently healthy adolescents and young adult female students of tripura.materials and methods : a cross - sectional study was conducted in a resource limited setup on 210 apparently healthy female adolescents and young adult students in tripura. mean (sd) of all parameters were compared (anova) to recognize significant independent (anthropometric measures) and dependent factors (blood pressure indices and so on). correlation (r) analysis was used to identify the better (p) indicator of blood pressure indices (dependent variable) and its impact was assessed by multiple regression analysis.results:blood pressure indices are comparatively higher in obese and overweight participants with statistically significant (95.5% confidence) mean differences. significant correlation with dependent factors is observed with bmi followed by wthr and waist circumference. impact of anthropometric measures with blood pressure indices is most significant for bmi (p 0.020) followed by wthr (p 0.500) and waist circumference (p 0.520).conclusion : bmi is a superior indicator of blood pressure indices and can identify participants at risk even in apparently healthy adolescent and young adult females. |
gastrointestinal stromal tumors (gists) are the most common mesenchymal tumors of the gastrointestinal tract. though initially labeled histologically as leiomyosarcomas, the identification of unique activating mutations in kit gene enabled classification of gist as a distinct entity (1). gist like other soft tissue sarcomas was conventionally associated with poor prognosis with a 5-year survival of 520% mainly due to resistance to conventional chemotherapy and radiotherapy (23). the discovery of imatinib mesylate which is a small molecule inhibitor of receptor tyrosine kinases (rtk) notably kit dramatically changed the outcome of patients with gist (4). in the last one and half decade following the discovery of kit and imatinib, several new mutations responsible for the pathogenesis of gist have been discovered. concurrently several tyrosine kinase inhibitors (tkis) many of them effective in gist have also been discovered and approved by the united states food and drug administration (4). in addition, better understanding of the biologic behavior of gist led to proposition of several risk prediction models to streamline management and surveillance strategies. in this manuscript, we will provide an up to date review of the current concepts on mutational taxonomy, risk stratification, targeted therapies and surveillance strategies in patients with gist emphasizing the role of radiologists. gastrointestinal stromal tumors characteristically express a type iii rtk, kit in more than 90% of cases which is used for characterizing them on immunohistochemistry (5). in 8085% of gists, the kit gene is mutated which leads to non - ligand dependent autonomous activation of down - stream signal pathways, a key step in the pathogenesis in gist (6). in another 510% cases of gist, a mutation in the platelet derived growth factor receptor alpha (pdgfra) gene encoding another similar rtk drives the pathogenesis of gist (6). several types of mutations have been discovered in kit and pdgfra and vary from point mutations to frame - shift insertions or deletions. mutations in kit can involve exon 9, 11, 13, and 17 (7). the most common of these are mutations in exon 11 occurring in greater than 60% cases, often in gastric gists (fig. exon 9 mutations on the other hand tend to occur in small bowel gists (fig. primary mutations in exon 13 and 17 are rare but can occur as secondary mutations as a mechanism of resistance (9). majority of pdgfra mutant gists are gastric in origin. in up to 1015% cases of gist, both kit and pdgfra genes carry wild type sequences (4). while some of these tend to be sporadic in origin, some are associated with clinical syndromes like neurofibromatosis, carney - stratakis syndrome and carney triad. recent studies have shown that some of the erstwhile wild - type gists have non - kit and non - pdgfra driver mutations like braf v600e and succinate dehydrogenase (sdh) gene mutations (10). among the previously wild - type gists, gists with mutations in sdh subunits have garnered interest due to unique epidemiologic, clinical and histopathologic features increased incidence of young, female patients, association with paragangliomas, gastric origin, epithelioid or mixed epithelioid and spindle cell type and lymphatic spread at histopathology (fig. sdh is a key enzyme in the mitochondrial citric acid pathway which has several subunits (a, b, c, and d). loss of function of sdh is seen in patient with carney - stratakis syndrome, carney triad, familial paraganglioma syndromes and pediatric gists (11). mutations of kit exon 11, pdgfra and sdh subunits are often gastric in origin where as gists with kit exon 9 and neurofibromatosis are often small bowel in origin (12). sdh - deficient gists tend to be multifocal and frequently metastasize to nodes in addition to liver and peritoneum (13). in addition, presence of extraadrenal paragangliomas and/or pulmonary chondromas in patients with gist can hint towards sdh - deficient gist in the setting of carney - stratakis syndrome or carney triad (11). the type of mutation in gist also predicts the risk of recurrence and long - term outcome in gist. deletions in exon 11 are associated with aggressive behavior where as point mutations in exon 11 portend a better prognosis (7). similarly, patients with sdh - deficient gists have an indolent course in spite of metastasis (fig. 1). exon 9 mutant gists, sdh - deficient gists and wild - type gists are inherently resistant to treatment with imatinib (fig. this type of resistance is usually encountered in the first 6 months of therapy and referred to as primary resistance (151618). exon 11 mutants can develop resistance after initial response to imatinib usually after 6 months referred to as secondary resistance (17). secondary resistance in exon 11 gists usually occurs due to secondary mutations in exons 13 and 17 (7). upfront knowledge of the type of mutation can help radiologists in prompt interpretation of primary resistance to treatment with imatinib. mutations of kit exon 11, pdgfra and sdh subunits are often gastric in origin where as gists with kit exon 9 and neurofibromatosis are often small bowel in origin (12). sdh - deficient gists tend to be multifocal and frequently metastasize to nodes in addition to liver and peritoneum (13). in addition, presence of extraadrenal paragangliomas and/or pulmonary chondromas in patients with gist can hint towards sdh - deficient gist in the setting of carney - stratakis syndrome or carney triad (11). the type of mutation in gist also predicts the risk of recurrence and long - term outcome in gist. deletions in exon 11 are associated with aggressive behavior where as point mutations in exon 11 portend a better prognosis (7). similarly, patients with sdh - deficient gists have an indolent course in spite of metastasis (fig. 1). exon 9 mutant gists, sdh - deficient gists and wild - type gists are inherently resistant to treatment with imatinib (fig. this type of resistance is usually encountered in the first 6 months of therapy and referred to as primary resistance (151618). exon 11 mutants can develop resistance after initial response to imatinib usually after 6 months referred to as secondary resistance (17). secondary resistance in exon 11 gists usually occurs due to secondary mutations in exons 13 and 17 (7). upfront knowledge of the type of mutation can help radiologists in prompt interpretation of primary resistance to treatment with imatinib. gastrointestinal stromal tumors have a complex biologic behavior which makes predicting their malignant potential difficult., efforts have been made over the years to design consensus criteria, which can enable stratifying gists according to risk of recurrence or metastasis. the national institute of health (nih) consensus criteria proposed in 2001 were based on two important features : mitotic count and tumor size (fig. 4) (19). subsequently miettinen and lasota (20) in a large series of gists arising from various sites of gastrointestinal tract found that in addition to tumor size and mitotic count, the site of origin also determined the risk of recurrence in gist and proposed the armed forces institute of pathology (afip) risk criteria in 2006 (fig. 4). in 2008, joensuu (21) proposed the modified nih consensus criteria taking tumor site and tumor rupture into account as tumor rupture either spontaneously or during surgery significantly increases recurrence risk (22). the memorial sloan kettering cancer center (mskcc) nomogram was developed in 2009 from 127 patients with gist to accurately predict individual recurrence - free survival (rfs) after resection of localized gist and validated in two other cohorts (25). the nomogram was based on points assigned in a continuous non - linear fashion for tumor size, site and mitotic count and the total number of points then used to determine the 2- and 5-year rfs. the nomogram had high predictive value for 5-year rfs than nih criteria or the afip criteria. another study designed to validate the mskcc nomogram and compare it with the nih, afip and joensuu criteria found that the mskcc nomogram and the afip criteria performed better than the other two criteria in estimating the rfs (26). the reason for the better performance of mskcc nomogram and afip criteria was hypothesized to be related to the greater emphasis on tumor site in these criteria compared to nih or joensuu criteria (26). in a pooled population - based cohort of 2560 patients with operable gist who did not receive adjuvant imatinib, an attempt was made to refine the existing risk stratification criteria (27). in this study, large tumor size, high mitotic count, non - gastric location, tumor rupture and male sex were found to have independent prognostic significance. while nih, afip, and joensuu criteria all predicted outcome accurately, the joensuu criteria was the best criteria to identify patients with highest risk of recurrence (27). the authors in this study also generated novel heat contour maps using tumor size and mitotic count as continuous non - linear variables along with tumor site and tumor rupture. these maps provided individualized patient outcomes and were more accurate than the existing criteria in estimating recurrence risk implying that treating mitotic count and tumor size as non - linear continuous variables is better than categorizing them (27). though mutational status affects the risk of recurrence, this information was available in very few patients and was not incorporated in analysis (27). several other factors like tumor necrosis, vascularity, invasion of adjacent viscera further refining of the risk stratification schemes using these additional factors can increase the accuracy of prediction models. recently there has been increase in interest in identifying imaging biomarkers which can predict long - term outcome in gist patients. both tumor size and mitotic count at histopathology can be subject to variations across institutions. furthermore, variability in expertise of subjective assessment of mitotic count, alteration of mitotic count following neoadjuvant imatinib therapy and possibility of non - representative biopsy samples due to tumor heterogeneity can be challenges which can be difficult to address (28). accordingly, predicting risk based on pre - operative imaging features can be alluring. in a study of 143 patients with gastric gist at our institute, pre - operative treatment nave ct morphologic features were predictive of metastasis in gist (28). on multivariate analysis, tumor size > 10 cm, irregular lobulated outline and enhancing solid component were independent predictors of metastasis (fig. these features were also associated with higher mitotic count and poor overall outcome. in tumors 10 cm or smaller in size, enhancing solid component and irregular / lobulated outline were associated with metastasis (28). while these ct imaging features can be anticipated to be incorporated in the risk stratification schemes in the future, they can help in management decisions in some patients who receive neoadjuvant therapy and closer surveillance of patients with small gists with enhancing component or irregular / lobulated outline (28). surgery is the treatment of choice for all resectable gists. however, targeted therapy with imatinib and other tkis is now widely used in the management of gist in various settings. neoadjuvant imatinib is used preoperatively to downsize the tumor to enable less morbid organ - sparing surgeries (29). after successful resection of gist, imatinib is administered in the adjuvant setting to prolong the progression free survival (pfs) (30). currently the duration of adjuvant imatinib is three years, although there are ongoing trials evaluating the advantage of 5-year adjuvant imatinib (3031). in the metastatic setting, imatinib is the first - line of treatment (32). in patients demonstrating primary or secondary resistance to imatinib, second - line sunitinib is the treatment of choice (33). resistance to sunitinib is currently managed with third - line regorafenib (34). in patients who are refractory to all lines of treatment, a recent study has shown that rechallenge with imatinib can slow the disease progression as some of the tumor clones tend to remain sensitive to imatinib (35). computed tomography (ct) is routinely used in monitoring the response to treatment in gist. both primary and metastatic gist have a unique morphologic response to imatinib on ct scan (figs. 1, 5) the heterogeneously enhancing primary and metastatic lesions in gist tend to show dramatic decrease in the enhancing component after treatment with little or no change in lesion size (fig. 5). a transient increase in size this atypical pattern of treatment response causes ambiguity when change in size according to response evaluation criteria in solid tumors (recist) is used for interpreting response. accordingly, alternate tumor response criteria incorporating changes in tumor attenuation along with size reduction were proposed by choi. according to the choi criteria, a 15% decrease in ct attenuation or 10% decrease in unidimensional size indicates response in contrast to 30% decrease in unidimensional size as per recist. in a study of 40 patients with gist treated with imatinib and evaluated with pre- and post - treatment ct and f - fluorodeoxy glucose (fdg) positron emission tomography (pet)/ct, choi criteria had greater sensitivity in identifying responders compared to recist although both had similar specificity and correlated better with disease - specific survival (37). given that both primary and metastatic tumors in gist tend to be irregular rather than spherical (as assumed by recist), it is often argued that volume is a better representation of the actual number of tumor cells than single longest diameter (38). accordingly, moderate changes in size can be detected better using volumetric analysis rather than recist (39). the utility of volumetric analysis in assessing response to treatment in hepatic metastasis from gist was attempted by schiavon. (40) in two independent studies consisting of 84 and 78 patients with hepatic metastases from gist who were treated with imatinib (41). while both choi criteria and volumetric criteria identified more number of imatinib responders compared to recist, only volumetric criteria had better correlation with overall survival (4041). they concluded that gist metastasis should be conceptualized mathematically as ellipsoidal lesions rather than spheroidal (4041). similarly, in the case of primary gists, in a study of 127 patients at our institute, we found that the actual tumor volume in primary gists can be replicated using the mathematical model for scalene ellipsoid which relies on three measured axes compared to single axis in spheroids (39). patients who fail to respond or develop resistance to imatinib are treated with second- and third - line tkis. it is not known if the dramatic density changes seen with imatinib occur when challenged with new tkis. few recent studies have attempted to study the various treatment response criteria in metastatic gist patients treated with second- and third - line tkis and correlate them with survival (424344). in the study by schramm. (42), recist, choi and volumetric criteria were compared in 20 patients with metastatic gist treated with second - line sunitinib at 3-months and 1-year intervals after start of treatment and were correlated with disease specific survival (dss). the authors found that though choi criteria classified more number of patients as partial responders on 3-month and 1-year scans, partial responders by choi criteria at 1-year follow - up had shorter dss than patients with stable disease (sd) or progressive disease (pd) (42). partial responders as per recist had the longest dss and patients with pd per recist had the shortest survival on both 3-month and 1-year scans (42). accordingly, the authors concluded that choi criteria may not be helpful in identifying patients who tend to have longer survival on follow - up scans while treating with second - line sunitinib (42). in another study performed at our institute, shinagare. (44) compared choi criteria, recist, recist 1.1 and world health organization (who) criteria in 20 patients with advanced gist treated with third - line regorafenib in a phase ii trial. similar to other studies, choi criteria identified more number of patients as partial responders. however, clinical benefit rate defined as complete or partial response or sd for 16 weeks were similar among all tumor response criteria (44). furthermore, the pfs was strongly concordant with overall survival by recist, recist 1.1 and who criteria but not by choi criteria (44). the authors therefore concluded that in the current scenario using recist 1.1 based response evaluation may be prudent especially in clinical trial setting as choi criteria due to high sensitivity would progress patients sooner than recist (44). the role of fdg - pet / ct in the management of gist is unclear. initial studies have shown that metabolic activity in gist treated with imatinib declines dramatically on fdg - pet / ct and therefore fdg - pet / ct can be used for determining efficacy of drugs early in the treatment course (454647). however the routine use of fdg - pet / ct in clinical practice does not have additional advantages over ct scan (30). the national comprehensive cancer network (nccn) guidelines do not recommend fdg - pet / ct in the routine management of gist (30). the european society for medical oncology (esmo) guidelines recommend fdg - pet / ct when targeted therapy is under investigation (48). fdg - pet / ct can be used to evaluate ambiguous findings encountered on ct or magnetic resonance imaging (mri) but has no role in surveillance. mri can be used as a problem solving tool for clarifying unusual responses on ct. increase in tumor density in some gist metastasis due to hemorrhage (especially with sunitinib) can mimic progression. mri due to better soft tissue resolution can help in such scenarios (4). the role of other advanced imaging techniques in the management of gist is under research. dual energy ct (dect) scan allows visualization and quantification of iodine - related attenuation (ira) and has the potential for accurate response assessment in gist (49). in a study of 17 patients with advanced gist treated with tkis, recist, choi criteria, and dect criteria patients were classified per dect criteria as non - responders in this study if there was > 20% increase in size and ira, > 50% increase in size or ira (49) both dect criteria and recist predicted pfs and overall survival (os) but only dect criteria were able to differentiate responders and non - responders according to pfs and os (49). volume ct perfusion (ctp) imaging is a novel imaging technique which determines tumor perfusion (50). a decrease in perfusion parameters following targeted therapy can confirm response in ambiguous cases of response (50). though complete surgical resection is feasible in a substantial proportion of gists, relapses are common, especially with high - risk gists (2). the two most common sites of recurrence are the liver and peritoneum. in patients with no evidence of disease, recurrences can be seen as new metastatic deposits in the liver and peritoneum where as patients with residual cystic metastases, recurrence can be seen as increase in size or density of cystic lesions or as new intratumoral nodules referred to as ' nodule within mass ' pattern of progression (fig. the optimal strategy for imaging surveillance is uncertain and can be guided by risk stratification using anatomic site of origin, tumor size and mitotic count. there are no established guidelines for the frequency of surveillance imaging in gist. in patients who have resectable localized gist, the nccn recommends performing ct arbitrarily at intervals of 36 months for 35 years and then annually in the adjuvant setting after resection of the primary with the aim of detecting local recurrences and distant metastases (30). (52) found that hazard adjusted follow - up ct recommendations can significantly decrease the number of scans by up to 30% compared to the nccn recommendations without affecting the efficacy of recurrent tumor. the esmo guidelines suggest tailoring the follow - up schedules according to the risk stratification (48). gists with very low - risk of recurrence are invariably cured by surgery and therefore do not need adjuvant imatinib or longitudinal imaging. while gists with low - risk (excluding tumors with high mitotic counts) can be followed with sparse imaging for 5 years at 612 month intervals, intermediate and high - risk gists need denser imaging for at least 13 years (48). the timing of scans for high - risk gists recommended by esmo includes 36 month intervals for the first three years during adjuvant imatinib, then every three months for 2 years and every 6 months for another 3 years after cessation of imatinib. the last few decades have seen tremendous advances in the understanding of the molecular taxonomy and biologic behavior of gist. there has been proportionate increase in the contribution of radiologists in the complex management strategies of these patients. upfront knowledge of the mutational taxonomy and familiarity with the risk stratification models can help radiologists in appropriate interpretation of scans. the field of tumor response assessment in gist continues to evolve with advances in imaging techniques. awareness of nccn and esmo guidelines can help in planning surveillance strategies in patients with gist. | the management of gastrointestinal stromal tumors (gists) has evolved significantly in the last two decades due to better understanding of their biologic behavior as well as development of molecular targeted therapies. gists with exon 11 mutation respond to imatinib whereas gists with exon 9 or succinate dehydrogenase subunit mutations do not. risk stratification models have enabled stratifying gists according to risk of recurrence and choosing patients who may benefit from adjuvant therapy. assessing response to targeted therapies in gist using conventional response criteria has several potential pitfalls leading to search for alternate response criteria based on changes in tumor attenuation, volume, metabolic and functional parameters. surveillance of patients with gist in the adjuvant setting is important for timely detection of recurrences. |
renal cell carcinoma (rcc) is the third most common genitourinary cancer site after prostate and bladder cancer and comprises 2 - 3% of all cancers in the united states. the incidence of rcc ranges from 5 to 10 cases/100,000 population, with the rate being 1.6 times higher in men than in women. incidence rates of rcc differ among various ethnic groups and are highest among african americans and lowest among asian americans and pacific islanders, and these reflect the rates of their countries of origin. on the other hand, mortality rates are dramatically higher for native americans, with asian americans and pacific islanders experiencing the lowest rates. the risk factors for rcc include cigarette smoking, obesity, hypertension, reproductive and hormonal factors such as parity, physical activity, dietary factors such as consumption of processed meat and alcohol, long - term dialysis and environmental and occupational factors such as exposure to asbestos, petroleum products and cadmium [3, 4 ]. rcc manifests in a number of ways with localized symptoms such as pain, hematuria or abdominal mass or systemic symptoms such as weight loss, anorexia and pyrexia or with a number of other paraneoplastic syndromes. the tumor is commonly large at presentation and symptoms may not occur until relatively late in the disease. if detected early, rcc can be treated surgically, and 5-year survival rates approaching 85% can be achieved for patients with organ - confined disease (stages t1, t2, and n0). 20 - 30% present with metastases and 20 - 30% relapse distantly after curative nephrectomy. only 7% of rccs metastasize to distant organs, the most frequent sites of metastasis being the lung, lymph nodes, liver, bone and adrenal glands. however, many unusual sites of metastasis of rcc have been cited in the literature, namely stomach, gallbladder, oropharynx, left ventricle, skin [13, 14 ], tonsils, spleen, pancreas, pituitary gland, thyroid [19, 20 ], breast, testicles, gastrocnemius muscle, ciliary body and small intestine [7, 8, 24, 25 ]. commonly, cancers metastasizing to the small bowel arise from the lung, head and neck, breast, esophagus and malignant melanomas and occurrences of metastatic deposits from rcc to the small intestine are exceedingly rare phenomena, with only 7.1% of all metastatic tumors to the small intestine being accounted for by rcc. here we present a rare clinical case of a 53-year - old gentleman with metastasis from rcc to the small intestine presenting with extensive polyposis and massive gastrointestinal bleeding which was successfully managed with intraoperative endoscopic polypectomy and segmental small bowel resection. the patient was a 53-year - old gentleman with a known history of coronary artery disease status post coronary stenting who started experiencing progressive shortness of breath and chest pain in early january 2011. he presented to the local emergency room with increasing chest pain and shortness of breath. however, at that point, he was found to be anemic with a hemoglobin (hg) of 7% and a hematocrit (hc) of 21%. a ct scan of the abdomen showed a large lesion in the right kidney consistent with primary rcc. the pathology revealed an 11 9 6 cm rcc (clear cell type) with renal vein and perinephric adipose metastasis. postoperatively, the patient had an uneventful hospital stay with a transient drop in hg / hc requiring 2 units of packed cell transfusion before discharge, a week after surgery. two weeks later, the patient again presented to his local emergency room with melena and severe symptomatic anemia requiring multiple transfusions. upper endoscopy revealed 2 small vascular polyps in the proximal duodenum, biopsy of which confirmed metastasis from rcc (fig. this was followed by mesenteric and celiac angiograms which did not show any evidence of tumor blush or active bleeding. one of the branches of the celiac artery thought to be supplying the proximal small bowel was embolized with coils. a repeat ct scan showed a small collection of fluid at the previous surgical nephrectomy site with no other abnormality. the patient continued to have melenotic stools off and on with a slow drop in hg / hc. three weeks later the patient was readmitted with repeated complaints of weakness and severe anemia requiring more transfusions. he underwent small bowel enteroscopy which showed multiple vascular polyps throughout the upper small bowel. he underwent capsule endoscopy, which confirmed extensive polyposis throughout the small bowel (fig. a surgical consultation initiated a tumor board discussion and a dilemma on appropriate surgical intervention. it was reasoned that resection of the entire small bowel would cause permanent short bowel syndrome with lifelong dependence on parenteral nutrition. the patient eventually underwent laparotomy with intraoperative endoscopy of the entire small bowel achieved by making two small openings in the small bowel. with the help of the surgeon, 4) totaling only 60 cm with extensive transmural involvement had to be resected with primary anastomosis. after 4 h of intraoperative endoscopy, the entire small bowel was clear of any polyps. he was eventually discharged on the 7th postoperative day with normal bowel function and a low but stable hg / hc. because of significant weight loss and poor appetite over the previous 3 months, the patient was sent home on parenteral nutrition. in the 2 months following his surgery he was started on oral sorafenib and is being followed closely by his oncologist. while his prognosis is guarded, this patient has significant improvement in his quality of life with no need of frequent hospitalizations and transfusions. very few cases have been documented regarding metastasis of rcc to different regions of the small intestine, namely duodenum [24, 25, 26 ], jejunum [7, 8, 27, 28, 29 ], and ileum. the common feature across all these case reports was the presence of a solitary metastatic lesion in the small intestine. these solitary lesions have been reported from after 6 months up to 13 years following initial nephrectomy [7, 8, 24, 25, 26, 27, 28, 29 ]. we report here the first case of rcc metastasizing to the small intestine in the form of extensive polyposis presenting as gastrointestinal bleeding. the possible etiology of extensive small bowel metastasis in the form of polyposis with sparing of the colon is puzzling. the likely possibility may be transient showering of multiple tumor emboli in the celiac and mesenteric arteries. the fact that our patient was already bleeding before his diagnosis suggests that these polyps had been present for a while. the decision of meticulously removing close to 100 polyps by intraoperative endoscopy prevented the patient from requiring total small bowel resection and lifelong dependence on parenteral nutrition. such endoscopic intervention was only possible by applying a great team approach to a patient with previously unreported metastatic rcc to the small bowel. in conclusion, gastrointestinal bleeding in a patient with known rcc should always trigger full gastrointestinal work - up including capsule endoscopy and, if necessary, double balloon enteroscopy. we present a treatment option of intraoperative therapeutic endoscopy that can be employed in any patient with extensive metastatic small bowel polyposis from any etiology. hopefully this case report will stimulate physicians and oncologists to think outside the box and continue to come up with innovative treatment ideas to help their patients. all authors declare that there are no potential conflicts (financial, professional, or personal) relevant to this paper. | we present here a rare clinical case of a 53-year - old gentleman with metastasis from renal cell carcinoma (rcc) to the small intestine presenting with extensive polyposis and massive gastrointestinal bleeding which was successfully managed with intraoperative endoscopic polypectomy and segmental small bowel resection. the patient presented with melena 2 weeks after right nephrectomy for rcc. capsule endoscopy found extensive polyposis throughout the small bowel, and the histological features confirmed the diagnosis of metastatic rcc. the patient eventually underwent laparotomy with intraoperative endoscopy of the entire small bowel. most of the polyps were removed by snare polypectomy. three segments of the small bowel with extensive transmural involvement had to be resected with primary anastomosis. in the 2 months following his surgery, the patient had no further evidence of gastrointestinal bleeding. the decision of meticulously removing close to 100 polyps by intraoperative endoscopy prevented the patient from requiring total small bowel resection and lifelong dependence on parenteral nutrition. in conclusion, gastrointestinal bleeding in a patient with known rcc should always trigger full gastrointestinal work - up including capsule endoscopy and, if necessary, double balloon enteroscopy. |
fms - like tyrosine kinase-3 (flt3), a member of type 3 receptor tyrosine kinase family (rtk), is normally expressed by primitive hematopoietic stem / progenitor cells and is lost during differentiation (1). under physiological conditions, flt3 signaling has an important role in hematopoietic stem cell differentiation (2), b - cell lineage commitment (3) and dendritic cell expansion (4). overexpression of flt3 mrna has been reported in patients with acute myeloid leukemia (aml), acute lymphoid leukaemia (all) and blast crisis of chronic myeloid leukemia (cml) (5). here aberrant signaling of flt3 results from overexpression of the wild - type form, activating mutations in its juxtamembrane (jm) or in its tyrosine kinase (tk) domain. segmental duplications occur in the form of repetition from 3 to over 400 base pairs (6) in the jm domain (exons 14 and 15) (7, 8) ; in the region containing amino acids residues y591-y597 (9). other major mutation site in flt3 is the conserved aspartate residue (d835) in the activation loop which may be replaced by valine (10), histidine (11), asparagine, glutamate, or tyrosine (12). mutant forms of flt3 have been reported in 25% of childhood all (10) and 30% of aml patients (13, 14), in myelodysplastic syndrome (mds) and chronic myelomonocytic leukemia (cmml) (15). they have a confirmed role in transformation of mds to aml (16), which results in a higher rate of aml relapse and poor response to stem cell transplantation therapy (17). flt3 signaling is likely involved in autoimmune diseases (18) such as rheumatoid arthritis (ra) (19) and diabetes (20). therefore, flt3 and its downstream signaling pathways have been considered as therapeutic targets in some of these disorders. flt3 is not sensitive to imatinib (21) due to the presence of a phenylalanine (phe) in the position 691 of flt3, as the gate - keeper residue, instead of a threonine in imatinib sensitive kinases such as stem cell factor receptor (c - kit), macrophage colony stimulating factor receptor (fms) and platelet - derived growth factor receptor (pdgfr) (22). ag1295 and these non - specific tyrosine kinase inhibitors (tkis) have been shown to inhibit autophosphorylation of flt3 and induce apoptosis in cells transfected with flt3-idt mutant (23, 24). some other inhibitors have also been discovered including cep701 (lestaurtinib), pkc412 (midostaurin) and su11248 (sunitinib) that were active in nm concentrations and have been shown to kill cell lines derived from leukemias. bis(1h-2-indolyl)-1-methanone derivatives such as d-64406 and d-65476 inhibited proliferation of growth factor - dependent ba / f3 cells transfected to express the oncogenic fusion protein tel - flt3 with ic50 value of 200 to 300 nm in the absence of the il-3, but more than 1000 nm in the presence of the il-3 (25). despite all efforts, no selective inhibitor has yet been developed for flt3 to be used as a drug. thus, a better understanding of downstream signaling pathways could provide new insights into the molecular mechanisms and alternative drug targets. although mutations in both jm and tk domains are believed to render the kinase domain constitutively active as reported for mutants of another type 3 rtk, kit (26), constitutive activation of the flt3 kinase domain may affect its protein trafficking. this can affect the downstream signaling pathways, and also drug response (27) in the cells harboring such flt3 mutations. identification of these differences may be useful in development of therapeutic strategy. in this study, growth promoting, drug response to cep701, pkc412 and sunitinib as well as protein trafficking of flt3 wild - type were compared with two different constitutively active mutants (flt3-itd and -d835y). the factor - dependent murine early myeloid cell line fdc - p1 was routinely maintained in dmem 10% fcs supplemented with murine granulocyte - macrophage colony stimulating factor (gm - csf) as previously described (28). the dna constructs for flt3 (wt, it d, and d835y) were supplied by dr. hitoshi kiyoi (nagoya university school of medicine, nagoya, japan) and subcloned into mscv - ires - gfp vector. the vector was prepared by removing fms from mscv - fms - ires - gfp supplied by dr ab lyons, (hanson institute, adelaide), with the consent of the originator dr m roussel, (st. jude children 's research hospital, memphis ; tn.). mscv - flt3-ires - gfp was introduced into phoenix (clontech, mountain view, ca) cells using lipofectamine 2000 (invitrogen, carlsbad, ca) and packaged as ecotropic retroviral particles. the transfected cells were incubated for 36 hr and the supernatant was collected and used for transduction of the fdc - p1 parent cells. dna was introduced into fdc - p1 cells by retrovirus - mediated gene transfer as previously described (29) with some modifications (centrifugation of transfection mix at 2000 g for 2 hr) to improve the infection rate as described for dendritic cells (dcs) (30). fdc - p1 cells expressing flt3-wt (fd - flt3-wt) were selected using flt3l as growth factor, while those expressing constitutively active mutant flt3 (fd - flt3-itd, and fd - flt3-d835y cells) selected in growth factor free medium. then fd - flt3 cells were sorted (facs aria, bd biosciences, ca) for expression of gfp to obtain a homogenous population. flt3 expression on sorted cells was confirmed by flow cytometry using flk2/flt3 (sc-19635) antibody (santa cruz biotechnology, santa cruz, ca) and sheep anti - mouse igg conjugated with pe (chemicon, billerica, ma) as secondary antibody. cell proliferation of fdc - p1 parent, fd - ev and fd - flt3 cell lines in growth factor - free media. all the cell lines were cultured for 48 hr and then the number of live cells in each well was determined by mts. mts solution (promega, madison, wi), 20 l, was added to each well, incubated for 2 hr and the absorbance at 590 nm was measured using a fluostar optima plate reader (bmg labtech, offenburg, germany). results were analyzed using graphpad prism 4.3 for windows, (graphpad software, san diego, ca). for cell proliferation inhibition assay, serial dilutions of inhibitors were prepared in 96-well tissue culture plates in dmem medium (gibco, invitrogen) containing flt3l and 10% fcs (jrh). fd - flt3 (wt, it d, and d835y) (4x10) cells were added to each well and the plates were incubated at 37c in a humidified 5% co2 in air atmosphere for 48 hr. the number of viable cells was assessed as described for cell proliferation assay using mts. results were analyzed using graphpad prism 4.3 and each ec50 value is calculated as the average of three independent experiments standard deviation and with four replicates in each experiment. in the phosphorylation inhibition assay, fd - flt3-d835y cell were pre - incubated with different concentrations (25, 5, 1, 0.2 and 0.04 nm) of cep701 for 30 min before pulsing with flt3l for 5 min. finally, the cells were washed once with cold pbs and lysed with ice - cold 1% np40 in tse (50 mm tris - hcl, 150 mm nacl, 1 mm edta ph 8.0) with complete protease inhibitor cocktail (roche, basel, switzerland), 5 mm sodium fluoride, 5 mm tetra sodium pyrophosphate, 5 mm sodium vanadate, and 1 mm phenylmethylsulfonyl fluoride (sigma). protein concentration in the cell lysates was determined by a microbca kit (pierce, rockford, il). flt3 protein was immunoprecipitated from the cell lysate containing 500 g total protein using 2 g of anti - flt3 sf-1.340 (santa cruz biotech) with protein - g sepharose beads (invitrogen) (30 l of 50% slurry). the immunoprecipitated flt3 resolved on 6% sds / polyacrylamide gels along with protein markers (fermentas or bio rad). proteins on the gels were transferred to hybond nitrocellulose membrane (amersham biosciences, pittsburgh, pa). the membranes were blocked with 2% bsa (sigma) for 30 min and one of them probed for phosphotyrosine using a cocktail of 4g10 (upstate, temecula, ca) and py20 (bd biosciences, san jose, ca) antibodies. the membrane was washed three times with tbst (10 mm tris ph8, 150 mm nacl and 0.1% tween-20 (all from sigma)) and incubated with secondary anti - mouse antibody conjugated with hrp (ge healthcare) for 45 min. the membranes were washed three times in tbst buffer and incubated with ecl - plus reagent (ge healthcare) for 1 min, and the fluorescent product was measured by a typhoon 9410. then the membrane was stripped and probed with flt3 (sc-479 antibody, santa cruz biotechnology) as described for phosphotyrosine. to evaluate proportions of intra- and cell surface flt3 receptor, fd - flt3 cells were fixed in 5% formaldehyde for 10 min and then divided into two parts and one of them was permeabilised using 100% methanol (15 min). both samples of the fixed and the fixed - permeabilised cells were immunostained for flt3. briefly, the samples (2x10 cells) of negative control (fdc - p1 parent cells), the fdc - p1 cells transduced to express flt3 wt, flt3-itd, and flt3-d835y were washed once with pba (pbs with 0.5% bsa and 0.05% sodium azide) (all chemicals from sigma). then cells were resuspended in 100 l of normal rabbit serum containing 2 g of flt3 antibody (fc1.340 (santa cruz biotechnology), vortexed and incubated on ice for 60 min, then washed three times with 2 ml pba. the cells were resuspended in 100 l pba containing 1 g secondary antibody (anti - mouse igg conjugated with pe (chemicon, billerica, ma) ; and incubated on ice for 30 min. finally, the cells were washed three times with 2 ml pba and fixed with cold facs - fix solution (0.5% paraformaldehyde in pbs) and analyzed using a facscalibur flow cytometer (bd biosciences). data for intensity of pe fluorescence (representing flt3 expression) and also gfp (as the reporter gene) were analyzed using cellquest software (bd biosciences). the geo mean of the fixed and fixed permeablized cells was considered as the representative of surface and total flt3 expression, respectively. the fd - flt3-wt, -itd and d835y cells, the geo mean values for flt3 were normalized to the gfp in each cell type and the intracellular flt3 was calculated by deduction of surface from total flt3. to compare the effects of flt3 (wt, it d and d835y mutants) expression on factor - dependent cell growth and survival, fdc - p1 parent cells were compared to those transduced with mscv - ires - gfp empty vector (ev), or mscv - flt3-ires - gfp (wt, it d and d835y). as shown in the figure 1, parental, ev and flt3-wt fdc - p1 cells did not proliferate in the growth factor (gf)-free medium while expression of flt3 mutants (it d and d835y) enabled proliferation. after 48 hr, the number of live cells was ~7 and ~21 times more for the fd - flt3-d835y and flt3-itd expressing cells, respectively. thus while both mutants conferred factor dependent growth, the flt3-itd appeared more efficient with > 3 fold more cells than the d835y mutant. in order to assess the effects of flt3 mutations on its susceptibility to inhibitors, cell proliferation assays were repeated on the fdc - p1 cells in the presence of cep701, dasatinib, imatinib, pkc412 and sunitinib. the experiments showed that cep701, pkc412 and sunitinib could inhibit the growth of fd - flt3-wt cells (table 1). however, dasatinib and imatinib were not effective at high nm concentrations (ec50 > 100 and 2000 nm, respectively). sunitinib with an ec50 value of 7.9 nm appeared to be the most potent among the five compounds tested in these experiments. comparing the response of fd - flt3-itd against fd - flt3-wt cells indicated similar sensitivities to the different compounds, apart from sunitinib which was three times less effective against the flt3-itd mutant than the flt3-wt (table 1). it was observed that the fd - flt3-d835y cells were 15 times more sensitive to cep701 and 30 times more sensitive to pkc412 compared to the flt3-wt and it d mutant cells, respectively (table 1). while the it d mutant appeared resistant to sunitinib, the response of the flt3-d835 mutant was almost equivalent to wt cells. to better understand the sensitivity of flt3-d835y to cep701, the phosphorylation status of flt3-d835y was investigated by western blot analysis. in figure 2, blotting flt3 immunoprecipitates against phosphotyrosine indicated that flt3-d835y is present as multiple bands, which possibly caused by different levels of posttranslational modification including phosphorylation and glycosylation. however, it 's fully glycosylated 160 kda form is missing, which indicates that very small proportion of flt3 can be mature to be detectable by flowcytometry (unpublished data), but not by western blotting. figure 2 also shows that the treatment of fd - flt3- d835y cells with different concentrations of kinase inhibitor, and cep701, resulted in diminished flt3 phosphorylation and increased intensity of flt3-reactive bands. there was a dose dependent inhibition of phosphorylation of flt3-d835y mutant ; in addition, cep701 was able to completely inhibit receptor phosphorylation at the concentration of 25 nm. to investigate the differences in the growth - promoting activities of the flt3-itd and d835y constructs, the system used to express each protein involved retroviral transduction of a polycistronic mrna with each flt3 cdna followed by an ires (internal ribosome entry site) and the cdna for gfp (green fluorescent protein). since both proteins are derived from the same transcript, gfp expression examination of gfp levels as geometric means (geo means) showed these to be almost equal in the fd - flt3-wt, -itd and d835y cells with values of 85, 72 and 70, respectively (table 2). the surface and total (both surface and intracellular) expression of flt3 was then determined comparing the values from fixed and fixed - permeabilised cells as described in the methods section. analysis of flt3 expression levels across the cell lines showed that the surface expression of flt3-wt was almost three times higher than the activating mutants (flt3-itd, d835y) (figure 3). the signal for pe (representing flt3) was similar for fixed and fixed - permeabilised samples for cells expressing flt3-wt or the flt3-itd mutant suggesting that all expressed flt3 could reach the cell surface. in contrast, the surface expression of flt3 in d835y mutants was less than those in it d. total flt3 signal was increased about 30% in the d835y after permeabilisation indicating a high percentage of flt3 protein trapped inside the cells. to compare the effects of flt3 (wt, it d and d835y mutants) expression on factor - dependent cell growth and survival, fdc - p1 parent cells were compared to those transduced with mscv - ires - gfp empty vector (ev), or mscv - flt3-ires - gfp (wt, it d and d835y). as shown in the figure 1, parental, ev and flt3-wt fdc - p1 cells did not proliferate in the growth factor (gf)-free medium while expression of flt3 mutants (it d and d835y) enabled proliferation. after 48 hr, the number of live cells was ~7 and ~21 times more for the fd - flt3-d835y and flt3-itd expressing cells, respectively. thus while both mutants conferred factor dependent growth, the flt3-itd appeared more efficient with > 3 fold more cells than the d835y mutant. in order to assess the effects of flt3 mutations on its susceptibility to inhibitors, cell proliferation assays were repeated on the fdc - p1 cells in the presence of cep701, dasatinib, imatinib, pkc412 and sunitinib. the experiments showed that cep701, pkc412 and sunitinib could inhibit the growth of fd - flt3-wt cells (table 1). however, dasatinib and imatinib were not effective at high nm concentrations (ec50 > 100 and 2000 nm, respectively). sunitinib with an ec50 value of 7.9 nm appeared to be the most potent among the five compounds tested in these experiments. comparing the response of fd - flt3-itd against fd - flt3-wt cells indicated similar sensitivities to the different compounds, apart from sunitinib which was three times less effective against the flt3-itd mutant than the flt3-wt (table 1). it was observed that the fd - flt3-d835y cells were 15 times more sensitive to cep701 and 30 times more sensitive to pkc412 compared to the flt3-wt and it d mutant cells, respectively (table 1). while the it d mutant appeared resistant to sunitinib, the response of the flt3-d835 mutant was almost equivalent to wt cells. to better understand the sensitivity of flt3-d835y to cep701, the phosphorylation status of flt3-d835y was investigated by western blot analysis. in figure 2, blotting flt3 immunoprecipitates against phosphotyrosine indicated that flt3-d835y is present as multiple bands, which possibly caused by different levels of posttranslational modification including phosphorylation and glycosylation. however, it 's fully glycosylated 160 kda form is missing, which indicates that very small proportion of flt3 can be mature to be detectable by flowcytometry (unpublished data), but not by western blotting. figure 2 also shows that the treatment of fd - flt3- d835y cells with different concentrations of kinase inhibitor, and cep701, resulted in diminished flt3 phosphorylation and increased intensity of flt3-reactive bands. there was a dose dependent inhibition of phosphorylation of flt3-d835y mutant ; in addition, cep701 was able to completely inhibit receptor phosphorylation at the concentration of 25 nm. to investigate the differences in the growth - promoting activities of the flt3-itd and d835y constructs, their relative expression levels were evaluated. the system used to express each protein involved retroviral transduction of a polycistronic mrna with each flt3 cdna followed by an ires (internal ribosome entry site) and the cdna for gfp (green fluorescent protein). since both proteins are derived from the same transcript, gfp expression examination of gfp levels as geometric means (geo means) showed these to be almost equal in the fd - flt3-wt, -itd and d835y cells with values of 85, 72 and 70, respectively (table 2). the surface and total (both surface and intracellular) expression of flt3 was then determined comparing the values from fixed and fixed - permeabilised cells as described in the methods section. analysis of flt3 expression levels across the cell lines showed that the surface expression of flt3-wt was almost three times higher than the activating mutants (flt3-itd, d835y) (figure 3). the signal for pe (representing flt3) was similar for fixed and fixed - permeabilised samples for cells expressing flt3-wt or the flt3-itd mutant suggesting that all expressed flt3 could reach the cell surface. in contrast, the surface expression of flt3 in d835y mutants was less than those in it d. total flt3 signal was increased about 30% in the d835y after permeabilisation indicating a high percentage of flt3 protein trapped inside the cells. to investigate the possible differences in growth promoting properties and drug responses, these flt3 mutants were expressed on murine factor dependent fdc - p1 cells. the fdc - p1 cell line needs either mouse il-3 or gm - csf for proliferation in dmem medium (31) which, however, can be abrogated by expression of oncogenic tyrosine kinases via induction of c - myc (32), or wild type tyrosine kinases in the presence of the specific ligand such as csf-1 (33). this makes fdc - p1 cells a suitable host for expression and studying these growth factor receptors and oncogenic kinases. in this study, we demonstrated that fdc - p1 cells became factor - independent by expression of human flt3-itd and -d835y mutants (figure 1). jm domain mutation (it d) relieves the autoinhibitory function of this domain or disrupts the steric hindrance preventing receptor dimerization in the absence of flt3l (6). similar to the kit d816 mutants (34), replacement of d835, on the other hand, may destabilize the inactive conformation via disruption of the h - bonds in the activation loop. comparing the homology models of flt3-d835y with flt3-wt structure (1rjb) showed that several h - bonds between side - chain of asp835 with met837 and ser838 in the activation loop would be eliminated if asp835 replaced with a val or tyr (unpublished data) which may contribute to the destabilization of inactive structure. in these fd - flt cells, the transcription of a polycystronic mrna for both gfp and flt3 is controlled by a single mscv promoter. therefore, it can be assumed that the expression of flt3 and gfp are equal in the mrna level. this was confirmed by the similarity of gfp expression in fd - flt3-wt, -itd and d835 mutants (table 2). however, the geo means for total flt3 expression in the fd - flt3-wt cells are about 70 and 62% higher than fd - flt3-itd and -d835y, respectively. this indicates that a significant proportion of the mutant flt3 protein disappears during posttranslational stages. constitutive activity of flt3 mutants results in its autophosphorylation, which are subjected to degradation via ubiquitin - binding (35) and also being detected as misfolded protein (36). misfolded proteins are retained in the er due to interaction with calnexin and calreticulin and may be subjected to degradation (37). therefore, it can be concluded that the majority of the expressed mutant flt3 was subjected to degradation as misfolded protein. it was reported that phosphorylation process affects maturation of the rtks including flt3 (36). treatment of fd - flt3-d835y cells with cep701 reduced flt3 phosphorylation dose - dependently and confirmed that phosphorylation inhibition is involved in the preservation of flt3 protein (figure 3). the minimum molecular weight of flt3 is 130 kd which can be increased up to 160 in mature protein. it was shown in figure 3 that the mature 160-kd flt3 form is missing in the western blot analysis ; but, inhibition of phosphorylation may protect the flt3 from degradation in the early stages. therefore, flt3 protein underwent more maturation and appeared with increased molecular weight bands in blotting for flt3. however, this needs more investigation as it seems that phosphorylation may have also adversely affected interaction of the flt3 with the antibody. this study also revealed that 40% of detected flt3 was intracellular in fd - flt3-d835y cell compared to 4 and 4.5% in fd - flt3- it d cells. this difference may result from the dynamics of the kinase domain activation between jm domain and activation loop mutants. in other words, it can be speculated that the inactive conformation of flt3-itd protein is more stable than flt3-d835y. therefore, it is more probable for flt3-itd to remain unphosphorylated to reach cell membrane for being activated via dimerization. it differs with flt3-d835y where high proportion of expressed protein may adopt the flt3 active conformation upon translation via a dimerization - independent mechanism as described for the kinase domain mutants of epidermal growth factor receptor (egfr) (38). as mentioned earlier, flt3-d835y mutant protein was retained intracellularly ; however, despite the report of flt3-itd retention in the er of cos-7 cells (36), our data did not show a significant retention of intracellular flt3-itd protein in the fd - flt3-itd cells (figure 2). this suggests that the protein degradation capacity of the fdc - p1 may be higher than cos-7 cells ; not allowing intracellular accumulation of constitutively active forms of flt3-itd. these degradation products, then, appear as smear in western blot analysis of the cell lysate (unpublished data). again, since greater proportion of flt3-d835y (40%) (compared to it d mutant) adopts active conformation, gets phosphorylated upon translation, and retained in the er as misfolded protein, which exceeds degradation capacity, it can be detected as intracellular flt3. fd - flt3-itd cells were three times more resistant to sunitinib than the fd - flt3-wt cells. since flt3-itd is a constitutively active mutant (24), the higher ec50 value is consistent with the finding in kit that sunitinib prefers the inactive conformation of the kinase domain (39). flt3-itd expression also increased the ec50 value of cep701, pkc412 and sunitinib by 15, 30 and 3.5 times compared to the flt3-d835 mutants. this is consistent with the frequent reports of drug resistant blast cells expressing flt3-itd in the patients (40). it was shown in this study that the number of viable fdc - p1 cells expressing flt3-itd was 3 to 4 times faster than those expressing flt3-d835y. as a result, it can be speculated that d835y mutation may have some adverse effects on cell proliferation / survival. this may explain the finding that the jm domain mutations are the most common genetic alteration in aml (41). lower proliferation / survival capacity and high sensitivity to kinase inhibitors may result from intracellular retention of flt3-d835y mutant protein because it was shown that protein retention can cause er stress, altered signaling pathways and activation of apoptotic signaling pathways (36, 42 - 44). a combination of preferential affinity of cep701 and pkc412 to the kinase domain compared to the jm domain mutants (29) and damaging effects of er stress may contribute to the high sensitivity of d835y to kinase inhibitors compared to the flt3-wt and it d mutant. therefore, studying these genetic variations can be applied to the determination of prognosis as well as designing of a therapeutic plan for the patients with flt3 mutations. this study demonstrates that even though both types of juxtamembrane and kinase domain mutations render the flt3 kinase constitutively active, but they have different sub - cellular effects and drug responses. therefore, identifying the mutation type is required for designing a personalized treatment strategy for the patients. | objective(s) : mutant forms fms - like tyrosine kinase-3 (flt3), are reported in 25% of childhood acute lymphoid leukemia (all) and 30% of acute myeloid leukemia (aml) patients. in this study, drug response, growth promoting, and protein trafficking of flt3 wild - type was compared with two active mutants (internal tandem duplication (it d)) and d835y. materials and methods : flt3 was expressed on factor - dependent cells (fdc - p1) using retroviral transduction. the inhibitory effects of cep701, imatinib, dasatinib, pkc412 and sunitinib were studied on cell proliferation and flt3 tyrosine phosphorylation. total expression and proportion of intracellular and surface flt3 was also determined. results : fdc - p1 cells became factor - independent after expression of human flt3 mutants (it d and d835y). fdc - p1 cells expressing flt3-itd grow 3 to 4 times faster than those expressing flt3-d835y. fd - flt3-itd cells were three times more resistant to sunitinib than the fd - flt3-wt cells. the geo means for surface flt3 expression in fd - flt3-itd and -d835y were 65 and 70% less than the fd - flt3-wt cells. about 40% of expressed flt3 was detected as intracellular in fd - flt3-d835y cell compared to 4 and 4.5% in fd - flt3-wt and -itd cells. conclusion : retention of d835y flt3 mutant protein may cause altered signaling, endoplasmic reticulum stress and activation of apoptotic signaling pathways leading to lower proliferation rate in fd - flt3-d835y than the flt3-wt and it d mutant., these may also also contribute, along with the preferential affinity, to the increased sensitivity of d835y of cep701 and pkc412. studying these genetic variations can help determining the prognosis and designing a therapeutic plan for the patients with flt3 mutations. |
traditionally laparoscopic cholecystectomy (lc) is performed under general anesthesia. with the advent of advanced laparoscopic surgical techniques, it has become possible to perform laparoscopic surgery of the gastrointestinal tract using epidural anesthesia. in the past decade, many surgeons published case reports of lc that was performed in pregnant and non - pregnant patients while they were under epidural anesthesia. ji hyun lee. have recently published their experience with lc being performed under epidural anesthesia in eleven patients. based on our experience with open cholecystectomy done under epidural anesthesia, we evaluated our experience of lc using epidural anesthesia. a total of 20 patients with asa status i or ii were included in the proposed study. inclusion criteria : patients with asa class i & ii and aged between 18 - 60 years were included in the study. exclusion criteria : patients below 18 years and above 60 years, those not willing to undergo lc, obese patients, patients with asa iii and above, patients presenting with acute symptomatology and suspected or proven gallbladder malignancy, patients having deranged bleeding parameters or vertebral column deformities, patients with proven or suspected common bile duct stones and having history of jaundice or gallstone pancreatitis, were excluded from the study. the patient was placed in the sitting position. continuous infusion was done with lactated ringer 's solution. under all aseptic and antiseptic precautions, the epidural space was identified using 17-gauge tuohy needle and loss of resistance technique, in the t9-t10 interspace or one or two spaces above or below this interspace when it was not possible in this space. the patient was then placed in the supine position and 3 ml of 2% lidocaine with adrenaline (1:200000) was given as a test dose followed by 10 ml of 0.5% bupivacaine, which was given via the epidural catheter. thereafter incremental doses of 3 ml of 0.5% bupivacaine was given till the desired level of block was reached. the anesthetic solution was prepared using 18 ml of lidocaine 2% plus epinephrine (1:200000) and 2 ml of sodium bicarbonate 8.4%. after negative aspiration, 3 ml of the solution was administered as a test dose followed by an additional 7 ml along with 50 g fentanyl and an additional 2 ml of the solution was administered incrementally to reach the desired level of segmental block. the upper and lower levels of sensory and motor block were assessed by a pinprick test and the bromage scale, respectively, and recorded every 5 minutes until the start of surgery and every 15 minutes postoperatively. intraoperative anxiety was treated with midazolam, 1 - 2 mg ; abdominal or referred shoulder pain with incremental fentanyl, 1 - 2 g / kg ; and hypotension with ephedrine, 5 - 10 mg ; all as intravenous (i.v.) boluses as required. the discomforts of the patient, during and after the procedure, were recorded (for example : pain, nausea, or itching). oxygen at the rate of 6 liter / minute was supplied via a face mask to all the patients while monitoring the end - tidal carbon - dioxide. surgery will be performed with the conventional four - port technique : one 10 mm trocar above the umbilicus, one 10 mm below the xiphoid, one 5 mm below the right costal margin at the mid - clavicular line and one more 5 mm trocar below the right costal margin at the anterior axillary line. pneumoperitoneum will be established with carbon dioxide at a maximum intra - abdominal pressure of 10 mm hg instead of the usual 15 mm hg, to avoid shoulder pain due to diaphragmatic irritation. operating table tilting to the left and also the patients head up which is the conventional position for lc will be kept to the minimum or none to avoid diaphragmatic irritation. simplified questionnaire forms were being developed for patients [table 1 ] and also for the operating team [table 2 ] to evaluate comments about the operation. the patients were asked to complete the questionnaire form on the first postoperative day. visual analog scale (vas) for pain bromage scale [table 3 ] was also noted during surgery to assess the intensity of motor blockade. continuous infusion was done with lactated ringer 's solution. under all aseptic and antiseptic precautions, the epidural space was identified using 17-gauge tuohy needle and loss of resistance technique, in the t9-t10 interspace or one or two spaces above or below this interspace when it was not possible in this space. the patient was then placed in the supine position and 3 ml of 2% lidocaine with adrenaline (1:200000) was given as a test dose followed by 10 ml of 0.5% bupivacaine, which was given via the epidural catheter. thereafter incremental doses of 3 ml of 0.5% bupivacaine was given till the desired level of block was reached. the anesthetic solution was prepared using 18 ml of lidocaine 2% plus epinephrine (1:200000) and 2 ml of sodium bicarbonate 8.4%. after negative aspiration, 3 ml of the solution was administered as a test dose followed by an additional 7 ml along with 50 g fentanyl and an additional 2 ml of the solution was administered incrementally to reach the desired level of segmental block. the upper and lower levels of sensory and motor block were assessed by a pinprick test and the bromage scale, respectively, and recorded every 5 minutes until the start of surgery and every 15 minutes postoperatively. intraoperative anxiety was treated with midazolam, 1 - 2 mg ; abdominal or referred shoulder pain with incremental fentanyl, 1 - 2 g / kg ; and hypotension with ephedrine, 5 - 10 mg ; all as intravenous (i.v.) boluses as required. the discomforts of the patient, during and after the procedure, were recorded (for example : pain, nausea, or itching). oxygen at the rate of 6 liter / minute was supplied via a face mask to all the patients while monitoring the end - tidal carbon - dioxide. surgery will be performed with the conventional four - port technique : one 10 mm trocar above the umbilicus, one 10 mm below the xiphoid, one 5 mm below the right costal margin at the mid - clavicular line and one more 5 mm trocar below the right costal margin at the anterior axillary line. pneumoperitoneum will be established with carbon dioxide at a maximum intra - abdominal pressure of 10 mm hg instead of the usual 15 mm hg, to avoid shoulder pain due to diaphragmatic irritation. operating table tilting to the left and also the patients head up which is the conventional position for lc will be kept to the minimum or none to avoid diaphragmatic irritation. simplified questionnaire forms were being developed for patients [table 1 ] and also for the operating team [table 2 ] to evaluate comments about the operation. the patients were asked to complete the questionnaire form on the first postoperative day. visual analog scale (vas) for pain bromage scale [table 3 ] was also noted during surgery to assess the intensity of motor blockade. hypotension was observed in ten patients during surgery which was treated successfully with intravenous ephedrine. significant bradycardia, with a heart rate below 50 bpm, occurred in three patients. eight patients experienced shoulder pain and five of them required intravenous fentanyl injection at a dosage of 50 g. two patients could not tolerate the shoulder pain and general anesthesia was given for them. the average total operation time was 44.4 minutes (range, 34 - 58 minutes) and total anesthesia time was 68.2 minutes (range, 52 - 89 minutes). all patients could ambulate 6 hours after the operation, and there were no complications or morbidity in the postoperative period. the mean hospital stay for the patients was one and a half day (range 1 - 3 days). the average patient 's satisfaction score assessed at 3 hours after the operation was 8.2 (range, 7 - 9) and the average pain score (vas) checked at 4 hours after operation was 2.1 (range, 1 - 3). surgeons did not have problems with relaxation of musculature, or the surgical technique, and answered that there was no difference between the technique and general anesthesia. patients can be awake and oriented at the end of the surgery and have less postoperative pain, nausea and vomiting. problems related to general anesthesia such as oral and teeth injury during laryngoscopy, and a sore throat and stomach inflation as a result of mask ventilation could be avoided in a regional anesthetic setting. for the successful completion of lc under regional anesthesia, neuraxial blockade must be performed to cover t6 level or above as demonstrated by lal. referred shoulder pain due to diaphragmatic irritation from carbon dioxide pneumoperitoneum was a significant intraoperative problem. eight patients (40%) experienced shoulder pain and five of them required intravenous fentanyl injection at a dosage of 50 g. sinha reported shoulder pain in 12.29% of patients, whereas pursnani. noted shoulder pain occurred in 2 of their 6 patients operated under epidural anesthesia, and was easily manageable with reassurance, no medical treatment, or simple analgesics. the higher incidence of shoulder pain in our patients might be due to relative inexperience of the operating surgeons as the procedure was carried out in our hospital for the first time. shoulder pain may be managed by using nitrous oxide, gentle surgical manipulation, nasogastric tube insertion for gastric decompression, irrigation of the right diaphragm with 2% lidocaine solution, phrenic nerve block and addition of non - steroidal anti - inflammatory drugs. no significant changes were noted in the respiratory parameters during epidural anesthesia in any patients similar to the findings by ciofolo. zhang. in their retrospective analysis of 100 patients undergoing lc under epidural anesthesia while compared with similar number of patients undergoing lc under general anesthesia has concluded that lc is feasible under epidural anesthesia and is a safe procedure in selected patients. most of the patients regarded epidural anesthesia as a comfortable procedure with lesser cost than those undergoing the same procedure under general anesthesia. ross. in their study of laparoendoscopic single - site (less) surgery for cholecystectomy under epidural anesthesia in 20 patients with a single incision around umbilicus has concluded that epidural anesthesia appears to be a preferable alternative to general anesthesia for patients undergoing less cholecystectomy with no operative or anesthetic conversions, and less postoperative pain at discharge. one of the most important problems of lc under regional anesthesia is inadequate relaxation of abdominal musculature but this problem was not encountered in our patients. our study has limitations in patient selection, but since it was our first venture in the procedure we selected relatively healthier patients without any acute inflammations. but this procedure might be more beneficial to those patients with higher risk for general anesthesia due to the presence of co - morbid conditions. our study has provided preliminary evidence about the efficacy of epidural anesthesia for performing lc. this procedure may be more beneficial for high - risk patients for general anesthesia. shoulder pain may be controlled by using nitrous oxide and local irrigation of diaphragm with anesthetics. | background : laparoscopic cholecystectomy (lc) is normally performed under general anesthesia. but of late this operation has been tried under regional anesthesia successfully without any added complications like epidural anesthesia.aims:the aim of the study was to study the feasibility of performing lc under epidural anesthesia in normal patients so that the benefits could be extended to those high - risk patients having symptomatic gallstone disease and compromised cardio - pulmonary status where general anesthesia is contraindicated.materials and methods : in all, 20 patients with the american society of anesthesiologist 's class i or ii were enrolled in the study. the level of epidural block and satisfaction score, both for the patient and the surgeon, were noted in the study.results:the lc was performed successfully under epidural anesthesia in all but two patients who had severe shoulder pain in spite of giving adequate analgesia and were converted to general anesthesia.conclusions:the lc can be performed safely under epidural anesthesia with understanding between patient and surgeon. however, careful assessment of complications in the patients should be done to make the procedure safer. |
this study compared the contents of prosapogenin depending on the extracting conditions of red ginseng to provide basic information for developing red ginseng - based functional foods. the content of ginsenoside rg3 reached their maximum value at 24 h of extraction, followed by 36 h and 72 h of extraction at 100c. |
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in the kinetoplastid parasite trypanosoma brucei clathrin - mediated endocytosis is essential for survival and aids immune evasion in the mammalian host. the formation of endocytic clathrin coated vesicles in t. brucei is via a unique mechanism owing to an evolutionarily recent loss of the adaptor protein (ap)2 complex, a central hub in endocytic vesicle assembly. despite this loss, recent studies examining endocytic clathrin coat assembly have highlighted a high degree of conservation between trypanosomes and their mammalian hosts. in particular phosphatidylinositol 4,5-bisphosphate (pi(4,5)p2) and its putative effectors, tbcalm and tbepsinr, are central to clathrin - mediated endocytosis in the trypanosome, just as they are in animal cells. in addition to providing insights into the cell biology of t. brucei, these studies also suggest an ancient, possibly pan - eukaryotic connection between ptdins(4,5)p2 and endocytosis. |
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intermediate hosts are typically sheep ; however, the disease can occur less frequently in humans. a history of direct contact with dogs is not available in all reported cases, and infection can be acquired by the consumption of contaminated food and milk. after ingestion of contaminated food, the embryos migrate through the portal system to the liver, and later the lungs. intracranial hydatid disease is rare, with a reported incidence of 1 - 2% of all cases with hydatid disease. cerebral hydatid cysts are more common in the pediatric population, with 80% of the affected patients being children ; moreover, the cysts are most frequently observed in the supratentorial region and parietal lobe. the high incidence in children is due to patent ductus arteriosus or other valve dysfunctions. the main treatment is the complete surgical removal of the cyst without rupture, and the most commonly used surgical technique is the dowling method modified by arana - iniguez. a 13-year - old male patient was admitted to the emergency department with headache, nausea, and vomiting. ct showed a solitary cystic lesion with a diameter of 10 cm, which was hypodense, sharply demarcated, without perifocal edema, and caused a shift of the midline structures to the contralateral side. solitary cystic lesion located in the right temporoparietal region because of the midbrain herniation signs, urgent operative intervention via a large craniotomy was performed. after visualization of the cyst wall, 0.9% saline solution was used to dissect the hydatid cyst from the surrounding brain to facilitate cyst removal (dowling method modified by arana - iniguez). the dura was closed in a watertight fashion after filling the remaining cavity with 0.9% saline solution [figure 2 ]. appearance of the hydatid cyst during removal by the dowling method modified by arana - iniguez. as the saline injection dissected the cyst from the surrounding brain, the cyst mass moved outside of the brain from its nest postoperative thorax and abdominal ct revealed no other foci. fifteen days after the operation, a left hemiparesis occurred and cranial ct scan revealed a brain compressing subdural hygroma [figure 3a ] ; therefore, we inserted a subdural - peritoneal shunt. based on cranial ct performed during follow - up, the subdural - peritoneal shunt was effective and no complaints were reported [figure 3b ]. in the same ct examination done for follow - up, we noted that the site from where the hydatid cyst was removed was flask - shaped, relaxed space, and filled with cerebrospinal fluid (csf) [figure 3b ]. however, in the follow - up at the fourth month after intracranial operation, we saw a distended cyst that had replaced the flask and relaxed csf - filled space from where the hydatid cyst was removed ; interestingly, we noted a jet - flow with flow void effect as an influx. this appearance suggested that a one - way valve mechanism had developed that filled the space [figure 4 ]. (b) postoperative ct determined regression of subdural hygroma with flask, relaxed space, filled with cerebrospinal fluid during follow - up, mri scans showed a porencephalic cyst at the site where the hydatid cyst was extirpated. interestingly, we noticed a thin outer membrane (black arrow) at the operation site around the porencephalic cavity. the jet - flow (white arrow) and flow void provided evidence of the one - way filling valve in figure 4, the jet - flow and flow void of the high - speed csf can be easily observed. this setup demonstrates the one - way valve mechanism of csf flow, which causes enlargement of the porencephalic cyst. the patient underwent a third surgical procedure, and fenestration of the cyst to the subdural space was performed. a postoperative follow - up cranial ct scan showed no signs of the porencephalic cyst [figure 5 ]. postoperative albendazole therapy was then applied for 4 months (10 mg / kg per day). the patient was discharged following normal neurological exams. currently, the patient is healthy and attending school without any epileptic seizures. the sulci around the porencephalic cyst were calm, and the operation cavity showed no tension or pressure to the surrounding tissue, thus providing evidence of decompression a history of direct contact with dogs is not available in all reported cases, and the infection can be acquired by consuming contaminated food and milk. a cerebral hydatid cyst is more common in the pediatric population, with 80% of the patients being children. this high incidence in children is primarily due to patent ductus arteriosus or other valve dysfunctions. intracranial hydatid cysts are more frequently located in the supratentorial region, with the parietal lobe being the most common site. hydatid cysts typically involve the middle cerebral artery territory, with other less common sites being the skull, cavernous sinus, eyeball, pons, cerebellum, ventricles, and interpeduncular cistern. cerebral hydatid cysts usually grow slowly and present clinical symptoms when they increase in size and produce a mass effect. this may be due to the elastic structure of the cranial bones, open sutures, and compressibility of the neural tissue. the growth rate of hydatid cysts in the brain has been variably reported at 1.5 - 10 cm per year. the following growth rates have been reported : evliyaoglu. reported a growth rate of 1 cm / month by assessing repeated examinations ; kemaloglu. reported hydatid cyst growth rate as 4.5 cm in 6 months based on ct findings ; pasaoglu. reported the growth rate of an untreated cyst as 10 cm / year based on follow - up ct ; kalelioglu. reported a hydatid cyst growth rate of 5 cm / year based on follow - up ct scans ; vaquera. patients with intracranial hydatid cysts usually present with features such as raised intracranial pressure due to the mass effect of the large - sized cyst. focal neurologic deficits, such as hemiparesis, visual disturbances, ataxia, seizures, or other manifestations depending on the cyst location have been reported. headache and vomiting are almost universally present along with bilateral papilledema. in our patient, headache, nausea, and vomiting were clinical symptoms and bilateral papilledema without neurologic deficits was present. a study using cranial ct showed fluid that was generally the same as the csf without perilesional edema ; however, yis., around the cyst in the presence of active inflammation. a differential diagnosis of cerebral hydatid cysts on ct, as with arachnoid cyst, porencephalic cyst, neurocysticercosis, abscess, and pilocytic astrocytoma, has been reported. magnetic resonance imaging (mri) typically shows a cystic lesion with a hypointense halo around the cyst capsule. ozkan. reported the usefulness of ct scans in the diagnosis of cerebral hydatid disease and in the planning of appropriate surgical management. we could not perform a cranial mri prior to the operation due to the poor condition of the patient. the patient underwent an urgent operation and ct showed hypodense cystic lesion that was sharply demarcated and without perifocal edema and fluid identical to csf. in emergencies, such as herniation in the presence of cystic lesions identified by ct, the possibility of hydatid cysts should be considered. otherwise anaphylactic shock, chemical meningitis, or recurrence may occur due to the rupture of the hydatid cyst. in literature review, we found two cases that received an urgent operation for a cerebral hydatid cyst. while onal. treated successfully a 13-year - old girl by puncturing the cyst, another patient died after operation. our patient, who also received emergency surgery due to the rapid deterioration of his neurological status, was discharged after three operations following normal neurological exams. the primary cysts are formed as a result of direct infestation of the larvae in the brain without the involvement of other organs. the secondary multiple cysts result spontaneously or from trauma or rupture during operation of the primary intracranial hydatid cyst. our patient 's thorax and abdominal ct showed no other foci for the hydatid disease. the most commonly used surgical technique is the dowling method modified by arana - iniguez. in addition, direct puncture and aspiration of the cyst fluid through a small hole in the cyst wall or expulsion of the cyst through a small cortical incision over the cyst using insufflation of air during contralateral ventricle techniques can be applied. a small number of reports showed complete disappearance of multiple intracranial hydatid cysts with albendazole therapy using a daily dose of 10 mg / kg taken three times a day for 4 months. in the literature, recurrence rates of 19% and a mortality of 10 - 12%, morbidity of 9.8%, and a preoperative mortality of 8.48% have been reported. when large cysts are removed in the pediatric population, subdural collections and porencephalic cysts have been reported. our patient received three operations for the treatment of hydatid cysts, subdural hygroma, and fenestration of the porencephalic cyst to subarachnoid space ; subsequently, postoperative albendazole therapy was applied for 4 months (10 mg / kg per day). hydatid cysts are known to enlarge slowly, rarely requiring emergency surgery.in case of emergencies, such as midbrain herniation and if cystic lesions identified by ct, the possibility of hydatid cysts should be considered where the infection is endemic.irrespective of the preoperative diagnosis, if there is apprehension in the concrete diagnosis and suspicion of a possibility of a hydatid cyst through ct, we highly recommend the team to be prepared to remove a hydatid cyst without rupture. hydatid cysts are known to enlarge slowly, rarely requiring emergency surgery. in case of emergencies, such as midbrain herniation and if cystic lesions identified by ct, the possibility of hydatid cysts should be considered where the infection is endemic. irrespective of the preoperative diagnosis, if there is apprehension in the concrete diagnosis and suspicion of a possibility of a hydatid cyst through ct, we highly recommend the team to be prepared to remove a hydatid cyst without rupture. | hydatid disease is a parasitic infection affecting the brain in about 2% of the cases. brain involvement is most commonly observed in children. here, we report a 13-year - old male patient who presented with headache, nausea, and vomiting. before cranial computed tomography (ct) was performed, the patient had generalized epileptic seizures. he was disoriented, and had anisocoria with dilatation of the right pupilla. ct showed a cystic lesion of 10-cm diameter in the right temporoparietal region that had caused a shift of the midline structures to the contralateral side ; an urgent operation was performed as there were signs of midbrain herniation. |
overall, a total of 13,971 male employees and their spouses, after 12 h of fasting, underwent health check - ups during a baseline survey performed in 2004 and 2005 at the hitachi health care center in ibaraki prefecture. of these, 2,655 men received a computed tomography scan. the final analysis involved 1,106 men from the initial study who participated in a 3-year follow - up survey performed in 2007 and 2008 (aged 3072 years in the baseline survey). the mean age of the men for whom follow - up data were available was younger than that of the men for whom follow - up data were not available. in addition, the mean age of the men who received a computed tomography examination in the baseline survey was greater than the mean age of the men who did not receive the computed tomography examination. no significant differences were seen between the other characteristics when age - adjusted comparisons were performed. self - reporting questionnaires were used to investigate whether the subjects were currently receiving medical treatment for hyperlipidemia, hypertension, or diabetes at the time of both surveys. body height and weight were measured using an automated scale (bf-220 ; tanita), and the bmi was defined as the weight in kilograms divided by the square of the height in meters. vfa, sfa, and waist circumference were measured using a computed tomography scanner and protocols previously described (20). in brief, single - slice imaging was performed at the umbilical level in a spine position using a redix turbo ct machine (hitachi medico). the imaging conditions were 120 kv, 50 ma, and a slice thickness of 5 mm. vfa, sfa, and waist circumference were calculated using fatpointer software (hitachi medico). triglyceride and hdl cholesterol levels were measured using the oxygen method with a hitachi 7600 device (sekisui medical). blood glucose levels were measured using the glucose electrode technique with an adams glucose ga-1170 device (arkrey). blood pressure was measured using an oscillometric method with a kentaro advance bp-203rv iii a / b device (colin) while the patient was in a sitting position and after the patient had rested for 3 min. informed consent was obtained from each examinee regarding the use of his or her data for research purposes. the current study was approved by the ethics review committee of the national center for global health and medicine. subjects with two or more of the following four factors, defined by the national cholesterol education program 's adult treatment panel iii guidelines (21), with the exception of waist circumference, were defined to have clustering of metabolic risk factors : 1) triglyceride 150 mg / dl (1.69 mmol / l), 2) hdl cholesterol 1,000 subjects being followed over a 3-year period. our study is thought to have a small measurement bias because vfa and sfa were measured using the same computed tomography machine, in the same region, and at the same state of expiration during both the baseline examination and the 3-year follow - up examination. a random measurement error could have diluted the relationship between vfa and the metabolic risk factors. the study subjects were limited to men, and further studies in women are needed. in conclusion, the current study of japanese men showed that changes in the vfa were associated with changes in metabolic risk factors. the vfa was significantly related to log triglyceride and hdl cholesterol and was independent of body weight and waist circumference, suggesting the importance of measuring the vfa repeatedly over time. the adoption of a lifestyle that does not result in an increase in vfa is important in preventing metabolic syndrome. | objectivethe effects of longitudinal changes in the visceral fat area (vfa), and other anthropometric indices, on the risk factors of metabolic syndrome were not studied. we calculated the changes in metabolic risk factors in relation to changes in certain anthropometric indices in a large - scale study of japanese men.research design and methodsthe subjects were 1,106 men participating in the hitachi health study who received a computed tomography examination in both 2004 and 2007. vfa, subcutaneous fat area (sfa), and waist circumference were measured using the computed tomography. we examined how longitudinal changes in each anthropometric index over a 3-year period influenced the value of each metabolic risk factor.resultschanges () over a 3-year period in body weight, sfa, and waist circumference strongly correlated, while the changes in body weight and vfa were weakly correlated. changes in the vfa were associated with changes in metabolic risk factors, especially changes in triglyceride and hdl ; we found these changes to be independent of the body weight and waist circumference.conclusionschange in body weight is not a precise surrogate marker of vfa, and repeated vfa measurements over time are useful. adopting a lifestyle that does not increase the vfa is important in preventing metabolic syndrome. |
the widespread uses of radiological imaging increased the number of incidentally detected renal tumors in asymptomatic patients. high frequency of small renal tumors detection is also associated with wide accessibility to ultrasonography, which has become the initial imaging technique during the diagnostic process in urology. 45% of these tumors are renal cell carcinoma (rcc) in stage t1n0m0, characterized by favorable prognosis after radical surgical treatment. retrospective studies have proved that the application of nephron - sparing surgery (nss) for these patients is as effective as radical nephrectomy. the use of nss techniques in small tumors is highly recommended in all patients due to increased risk of development of chronic renal disease after radical nephrectomy [5, 6 ]. however, taking into account the increasing incidence of kidney cancer, partial nephrectomy has become an inconvenient method of treatment. the main disadvantages of partial nephrectomy are the long learning curve, duration of surgery and high costs of the procedure. ablative treatment is an alternative method which can successfully replace partial nephrectomy in many cases. ablative techniques are also recommended for removal of renal tumors in patients with von hippel - lindau syndrome and with one active kidney. the most commonly used ablative techniques in urology are cryoablation and radiofrequency ablation (rfa). the next generation ablative techniques utilizing microwaves and ultrasound for tissue necrosis are under clinical trials. according to eau (european association of urology) guidelines, renal tumors smaller than 4 cm (t1a) are the only suitable indication for ablative therapy. the primary factors for further eligibility of patients are the favorable localization of the lesion and the number of tumors. the secondary indications are age and life expectancy after surgery. given the controversial radicality of ablation therapies this treatment there is a need for long - term follow - up to compare the recurrence rate in patients after ablation and partial nephrectomy. results of this experiment should give a definitive answer to the question of the effectiveness of the use of ablation in young patients and establish the place of ablative techniques in the therapeutic process. this is particularly important taking into account the tendency for detection of renal tumors in young people without other diseases. ablation of renal tumors is mainly performed percutaneously nowadays under ultrasound, ct (computed tomography) or mri (magnetic resonance imaging) imaging in short - term general anesthesia. the choice of ablation and imaging method should be adopted by a team consisting of a urologist and an invasive radiologist. nevertheless, percutaneous ablation is preferred for removing small tumors localized in the renal cortex. more deeply located lesions should be ablated by laparoscopic access which allows one to protect the ureter, blood vessels and intestinal wall from iatrogenic injury during the procedure. the main advantage of laparoscopy is the possibility to take samples of pararenal fat for histopathological examination. the lack of pathological verification of the tumor type requires careful and accurate postoperative imaging of ablation lesions. ct or mri imaging in the follow - up of patients after renal tumor ablation is recommended. the first radiological evaluation should be done within 3 months after surgery and the second one after 9 months. these studies concerned the treatment of primary tumors of the liver. until the 1990s only a few introductory studies were published whose authors proposed cryoablation for experimental treatment for renal tumors. in 1995, uchida reported the first use of percutaneous renal cryoablation. the main limitation was the large size of cryoprobes that made it difficult to precisely place them in the specified part of the kidney. the advances that have been made in the field of cryoablation equipment production led to the development of needle - shaped cryoprobes with a diameter of 2 millimeters. such cryoprobes allow one to perform percutaneous surgery and are suitable to remove renal tumors. the effectiveness of tissue destruction during cryoablation depends on the cooling rate and the number of cooling cycles. cooling the normal tissue to 20c triggers apoptosis in all cells and results in their sudden death. only cooling of the tumor mass to 40c guarantees the death of all cancer cells. the cooling rate of tissue in the range of 10c / h results in water crystallization in the extracellular space. ice crystals disrupt the cell membrane and increase at the same time the extracellular osmolarity. cooling at a rate of 100c / h initiates water crystallization in the cell cytoplasm. the latest devices for cryoablation allow one to cool the tissue at the rate of 50c per hour. apart from mechanical injury caused by ice crystals, the devastating effect of low temperature on tumor tissue is a consequence of damage to endothelium of blood vessels. this leads to abnormal tumor tissue perfusion and in turn extends the area of necrosis. additionally, even a small decrease of temperature triggers activation of heat shock protein, which may initiate apoptosis. the area of frozen tissue depends on the size of the cryoprobe, the probe core temperature and the degree of tissue vascularization. the formed ice ball has an average temperature of 50c ; this is sufficient to freeze a tumor and initiate necrosis. some researchers suggest using higher temperatures of 30c to 40c in order to reduce the risk of bowel or ureter injury. the correct placement of the cryoprobe allows one to obtain at least 5 millimeters of tissue margin around the tumor, which is frozen up to 20c. the state of art is to perform double - cycle freezing with ultrasound evaluation of 1 cm area from the tumor 's borders.. followed up 48 patients after laparoscopic cryoablation for 36110 months (median 5 years). cancer - free survival was in this study 87.5%. a local cancer recurrence required repeat of the procedure and was successful in 97.5%. according to published data renal tumors more advanced than stage t1a are characterized by irregular shape and they require more than one cryoprobe to freeze them. in 1997, zlotta. first described the use of radiofrequency ablation (rfa) to treat renal tumors in three patients. currently, rfa is used as a routine method of ablation in many centers and it is mainly done percutaneously under ultrasound as well as ct or mri imaging. the concept of this technique is passing a high - frequency (460500 khz) alternating electrical current through the tissue surrounding an electrode. the passing current generates heat proportional to the current density ; therefore the highest temperature is generated in the tissues adjacent to the electrode. the death of cells is a result of protein denaturation and damage of the cell membrane by high temperature. the requirement for successful ablation using rfa is to obtain a temperature of 50100c throughout the tumor and maintain it for 35 min. in modern devices the temperature in the area surrounding the probe it reduces the effectiveness of ablation, because there is no circuit between the probe and the ground. several probes or a single probe with branches like an umbrella needs to be used in cases of renal tumors of size greater than 2 cm. less than 3 cm local recurrence was not observed in any case. on the other hand, in patients with tumors larger than 4 cm the efficacy of therapy decreased to 80%. gervais. did not report any local recurrence in 100 patients after rfa renal tumors smaller than 4 cm. the average follow - up in studies concerning clinical use of ablative methods is 2.5 years (fig. 2). thermoablation of renal tumor, using a probe with multiple tines (open method) state after unsuccessful rfa removal of renal mass. the arrows indicate the place of recurrence localization of tumor is another crucial factor that has an impact on ablation effectiveness. firstly, in this case there is a high risk of accidental damage of the renal hilum vessels. secondly, these vessels work as heat exchangers that decrease the temperature generated by the rfa probe. in overall clinical usefulness rfa the main challenge is to accurately place the rfa needle just in the middle of the long axis of the tumor. the renal mass before thermoablation (picture a). the same tumor after thermoablation (picture b) percutaneous application of high - intensity focused ultrasound (hifu) is an attractive method to treat small renal tumors. the hifu procedure is image guided using mri or ultrasound to target the ultrasound beam on the tumor in the most optimum way. the high - energy ultrasound waves are generated by a cylindrical piezoceramic element with a parabolic reflector. the computer automatically integrates data from the imaging device with the movements of the robotic arm where the ultrasound generator is situated, to precisely focus the ultrasound beam on the tumor and deposit specific energy within it. he observed interstitial hemorrhages, fiber rupture, shrinkage of collagen fibers and coagulation necrosis. in clinical practice the risk of accidental damage to healthy tissue is very low due to use of effective imaging guidance methods. currently hifu therapy for renal tumors is under clinical trials but it is gaining popularity very fast and in many centers hifu is already used routinely. published the results of ablating three tumors in one kidney by hifu. in this case, two tumors in the lower pole were successfully cured, but the third one localized in the upper pole was intact because the ultrasound waves were absorbed by the ribs. the failure of hifu therapy against renal tumors is associated with difficulties in the appropriate setting of hifu generator parameters. the histological heterogeneity of small renal tumors affects diffusion of the ultrasound beam in the tumor mass. the disturbances in the propagation of ultrasound waves have an impact on the effectiveness of hifu ability to cause necrosis. the difficulties in proper energy deposition within tissue lead to side effects often observed in patients undergoing hifu therapy. a long persistent hematuria and renal pain are most commonly observed. hifu is an experimental method today due to the limited therapeutic success confirmed in many clinical studies. further improvements of this method are absolutely necessary for the wide treatment of renal tumors in clinical practice. it generates an alternating magnetic field which induces rotation of water molecules and thermal energy flow. microwave ablation (ma) kills the cells in a similar mechanism as rfa does. routinely, renal tumors are treated using microwaves only in japan. in japan access to new medical technologies nevertheless, microwave ablation is the therapy of the future. according to the results of the preliminary research, it is an equally effective method as other ablative procedures presented above ; however, it is much faster and easier to perform. on the other hand, the number of studies is not sufficient to compare its efficacy to well constituted ablation techniques. the treatment of renal tumors using ma requires a single session and takes 5 min. in comparison with rfa, additionally, microwaves penetrate very easily through the tumor and the temperature is more equally distributed. studies have shown that microwave ablation can be used effectively for small renal tumors in stage t1. liang. reported 12 cases of renal tumors which were removed using microwave ablation. the follow - up was 11 months long and no recurrence was noted in any cases. based on the results of different studies, the risk of recurrence after renal tumor ablation using microwaves is calculated at 5%. | ablative therapies of renal tumors are steadily gaining popularity in clinical practice due to the many benefits they offer to patients. moreover, ablative procedures hold promise in the field of uro - oncology for the best compromise between low invasiveness, high efficacy and advantages in terms of procedural costs. reported outcomes with ablative therapies for small renal tumors are excellent and without significant differences for surgical procedures based on nephron - sparing surgery. nevertheless, these methods for treatment of small renal tumors should still be confined to carefully selected patients. this review discusses the currently used ablative techniques in urology. |
is mediated by clonally distributed t and b lymphocytes, namely, humoral and cellular immunity, and is characterized by specificity and memory. a basic mathematical model describing hiv-1 infection dynamics model with humoral immunity was introduced by murase. as (1)dttdt=dttttvt, ditdt=ttvtit, dvtdt = nitcvtqbtvt, dbtdt = gbtvtbt, where t(t), i(t), v(t), and b(t) represent the densities of uninfected cells, infected cells, virus, and b cells at time t, respectively. is the infection rate, n is the average number of virus particles produced over the lifetime of a single infected cells, and is the death rate of infected cells ; c is the death rate constant of the virus, g and are the recruited rate and death rate constants of b cells, and q is the b cells neutralization rate. mathematical models for virus dynamics with antibody immune response has drawn much attention of researchers (see, e.g., [113 ] and the reference therein). recently many studies have been done to improve the model (1) by introducing delays and changing the incidence rate according to different practical background. these studies used different delayed models with different forms of incidence rate ; see, for example, [6, 911 ] for discrete delays and [5, 13 ] for distributed delays. in the present paper, motivated by the works of [1, 5, 13 ], we propose the following model with a general incidence rate and distributed delays and humoral immunity : (2)dttdt=dttftt, vtvt, ditdt=0h1p1em1ftt,vt vtdit, dvtdt = n0h2p2em2itd cv(t)qb(t)v(t),dbtdt = gbtvtbt, where the parameters in system (2) have the same meanings as in system (1). it is assumed in (2) that the uninfected cells that are contacted by the virus particles at time t become infected cells at time t, where is distributed according to p1() over the interval [0, h1 ], where h1 is the limit superior of this delay. the constant m1 (m1 > 0) is assumed to be the death rate for infected cells during time period [t, t ] but not yet virus - producing cells and the term e denotes the surviving rate of infected cells during the delay period. on the other hand, it is assumed in (2) that a cell infected at time t starts to yield new infectious virus at time t, where is distributed according to a probability distribution p2() over the interval [0, h2 ] and h2 is limit superior of this delay. the factor e accounts for the probability of surviving infected cells during the time period of delay, where m2 is constant. in (2), the probability distribution functions pi(), i = 1,2, are assumed to satisfy pi() > 0, i = 1,2 and (3)0hipi()d=1, 0hipi()d 0, for all t > 0 and v 0,(h3)f(t, v)/v 0, for all t > 0 and v 0.(h4)(f(t, v)v)/v 0, for all t, v > 0. f(0, v) = 0, for all v 0, f(t, v)/t > 0, for all t > 0 and v 0, f(t, v)/v 0, for all t > 0 and v 0. the biological meaning of hypothesis (h1) to (h4) is given in. the incidence rate f(t, v)v given in (2) generalizes many common forms such as [5, 9, 13 ] (see section 6). the distributed delay is more general than the discrete one and it is more adapted to biological phenomena. h 1 or h2 can be infinity., we establish the nonnegativity and boundedness of solutions and we derived the basic reproduction ratios for viral infection and humoral immune response r0 and r1, respectively. in section 3, the existence of a possible three positive equilibria, an infection - free equilibrium e0, an infected equilibrium without b cells response e1, and an infected equilibrium with b cells response e2, is established. in sections 4 and 5, we show that the global asymptotic stability of these equilibria depend only on the basic reproduction numbers under some hypotheses on the incidence function. in section 6, the initial conditions of (2) are given as (4)t=1, i=2, v=3,b=4,i0, i=1,2,3,4,h,0, h = maxh1,h2, where = (1, 2, 3, 4) c, here c = c(h, 0 ], +), with c(h, 0 ],) ; denotes the banach space of continuous functions mapping the interval h, 0 ] into. theorem 1. under the initial conditions (4), all solutions (t(t), i(t), v(t), b(t)) of system (2) are nonnegative on [0, + and bounded. under the initial conditions (4), all solutions (t(t), i(t), v(t), b(t)) of system (2) prooflet us put system (2) in a vector form by setting z = (t, i, v, b) and(5)gz = g1zg2zg3zg3z=dttftt, vtvt0h1p1em1ftt,vtvtditn0h2p2em2itdcvtqbtvtgbtvtbt, where g : c+ ir and c+ = { = (1, 2, 3, 4) : c([h, 0 ], ir+)}. it is easy to check that gi(z)zi=0 0, i = 1,2, 3,4. due to [14, lemma 2 ], any solution of (2) with z() c+, say z(t) = z(t, z()), is such that z(t) ir+ for all t 0. it follows from the first equation of (2) that dt / dt dt(t). suptt(t) /d, so t(t) is bounded.let (6)ft=0h1p1em1ttd+it. then (7)dftdt=0h1p1em1dd0h1p1em1ttd itft, where = min{d, } and thus lim suptf(t) /. this implies that f(t) is bounded and so is i(t). thus, there exists a > 0 such that i(t). it follows from the third equations in (2) that (8)dvtdtncvt, and consequently v(t) is bounded. on the other hand, we have dh(t)/dt n h(t), where = min{c, } ; this implies that h(t) is bounded so also for b(t). finally, all the solutions of system (2) are bounded. let us put system (2) in a vector form by setting z = (t, i, v, b) and(5)gz = g1zg2zg3zg3z=dttftt, vtvt0h1p1em1ftt,vtvtditn0h2p2em2itdcvtqbtvtgbtvtbt, where g : c+ ir and c+ = { = (1, 2, 3, 4) : c([h, 0 ], ir+)}. it is easy to check that gi(z)zi=0 0, i = 1,2, 3,4. due to [14, lemma 2 ], any solution of (2) with z() c+, say z(t) = z(t, z()), is such that z(t) ir+ for all t 0. it follows from the first equation of (2) that dt / dt dt(t). let (6)ft=0h1p1em1ttd+it. then (7)dftdt=0h1p1em1dd0h1p1em1ttd itft, where = min{d, } and thus lim suptf(t) /. this implies that f(t) is bounded and so is i(t). thus, there exists a > 0 such that i(t). it follows from the third equations in (2) that (8)dvtdtncvt, and consequently v(t) is bounded. on the other hand, we have dh(t)/dt n h(t), where = min{c, } ; this implies that h(t) is bounded so also for b(t). we note that (11)k1=0h1p1em1d, k2=0h2p2em2d. global behaviour of system (2) may depend on the basic reproduction numbers r0 and r1 given by (12)r0=nk1k2ft0,0c, where t0 = /d and (13)r1=nk1k2f(m,/g)c, with, m = t0 (c / dngk1k2). here, r0 and r1 are the basic reproduction ratios for viral infection and humoral immune response of system (2), respectively. based on the hypotheses (h2) and (h3) it is clear that r1 1, then system (2) has an infected equilibrium without b cells response of the form e1 = (t1, i1, v1, 0) with t1 (0, t0).(2)if r1 > 1, then system (2) has an infected equilibrium with b cells response of the form e2 = (t2, i2, v2, b2) with t2 (0, m). suppose that the conditions (h1)(h3) are satisfied.(1)if r0 > 1, then system (2) has an infected equilibrium without b cells response of the form e1 = (t1, i1, v1, 0) with t1 (0, t0).(2)if r1 > 1, then system (2) has an infected equilibrium with b cells response of the form e2 = (t2, i2, v2, b2) with t2 (0, m). if r0 > 1, then system (2) has an infected equilibrium without b cells response of the form e1 = (t1, i1, v1, 0) with t1 (0, t0). if r1 > 1, then system (2) has an infected equilibrium with b cells response of the form e2 = (t2, i2, v2, b2) with t2 (0, m). proofthe steady states of system (2) satisfy the following equations : (14)dtft, vv=0,k1ft, vvi=0,nk2icvqbv=0,gbvb=0. from the last equation of (14), we have (15)(gv)b=0. equations (15) has two possible solutions, b = 0 or gv = 0.if b = 0, (14)3 yields i = (c / nk2)v.by substituting this into (14)2, we obtain that (16)k1ft, vcnk2v=0, which gives v = 0 or k1f(t, v) (c / nk2) = 0.if v = 0, we obtain the infection - free equilibrium e0(/d, 0,0, 0).if v 0, (14)1 and (14)2 yields (17)i = k1dtit. by substituting this into (14)3, we obtain (18)v = nk1k2(dt)cv(t). since i 0 and v 0, this implies that t /d.now, from (h1), (h2), and (h3), the following functional (19)kt = k1ft, vtcnk2, satisfies (20)k0kd=cnk22(r01)1,k(t)=k1ftndk1k2cfv>0. hence, we obtain the infected equilibrium without b cells response (21)e1=t1,i1,v1,0=t1,it1,vt1,0, where t1 is the unique zero in (0, t0) of k and i and v are given by (17) and (18).if b 0, from (15), we obtain (22)v=gv2, and from the first and second equation of (14), we have (23)i = i(t)=k1dt0. by substituting this into (14)3, we obtain (24)b = b(t)=dngk1k2(mt)q0, which implies that t m.now, from (14)1 the functional (25)l(t)dtft, v2v2=0 satisfies (26)l0lm=cngk1k21r11l(t)=gftd1,k(t)=k1ftndk1k2cfv>0. hence, we obtain the infected equilibrium without b cells response (21)e1=t1,i1,v1,0=t1,it1,vt1,0, where t1 is the unique zero in (0, t0) of k and i and v are given by (17) and (18). if b 0, from (15), we obtain (22)v=gv2, and from the first and second equation of (14), we have (23)i = i(t)=k1dt0. by substituting this into (14)3, we obtain (24)b = b(t)=dngk1k2(mt)q0, which implies that t m. now, from (14)1 the functional (25)l(t)dtft, v2v2=0 satisfies (26)l0lm=cngk1k21r11l(t)=gftd 0, and u0 = 0 at e0. the time derivative of u0(t) along the solutions of system (2) is given by (28)u0=1ft0,0ft,0dtft, vv + 1k10h1p1em1ft,vvdk1i + 1nk1k2n0h2p2em2idcvqbv + qngk1k2gbvb + 1k10h1p1em1ft, vvft,vvd + k1k20h2p2em2iid, with t = t(t), t = t(t), i = i(t), i = i(t), v = v(t), v = v(t), and b = b(t).at e0, using = dt0, we obtain (29)u0=1ft0,0ft,0dt0dt+ft, vvft0,0ft,0 cnk1k2vqngk1k2b = d1ft0,0ft,0t0t+cnk1k2 ft, vft,0nk1k2ft0,0c1vqngk1k2b. from (h2) and (h3) we have, respectively, (30)1f(t0,0)f(t,0)t0t0,ft, vft,0nk1k2ft0,0cft,0ft,0nk1k2ft0,0c = nk1k2ft0,0c = r0. then, r0 1 ensures that u00, for all t, i, v, b 0, u0=0 holds only for t = t0, v = b = 0, and from (2)2 we obtain i = 0. it follows that { e0 } is the largest invariant set in t, i, v, bu0=0. it follows from lasalle invariance principle that the infection - free equilibrium e0 is globally asymptotically stable. define a lyapunov functional : (27)u0t=t0t1ft0,0f,0d+1k1i+1nk1k2v + qngk1k2b+1k10h1p1em1 ttft,vvdd + k1k20h2p2em2ttidd, where k1 and k2 are given in (11). it is obvious that u0 is defined and continuously differentiable for all t, i, v, b > 0, and u0 = 0 at e0. the time derivative of u0(t) along the solutions of system (2) is given by (28)u0=1ft0,0ft,0dtft, vv + 1k10h1p1em1ft,vvdk1i + 1nk1k2n0h2p2em2idcvqbv + qngk1k2gbvb + 1k10h1p1em1ft, vvft,vvd + k1k20h2p2em2iid, with t = t(t), t = t(t), i = i(t),), v = v(t), and b = b(t). at e0, using = dt0, we obtain (29)u0=1ft0,0ft,0dt0dt+ft, vvft0,0ft,0 cnk1k2vqngk1k2b = d1ft0,0ft,0t0t+cnk1k2 ft, vft,0nk1k2ft0,0c1vqngk1k2b. from (h2) and (h3) we have, respectively, (30)1f(t0,0)f(t,0)t0t0,ft, vft,0nk1k2ft0,0cft,0ft,0nk1k2ft0,0c = nk1k2ft0,0c = r0. then, r0 1 ensures that u00, for all t, i, v, b 0, u0=0 holds only for t = t0, v = b = 0, and from (2)2 we obtain i = 0. it follows that { e0 } is the largest invariant set in t, i, v, bu0=0. it follows from lasalle invariance principle that the infection - free equilibrium e0 is globally asymptotically stable. in this section, we study the global stability of the infected equilibrium without b cells response e1 and the infected equilibrium with b cells response e2 of system (2) by the lyapunov direct method. we set (31)gx = x1lnx, for x0,. it is clear that for any x > 0, g(x) 0 and g(x) has the global minimum x = 1, with g(1) = 0.. then the equilibrium e1 is globally asymptotically stable if r1 1 0 for t (t1,), and ut1=0, so u(t) 0. consequently u1 is nonnegative defined with respect to the endemic equilibrium e1, which is a global minimum.we now prove that the time derivative of u1 is nonpositive. calculating the time derivative of w1 along the positive solutions of (2), we obtain (35)w1=1f(t1,v1)f(t, v1)t+1k1(1i1i)i + 1nk1k21v1vv+qngk1k2b=1ft1,v1ft, i1,v1(dtf(t, v)v) + 1k11i1i 0h1p1()em1f(t,v)vdi + 1nk1k21v1v n0h2p2em2idcvqbv + qngk1k2gbvb. at e1, by using = dt1 + f(t1, v1)v1 and c = nk2i1/v1 and /k1i1 = f(t1, v1)v1, we have (36)w1=1f(t1,v1)f(t, v1)dt1dt + f(t1,v1)v11f(t1,v1)f(t, v1) f(t, v)v+ft1,v1ft, v1f(t, v)v + 1k10h1p1em1ft,vvdk1i 1k1i1i0h1p1em1ft,vvd + k1i1+k1k20h2p2em2idi1k1v1v k1k2v1v0h2p2()em2id+k1i1 + qnk1k2v1gb. calculating the time derivative of w2, we obtain (37)w2=1k1ft1,v1v10h1p1em1 ft, vvft1,v1v1ft,vvft1,v1v1 hhh+lnft,vvft, vvd + k1k2i10h2p2em2ii1ii1+lniid=ft, vv1k10h1p1em1ft,vvd + 1k1ft1,v1v10h1p1em1lnft,vvft, vvd + k1ik1k20h2p2em2id + k1k2i10h2p2em2lniid. combining (36) and (37) and by using (/k1)i1 = f(t1, v1)v1, we obtain (38)u1=1f(t1,v1)f(t, v1)dt1dt + f(t1,v1)v11f(t1,v1)f(t, v1) + f(t1,v1)f(t, v1)f(t, v)v 1k1i1i0h1p1()em1f(t,v)vd + f(t1,v1)v1f(t1,v1)v 1k2ft1,v1v1i1v1v0h2p2()em2id + f(t1,v1)v1+1k1f(t1,v1)v1 0h1p1()em1lnf(t,v)vf(t, v)vd + 1k2f(t1,v1)v10h2p2()em2lniid + qnk1k2v1gb=1ft1,v1ft, v1dt1dt + ft1,v1v1ft, v1ft, vvv1 1ft, vft, v1+ft1,v1v1 1ft1,v1ft, v1+lnft1,v1ft, v1 + ft1,v1v11ft, v1ft, v+lnft, v1ft, v + 1k1f(t1,v1)v10h1p1()em1 1ft,vvi1ft1,v1v1i+lnft,vvi1ft1,v1v1id + 1k2f(t1,v1)v10h2p2()em2 1iv1i1v+lniv1i1vd+qnk1k2v1gb = d1f(t1,v1)f(t, v1)t1t + ft1,v1v1ft, v1ft, vvv11ft, vft, v1 f(t1,v1)v1g(ft1,v1ft, v1)f(t1,v1)v1g ft, v1ft, v1k1f(t1,v1)v1 0h1p1()em1g(f(t,v)vi1f(t1,v1)v1i)d 1k2f(t1,v1)v10h2p2()em2giv1i1vd + qnk1k2v1gb. from (h2), we have (39)1f(t1,v1)f(t, v1)t1t0, and from (h3) and (h4) we have (40)ft, v1ft, vvv11ft, vft, v10, and as g is positive, we have (41)u1qnk1k2v1gb. from remark 3 it is easy to verify that from (38), the largest invariant set in t, i, v, bu1t=0 is the singleton { e1}. using lasalle invariance principle, if r1 1 0 for t (t1,), and ut1=0, so u(t) 0. consequently u1 is nonnegative defined with respect to the endemic equilibrium e1, which is a global minimum. we now prove that the time derivative of u1 is nonpositive. calculating the time derivative of w1 along the positive solutions of (2), we obtain (35)w1=1f(t1,v1)f(t, v1)t+1k1(1i1i)i + 1nk1k21v1vv+qngk1k2b=1ft1,v1ft, i1,v1(dtf(t, v)v) + 1k11i1i 0h1p1()em1f(t,v)vdi + 1nk1k21v1v n0h2p2em2idcvqbv + qngk1k2gbvb. at e1, by using = dt1 + f(t1, v1)v1 and c = nk2i1/v1 and /k1i1 = f(t1, v1)v1, we have (36)w1=1f(t1,v1)f(t, v1)dt1dt + f(t1,v1)v11f(t1,v1)f(t, v1) f(t, v)v+ft1,v1ft, v1f(t, v)v + 1k10h1p1em1ft,vvdk1i 1k1i1i0h1p1em1ft,vvd + k1i1+k1k20h2p2em2idi1k1v1v k1k2v1v0h2p2()em2id+k1i1 + qnk1k2v1gb. calculating the time derivative of w2, we obtain (37)w2=1k1ft1,v1v10h1p1em1 ft, vvft1,v1v1ft,vvft1,v1v1 hhh+lnft,vvft, vvd + k1k2i10h2p2em2ii1ii1+lniid=ft, vv1k10h1p1em1ft,vvd + 1k1ft1,v1v10h1p1em1lnft,vvft, vvd + k1ik1k20h2p2em2id + k1k2i10h2p2em2lniid. combining (36) and (37) and by using (/k1)i1 = f(t1, v1)v1, we obtain (38)u1=1f(t1,v1)f(t, v1)dt1dt + f(t1,v1)v11f(t1,v1)f(t, v1) + f(t1,v1)f(t, v1)f(t, v)v 1k1i1i0h1p1()em1f(t,v)vd + f(t1,v1)v1f(t1,v1)v 1k2ft1,v1v1i1v1v0h2p2()em2id + f(t1,v1)v1+1k1f(t1,v1)v1 0h1p1()em1lnf(t,v)vf(t, v)vd + 1k2f(t1,v1)v10h2p2()em2lniid + qnk1k2v1gb=1ft1,v1ft, v1dt1dt + ft1,v1v1ft, v1ft, vvv1 1ft, vft, v1+ft1,v1v1 1ft1,v1ft, v1+lnft1,v1ft, v1 + ft1,v1v11ft, v1ft, v+lnft, v1ft, v + 1k1f(t1,v1)v10h1p1()em1 1ft,vvi1ft1,v1v1i+lnft,vvi1ft1,v1v1id + 1k2f(t1,v1)v10h2p2()em2 1iv1i1v+lniv1i1vd+qnk1k2v1gb = d1f(t1,v1)f(t, v1)t1t + ft1,v1v1ft, v1ft, vvv11ft, vft, v1 f(t1,v1)v1g(ft1,v1ft, v1)f(t1,v1)v1g ft, v1ft, v1k1f(t1,v1)v1 0h1p1()em1g(f(t,v)vi1f(t1,v1)v1i)d 1k2f(t1,v1)v10h2p2()em2giv1i1vd + qnk1k2v1gb. from (h2), we have (39)1f(t1,v1)f(t, v1)t1t0, and from (h3) and (h4) we have (40)ft, v1ft, vvv11ft, vft, v10, and as g is positive, we have (41)u1qnk1k2v1gb. from remark 3 it is easy to verify that from (38), the largest invariant set in t, i, v, bu1t=0 is the singleton { e1}. using lasalle invariance principle, if r1 1 0 for t (t2,), and ut2=0, so u(t) 0. consequently u2 is nonnegative defined with respect to the endemic equilibrium e2, which is a global minimum.we now prove that the time derivative of u1 is nonpositive. calculating the time derivative of w1 along the positive solutions of (2), we obtain (46)w3=1ft2,v2ft, v2t+1k11i2ii + 1nk1k21v2vv+qngk1k21b2bb=1ft2,v2ft, i2,v2dtft, vv + 1k11i2i0h1p1em1ft,vvdi + 1nk1k21v2vn0h2p2em2idkkkkkkkkkkkkkkkkkkkcvqbv0h2 + qngk1k21b2bgbvb. at e2, by using = dt2 + f(t2, v2)v2, c = (nk2i2/v2) qb2, and v2 = /g, we have (47)w3=1ft2,v2ft, v2dt2dt + ft2,v2v21ft2,v2ft, v2ft, vv + ft2,v2ft, v2ft, vv+1k10h1p1em1 ft,vvdk1i+ft2,v2ft, v2ft, vv + 1k10h1p1em1ft,vvdk1i 1k1i2i0h1p1em1ft,vvd+k1i2 + k1k20h2p2em2idqnk1k2bv k1k2v2v0h2p2em2id+qv2nk1k2b 1nk1k2vv2nk2i2v2qb2 + qnk2k2bb2vv2=1ft2,v2ft, v2dt2dt + ft2,v2v21ft2,v2ft, v2ft, vv + ft2,v2ft, v2ft, vv+1k10h1p1em1 ft,vvdk1i+ft2,v2ft, v2ft, vv + 1k10h1p1em1ft,vvdk1i 1k1i2i0h1p1em1ft,vvd+k1i2 + k1k20h2p2em2idqnk1k2bv k1k2v2v0h2p2em2id+qv2nk1k2b i2k1v2v+qb2nk1k2v+k1i2qv2b2nk1k2 + qnk1k2bvqv2nk1k2bqb2nk1k2v+qb2v2nk1k2=1ft2,v2ft, v2dt2dt + ft2,v2v21ft2,v2ft, v2 ft, vv+ft2,v2ft, v2ft, vv + 1k10h1p1em1ft,vvdk1i 1k1i2i0h1p1em1ft,vvd + k1i2+k1k20h2p2em2id i2k1v2vk1k2v2v0h2p2()em2id+k1i2. calculating the time derivative of w4, we obtain (48)w4=1k1ft2,v2v20h1p1em1 ft, vvft2,v2v2ft,vvft2,v2v2kkkkkk+lnft,vvft, vvd + k1k2i20h2p2em2ii2ii2+lniid=ft, vv1k10h1p1em1ft,vvd + 1k1ft2,v2v20h1p1em1lnft,vvft, vvd + k1ik1k20h2p2em2id + k1k2i20h2p2()em2lniid. combining (47) and (48) and by using (/k1)i2 = f(t2, v2)v2, we obtain (49)u2=1ft2,v2ft, v2dt2dt + ft2,v2v21ft2,v2ft, v2 + ft2,v2ft, v2ft, vv1k1i2i0h1p1em1 ft,vvd+ft2,v2v2 ft2,v2v1k2ft2,v2v2i2v2v 0h2p2em2id+ft2,v2v2 + 1k1ft2,v2v20h1p1em1 lnft,vvft, vvd+1k2ft2,v2v2 0h2p2em2lniid=1ft2,v2ft, v2dt2dt + ft2,v2v2ft, v2ft, vvv2 1ft, vft, v2+ft2,v2v2 1ft2,v2ft, v2+lnft2,v2ft, v2 + ft2,v2v21ft, v2ft, v+lnft, v2ft, v + 1k1ft2,v2v20h1p1em1 1ft,vvi2ft2,v2v2i+lnft,vvi2ft2,v2v2id + 1k2ft2,v2v20h2p2em2 1iv2i2v+lniv2i2vd=d1ft2,v2ft, v2t2t+ft2,v2v2 ft, v2ft, vvv21ft, vft, v2 ft2,v2v2gft2,v2ft, v2 ft2,v2v2gft, v2ft, v 1k1ft2,v2v20h1p1em1 gft,vvi2ft2,v2v2id 1k2f(t2,v2)v20h2p2()em2giv2i2vd. from (h2), we have (50)1f(t2,v2)f(t, v2)t2t0, and from (h3) and (h4) we have (51)f(t, v2)f(t, v)vv2(1f(t, v)f(t, v2))0, and as g is positive, we have (52)u20. thus, the equilibrium e2 is stable. in this case, note that u2=0 if and only if t = t2, i = i2, and v = v2 and using the third equation of (2), we obtain b = b2. therefore, it follows from lasalle 's invariance principal that the infected equilibrium with b cells response e2 is globally asymptotically stable. define a lyapunov functional (42)u2=w3+w4, where (43)w3=t2t1ft2,v2f,v2d+1k1i2gii2 + v2nk1k2gvv2+qngk1k2b2gbb2,(44)w4=1k1ft2,v2v20h1p1em1 ttgft,vvft2,v2v2dd + k1k2i20h2p2em2ttgii2dd, where k1 and k2 are given in (11). t2(1 (f(t2, v2)/f(, v2)))d verifies (45)u(t)=1f(t2,v2)f(t, v2). from (h2), we have u(t)0 for t (t2,), and ut2=0, so u(t) 0. consequently u2 is nonnegative defined with respect to the endemic equilibrium e2, which is a global minimum. we now prove that the time derivative of u1 is nonpositive. calculating the time derivative of w1 along the positive solutions of (2), we obtain (46)w3=1ft2,v2ft, v2t+1k11i2ii + 1nk1k21v2vv+qngk1k21b2bb=1ft2,v2ft, i2,v2dtft, vv + 1k11i2i0h1p1em1ft,vvdi + 1nk1k21v2vn0h2p2em2idkkkkkkkkkkkkkkkkkkkcvqbv0h2 + qngk1k21b2bgbvb. at e2, by using = dt2 + f(t2, v2)v2, c = (nk2i2/v2) qb2, and v2 = /g, we have (47)w3=1ft2,v2ft, v2dt2dt + ft2,v2v21ft2,v2ft, v2ft, vv + ft2,v2ft, v2ft, vv+1k10h1p1em1 ft,vvdk1i+ft2,v2ft, v2ft, vv + 1k10h1p1em1ft,vvdk1i 1k1i2i0h1p1em1ft,vvd+k1i2 + k1k20h2p2em2idqnk1k2bv k1k2v2v0h2p2em2id+qv2nk1k2b 1nk1k2vv2nk2i2v2qb2 + qnk2k2bb2vv2=1ft2,v2ft, v2dt2dt + ft2,v2v21ft2,v2ft, v2ft, vv + ft2,v2ft, v2ft, vv+1k10h1p1em1 ft,vvdk1i+ft2,v2ft, v2ft, vv + 1k10h1p1em1ft,vvdk1i 1k1i2i0h1p1em1ft,vvd+k1i2 + k1k20h2p2em2idqnk1k2bv k1k2v2v0h2p2em2id+qv2nk1k2b i2k1v2v+qb2nk1k2v+k1i2qv2b2nk1k2 + qnk1k2bvqv2nk1k2bqb2nk1k2v+qb2v2nk1k2=1ft2,v2ft, v2dt2dt + ft2,v2v21ft2,v2ft, v2 ft, vv+ft2,v2ft, v2ft, vv + 1k10h1p1em1ft,vvdk1i 1k1i2i0h1p1em1ft,vvd + k1i2+k1k20h2p2em2id i2k1v2vk1k2v2v0h2p2()em2id+k1i2. calculating the time derivative of w4, we obtain (48)w4=1k1ft2,v2v20h1p1em1 ft, vvft2,v2v2ft,vvft2,v2v2kkkkkk+lnft,vvft, vvd + k1k2i20h2p2em2ii2ii2+lniid=ft, vv1k10h1p1em1ft,vvd + 1k1ft2,v2v20h1p1em1lnft,vvft, vvd + k1ik1k20h2p2em2id + k1k2i20h2p2()em2lniid. combining (47) and (48) and by using (/k1)i2 = f(t2, v2)v2, we obtain (49)u2=1ft2,v2ft, v2dt2dt + ft2,v2v21ft2,v2ft, v2 + ft2,v2ft, v2ft, vv1k1i2i0h1p1em1 ft,vvd+ft2,v2v2 ft2,v2v1k2ft2,v2v2i2v2v 0h2p2em2id+ft2,v2v2 + 1k1ft2,v2v20h1p1em1 lnft,vvft, vvd+1k2ft2,v2v2 0h2p2em2lniid=1ft2,v2ft, v2dt2dt + ft2,v2v2ft, v2ft, vvv2 1ft, vft, v2+ft2,v2v2 1ft2,v2ft, v2+lnft2,v2ft, v2 + ft2,v2v21ft, v2ft, v+lnft, v2ft, v + 1k1ft2,v2v20h1p1em1 1ft,vvi2ft2,v2v2i+lnft,vvi2ft2,v2v2id + 1k2ft2,v2v20h2p2em2 1iv2i2v+lniv2i2vd=d1ft2,v2ft, v2t2t+ft2,v2v2 ft, v2ft, vvv21ft, vft, v2 ft2,v2v2gft2,v2ft, v2 ft2,v2v2gft, v2ft, v 1k1ft2,v2v20h1p1em1 gft,vvi2ft2,v2v2id 1k2f(t2,v2)v20h2p2()em2giv2i2vd. from (h2), we have (50)1f(t2,v2)f(t, v2)t2t0, and from (h3) and (h4) we have (51)f(t, v2)f(t, v)vv2(1f(t, v)f(t, v2))0, and as g is positive, we have (52)u20. thus, the equilibrium e2 is stable. in this case, note that u2=0 if and only if t = t2, i = i2, and v = v2 and using the third equation of (2), we obtain b = b2. therefore, it follows from lasalle 's invariance principal that the infected equilibrium with b cells response e2 is globally asymptotically stable. in this section, we give some particular examples. in (2), if f(t, v) = (t/(1 + v)) we obtain the following model : (53)dttdt=dttttvt1+vt, ditdt=0h1p1em1ttvt1+vtdit, dvtdt = n0h2p2em2itdkkkkkkkkcvtqbtvt, db(t)dt = gb(t)v(t)b(t), the global dynamics of model (53) is studied by elaiw.. so the work presented in is a particular case of (2) because the function (t/(1 + v)) satisfies the hypothesises (h1)(h4). another particular case of (2), if f(t, v) = (t/(1 + av + bt)) and h1 = h2 =, we obtain the following model which is presented by yang. : (54)dttdt=dttttvt1+avt+btt, ditdt=0p1em1ttvt1+avt+bttd it, dvtdt = n0p2em2itdcvtkkkkkkkkqbtvt, db(t)dt = gbtvtbt. the global asymptotic stability of possible equilibrium of (54) is established in. a last example, in (2), if f(t, v) = (t/(1 + v)) and p1() = p2() = (), where () is the dirac delta function, we obtain the results presented in. in the current paper, we have studied an hiv-1 infection model with humoral immune response and intracellular distributed delays and general incidence rate. the model has two distributed time delays describing time needed for infection of cell and virus replication. the global stability of our model is studied by employing the method of lyapunov functionals which are motivated by mccluskey for delayed epidemic models. this general incidence represents a variety of possible incidence functions that could be used in virus dynamics model as well as epidemic models. we establish that the global dynamics are determined by two threshold parameters, the basic reproduction ratios for viral infection and humoral immune response r0 and r1, respectively, which depend on the incidence function and the delay. we have proved that the infection - free equilibrium e0 is globally asymptotically stable if the basic reproduction ratios viral infection r0 1. in this case, the virus is cleared up. the hypotheses on the general incidence function are used to assure the existence of infected equilibrium without b cells response e1 and infected equilibrium with b cells response e2. we prove that if r1 1 1, the infected equilibrium with b cells response e2 is globally asymptotically stable. | in this work an hiv-1 infection model with nonlinear incidence rate and distributed intracellular delays and with humoral immunity is investigated. the disease transmission function is assumed to be governed by general incidence rate f(t, v)v. the intracellular delays describe the time between viral entry into a target cell and the production of new virus particles and the time between infection of a cell and the emission of viral particle. lyapunov functionals are constructed and lasalle invariant principle for delay differential equation is used to establish the global asymptotic stability of the infection - free equilibrium, infected equilibrium without b cells response, and infected equilibrium with b cells response. the results obtained show that the global dynamics of the system depend on both the properties of the general incidence function and the value of certain threshold parameters r0 and r1 which depends on the delays. |
the sample comprised 373 men aged 3069 years (mean 488 (sd 115) years) who participated in the physical activity and fitness for health promotion study, which was designed to investigate japanese physical activity and fitness as well as their association with other risk factors for lifestyle - related diseases. none of the subjects had any chronic diseases or were taking any medications that could affect the study variables. this study was conducted according to the guidelines laid down in the declaration of helsinki and all procedures involving human participants were approved by the ethical committee of the national institute of health and nutrition. written informed consent was obtained from all participants. dietary habits during the previous month were assessed with a brief self - administered diet history questionnaire (bdhq) for japanese adults. the bdhq is a four - page structured questionnaire that enquires about the consumption frequency of a total of fifty - six food and beverage items, with specified serving sizes described in terms of the natural portion or the standard weight and volume measurement of servings commonly consumed in general japanese populations. the bdhq for adults was developed based on a comprehensive (sixteen - page) version of a validated self - administered diet history questionnaire. estimates of dietary intake for the fifty - six food and beverage items, energy and selected nutrients were calculated using an ad hoc computer algorithm for the bdhq, which was based on the standard tables of food composition in japan. the validity of the bdhq using 16-d weighed dietary records as the gold standard is described elsewhere. nutrient variables were energy adjusted using the nutrient density method (amount of nutrient intake per 4184 kj (1000 kcal)), to reduce the measurement error common with dietary assessment questionnaires and to avoid biased grouping due to variation in body size and energy requirement. adequacy of nutrient intake was examined using the reference values given in the dri for japanese people as a temporal gold standard. of the eighteen micronutrients with estimated average requirement presented in the dri, five nutrients (cr, mo, se, iodine and na) were excluded from this study because the intake of these nutrients (cr, mo, se and iodine) can not be reliably assessed by the bdhq or because for na, a tentative dietary goal for the prevention of lifestyle - related diseases is more important than the estimated average requirement in the dri. the thirteen selected micronutrients were vitamin a, thiamin, riboflavin, niacin, vitamin b6, vitamin b12, vitamin c, folate, ca, mg, fe, zn and cu. for each of these nutrients, a nutrient adequacy score of 1 was allocated if the nutrient (amount of nutrient intake per 4184 kj) met or exceeded the estimated average requirement (amount of nutrient intake per 4184 kj) given in the dri, and a 0 if it did not meet the estimated average requirement. as a measure of overall micronutrient adequacy, an overall nutrient adequacy score (onas) was constructed by summing the scores of all nutrients. overall micronutrient adequacy was defined as low (onas < 10), moderate (onas = 10 or 11), or high (onas = 12 or 13) according to the tertile of onas. crf was defined as o2max during a maximal graded exercise test with bicycle ergometers (lode excalibur, lode bv ; monark ergomedic 828e). the methodology and equipment used in taking this measurement have been described in detail elsewhere. achievement of o2max was accepted if at least two of three criteria were met : (1) a plateau in o despite increasing the work rate ; (2) maximal rer 110 ; and (3) maximal heart rate was not less than 95 % of the age - predicted maximum (220 age). low (unfit), moderate and high crf were defined according to the lowest 25 %, the middle 50 % and the upper 25 %, respectively, of the age - specific distribution of o2max in all participants. we measured physical activity using the activity monitor (kenz lifecorder ; suzuken co. ltd). an activity monitor attached to a waist belt on the left side of the body was used to collect step count data for seven consecutive days in all participants. body mass was measured using an electronic scale with the subjects wearing light clothing and no shoes and was determined to the nearest 01 kg. bmi was calculated by dividing the body mass in kilograms by the square of height in metres (kg / m). cigarette smoking status was assessed by self - report on a lifestyle and health history questionnaire. subject characteristics (reported as mean values and standard deviations, or percentages) were contrasted between o2max tertiles using a one - way anova for age, height, body mass, bmi, o2max and step counts and for smoking status using the test (table 1). the test was also used to identify significant differences in the prevalence of participants with inadequate micronutrient intake across levels of crf. ancova was used to identify significant differences in the micronutrient intake across levels of crf after adjustment for the important confounding factors. the relationship of overall micronutrient intake adequacy with crf was examined by multiple regression models. logistic regression analysis was used to determine the or of being unfit (the lowest 25 % of the age - specific distribution of o2max) associated with each tertile of onas adjusted for age, bmi, smoking status (model 1) and further adjusted for physical activity (model 2). all statistical analyses were performed using spss statistical software version 19 (ibm japan ltd). statistical significance was set at p < 005 (two - sided). table 1.characteristics of participants(mean values and standard deviations, or percentages)all (n 373)low fitness (n 75)moderate fitness (n 208)high fitness (n 90)variablesmean sd mean sd mean sd mean sd p for trendage (years)4881154861144891164871160973height (cm)1700601700561703621693590480body mass (kg)669856839067586642700001bmi (kg / m)232272373123327225230004o2max (mk / kg per min)34877270483405243165<0001step counts (steps per d)887533897458267587633372103133435<0001smoking status (%) 0004non - smoker364270368439former smoker410392420402current smoker226338212158 characteristics of participants (mean values and standard deviations, or percentages) significant inverse trends were observed for body mass (p value for trend = 0001), bmi (p value for trend = 0004), and smoking status (p value for trend = 0004) across incremental crf categories. significant positive trends across incremental crf categories were observed for step counts (low fitness group 7458, medium fitness group 8763 and high fitness group 10313 steps per d, respectively). as shown in table 2, the average prevalence of adequacy calculated across all thirteen micronutrients was 789 % for japanese men. overall, the subjects reported diets with reasonable adequacy of intakes for niacin, vitamin b12, folate, fe and cu (965100 %). however, the prevalence of potentially inadequate intakes was 2535 % for mg, ca and zn, and may also be relatively high for vitamin a (611 %) and thiamin (810 %). table 2.micronutrient intake (unit/4184 kj) and proportion of participants with inadequate micronutrient intakes presenting a nutrient intake below the estimated average requirement (ear) in the low, moderate and high fitness tertiles(mean values, standard errors and percentages)all (n 373)low fitness (n 75)moderate fitness (n 208)high fitness (n 90)mean se % mean se % mean se % mean se % p p vitamin a (g re/4184 kj)3819971611335917837203800132061542432086511003001thiamin (mg/4184 kj)04000810040018000400183204001767025053riboflavin (mg/4184 kj)0700112106002120070011440700267003025niacin (mg ne/4184 kj)9101113930291390015148902111069089vitamin b6 (mg/4184 kj)0700162070025307001870700211054020vitamin b12 (g/4184 kj)4701103500291347015004501900039012folate (g/4184 kj)177628816171958400176338724185461711027064vitamin c (mg/4184 kj)57112713957730516056517216358223567074007ca (mg/4184 kj)2683465316246893645327026652982819837244004001 mg (mg/4184 kj)1330127268131326826713251722691356261267039100fe (mg/4184 kj)3900535390960039006584101011027084zn (mg/4184 kj)41003349410063604100435142006333028072cu (mg/4184 kj)0600100060010006001000600100058onas103010100020101015107017003onas, overall nutrient adequacy score ; re, retinol equivalents ; ne, niacin equivalents.p value for ancova adjusted for age, bmi and smoking habits.p value for trend by the test.1 g re = retinol (g) + -carotene (g) 1/12 + -carotene (g) 1/24 + -cryptoxanthin (g) 1/24 + other provitamin a carotenoids (g) 1/24.ne were computed as niacin (mg) + protein (mg)/6000 according to the dietary reference intake for the japanese. micronutrient intake (unit/4184 kj) and proportion of participants with inadequate micronutrient intakes presenting a nutrient intake below the estimated average requirement (ear) in the low, moderate and high fitness tertiles (mean values, standard errors and percentages) onas, overall nutrient adequacy score ; re, retinol equivalents ; ne, niacin equivalents. p value for trend by the test. 1 g re = retinol (g) + -carotene (g) 1/12 + -carotene (g) 1/24 + -cryptoxanthin (g) 1/24 + other provitamin a carotenoids (g) 1/24. ne were computed as niacin (mg) + protein (mg)/6000 according to the dietary reference intake for the japanese. we assessed differences of individual micronutrient intake and overall micronutrient intake status across levels of crf by ancova (table 2). participants in the high crf category had the highest vitamin a, riboflavin and ca intake and the highest onas compared with those in participants in the lower crf categories (p value for ancova < 005, after adjustment for age, bmi and smoking status). these differences remain after further adjustment for step counts (vitamin a, p = 002 ; riboflavin, p = 008 ; ca, p = 006). we also observed a significant inverse trend for the prevalence of inadequacy for the intake of vitamin a (p value for trend = 0006) and ca (p value for trend = 0005) across incremental crf categories (table 2). in a multiple regression analysis with age, bmi, smoking status and onas as independent variables and crf as the dependent variable, onas was the significant determinant of the variance in crf in terms of absolute (= 011, p < 005) and relative o2max (= 010, p < as seen in models 2 and 4, after further adjustment for step counts, onas remains the significant determinant of the variance in crf in terms of absolute (= 010, p < 005) and relative o2max (= 009, p < 005). table 3.results of the multiple regression analyses between overall micronutrient intake status (overall nutrient adequacy score ; onas) and cardiorespiratory fitness (n 373)o2max (litres / min)model 1model 2independent variable b p r b p r onas003011001033003010002038o2max (ml / kg per min)model 3model 4independent variable b p r b p r onas039010002027035009004034b, unstandardised regression coefficients ;, standardised regression coefficients.model 1 is adjusted for age, bmi and smoking habits. model 3 is adjusted for age and smoking habits.model 4 is adjusted for all covariates in model 3 plus step counts. results of the multiple regression analyses between overall micronutrient intake status (overall nutrient adequacy score ; onas) and cardiorespiratory fitness (n 373) b, unstandardised regression coefficients ;, standardised regression coefficients. we fitted logistic regression models to assess the association between onas (tertile) and being unfit, adjusting for age, bmi and smoking status, with the lowest tertile used as the referent (fig. individuals in the top tertile of onas had 52 % decreased odds of being unfit (or 048 ; 95 % ci 024, 097), compared with those whose onas were in the lowest tertile. the or of being unfit in the intermediate tertile compared with the lowest tertile was 057 (95 % ci 031, 1.05). the or for being unfit remained strong comparing the top tertile with the lowest tertile (or 049 ; 95 % ci 024, 099) when we further adjusted the models for step counts. after the further adjustment, the or for being unfit in the intermediate tertile compared with the lowest tertile was 056 (95 % ci 030, 104). similar results were observed when step counts were substituted with minutes of moderate- to vigorous - intensity physical activity as the adjusted variable (data not shown). 1.odds of being unfit (low cardiorespiratory fitness) by overall nutrient intake status categories (overall nutrient adequacy score (onas) tertiles)., lowest tertile (reference) ;, intermediate tertile ;, highest tertile. odds of being unfit (low cardiorespiratory fitness) by overall nutrient intake status categories (overall nutrient adequacy score (onas) tertiles)., lowest tertile (reference) ;, intermediate tertile ;, highest tertile. adjusted for age, bmi and smoking status. adjusted for age, bmi, smoking status and step counts. the main finding of the present study was that a number of dietary micronutrients and an overall micronutrient intake score representing the overall micronutrient adequacy for thirteen micronutrients were positively associated with crf in a group of japanese men. these associations were independent of physical activity and other potential confounding variables including age, bmi and smoking status, suggesting that physical activity does not confound the association between micronutrient intake and crf in our population sample of japanese men. furthermore, these results show that participants who had a poor overall micronutrient intake status have a significantly higher risk of being unfit compared with men with a good micronutrient intake status. micronutrients most commonly function as essential co - enzymes and co - factors for metabolic reactions (as structural components of enzymes and mitochondrial cytochromes and as active electron and proton carriers in the atp - generating respiratory chain) and thus help support basic cellular reactions (i.e. glycolysis, the citric acid cycle, lipid and amino acid metabolism) required to maintain energy production and life. although micronutrients probably play important roles in physical work capacity and therefore performance through different biological pathways, the relationship between dietary micronutrients and crf is not well studied in the population sample of adults, especially in large sample sizes. chatard. examined the association between micronutrient intake (eleven nutrient density variables) and crf in eighteen sportsmen aged 5672 years. diet was assessed with a 6-d diet recall and crf (o2max) was objectively measured using a monark cycle ergometer. stepwise regression analyses indicated that vitamin c intake (expressed per 1000 kj of energy intakes) was the only determinant to have a relationship with o2max. by contrast, butterworth. studied a group of 2040-year - old women (n 34) who varied widely in levels of physical activity and found no significant relationship between micronutrient intake (nutrient density) and o2max, as assessed by a maximal graded treadmill test. however, they did not adjust for the important confounding factors. to our knowledge, only one study examined individual micronutrient intake in relation to crf in a large sample of men and women. brodney. investigated nutrient intakes of 7959 men and 2453 women aged 2087 years across low, moderate and high fitness categories. crf was assessed using estimated o2max from treadmill time, and diet was assessed with a 3-d food record. the authors found that there was a significant difference across low, moderate and high fitness for both macronutrients and micronutrients (including vitamins a, b6, b12, c and e, folate and ca), after adjusting for age, smoking status, health status and bmi in ancova. although physical activity is usually positively associated with healthy nutritional intake, the aforementioned studies do not appear to consider the independent effects of these lifestyle factors on crf. our results are the first to confirm that micronutrient intake is correlated with crf independent of physical activity level (table 2). a unique contribution of the present study is that associations between individual and overall micronutrient adequacy and crf were examined in a sample of japanese men. our results showed a significant inverse trend for prevalence of inadequacy for the intake of vitamin a and ca across incremental crf categories (table 2). as in vivo biological activities of nutrients are interdependent, the combination of inadequate micronutrient intakes may have a greater impact on functional status than inadequate intake of individual micronutrients. we therefore also examined relationships between overall micronutrient intake status (onas, overall micronutrient adequacy for thirteen selected micronutrients) and crf. as shown in table 3, for each 1-score increase in onas, the value of o2max increased by 003 litres / min or 035 ml / kg per min after adjusting for step counts and other potential confounding variables. individuals who had an inadequacy intake of a single micronutrient (the top tertile of onas) had a 51 % decreased odds of being unfit compared with those whose intake of more than four micronutrients was inadequate (the lowest tertile) even after adjusting for age, bmi, smoking status and physical activity. these results extend the findings of a previous study of males in the netherlands, in which the authors carried out a double - blind intervention examination of the combined restriction of thiamin, riboflavin and vitamins b6 and c in relation to o2max in eleven men. they found that a combined restricted intake of these micronutrients caused a 98 % decrease in o2max within a few weeks. the aforementioned results indicate that it is necessary to incorporate both dietary and physical activity advice into fitness promotion counselling. the study participants consisted of only men aged 3069 years and are not representative of the entire japanese population, and thus the present results may have limited generalisability. our study was a cross - sectional study, and can not provide causal evidence on the association between micronutrient intake status and crf. despite its limitations, the present study has some strengths, including the relatively large population sample of japanese men, the objective measures of crf and physical activity and the examination of the micronutrient intake status (based on adherence to dris)crf relationship and an important confounding variable (physical activity). in conclusion, both several individual micronutrients ' intake and overall micronutrient intake status were found to be independently and positively associated with crf in japanese men. | previous studies have demonstrated that meeting the dietary recommendations for macronutrients was significantly associated with higher cardiorespiratory fitness (crf) levels in adults. however, the relation between the status of micronutrient intake and crf still remains unclear. this study examined the association between micronutrient intake status (based on adherence to the dietary reference intakes (dri)) and crf in japanese men. the study comprised 373 japanese men aged 3069 years. dietary intake was assessed with a self - administered diet history questionnaire. overall micronutrient intake status was quantified using an overall nutrient adequacy score (onas) for thirteen selected micronutrients. onas was calculated based on adherence to the dri for japanese. crf was defined as o2max during a maximal incremental test on a bicycle ergometer. physical activity was measured using accelerometer - based activity monitors for seven consecutive days. we observed a significant inverse trend for the prevalence of inadequacy for the intake of vitamin a and ca across incremental crf categories (p < 005). in a multivariate model, the onas was positively associated with absolute (= 010, p = 002) and relative o2max (= 009, p = 004), independent of physical activity. the or for being unfit (the lowest 25 % of the age - specific distribution of o2max) in the third onas tertile compared with the first onas tertile was 052 (95 % ci 028, 096). these results demonstrated that the intake of several individual micronutrients and overall micronutrient intake status are independently and positively associated with crf in japanese men. |
wrd wielu problemw sygnalizowanych przez dorastajcych pacjentw z wrodzonymi wadami serca (grown - up congenital heart przekonanie o bliej nieokrelonym ograniczeniu oglnej sprawnoci zwizanym z wad serca i jej wczeniejszym leczeniem. poprawie aktywnoci fizycznej pacjentw z guch moe suy odpowiednio przygotowany program kompleksowej rehabilitacji kardiologicznej (krk - guch) stanowicy kolejny, odlegy etap leczenia. ocena wpywu krk - guch na aktywno fizyczn pacjentw w odlegym okresie po chirurgicznej korekcji wrodzonych wad serca. do badania wczono 57 pacjentw (30 kobiet, 27 mczyzn) w wieku 23,7 4,1 roku, bdcych minimum 12 miesicy po korekcji operacji ubytku w przegrodzie midzykomorowej (ventricular septal defects vsd) lub w przegrodzie midzyprzedsionkowej (atrial septal defects asd). grupa a (n = 31), oraz nieuczestniczc w programie rehabilitacji grupa b (n = 26). u wszystkich pacjentw wykonano wstpne badania czynnociowe, po czym wdroono program krk - guch. po 30 dniach od badania wstpnego ponownie oceniono pacjentw z obu grup, stosujc takie same narzdzia badawcze jak we wstpnym badaniu. u pacjentw z grupy nierehabilitowanej (grupa b) stwierdzono istotnie nisz aktywno fizyczn po miesicu od badania wstpnego ni w grupie pacjentw rehabilitowanych w ramach programu krk - guch (grupa a). i tym samym najprawdopodobniej jako ycia pacjentw w pnym okresie po chirurgicznej korekcji wrodzonych wad serca. zaproponowany program krk - guch moe stanowi uzupenienie holistycznej opieki w opisywanej grupie pacjentw po korekcji wrodzonych wad serca. adolescents and young adults after surgery of grown - up congenital heart (guch) defects constitute a growing group of patients requiring cardiac care and multifaceted support. the reduction of early mortality and the prolongation of survival among patients with congenital heart defects (chds) may appear as a tangible success [1, 2 ]. however, apart from good short - term results and long - term survival, to fully assess the efficacy of the interventional treatment of cardiac defects, a broadly understood quality of life evaluation of adolescent patients with cardiac issues that were successfully treated during childhood is needed. among the many issues raised by adolescent guch patients, the widespread belief concerning unclearly defined restrictions in general fitness, associated with congenital heart defects and their previous treatment, appears to be fundamental. it directly impacts the patients everyday life and reduces their physical activity. undergoing cardiac surgery in childhood leads to a decrease in expectations, which naturally reduces the readiness of the patients themselves to take up various forms of physical activity. notwithstanding, the physical activity of guch patients may be improved by a properly prepared comprehensive cardiac rehabilitation (ccr - guch) program, serving as another stage of treatment with proven influence on exercise tolerance and quality of life. the present study evaluates the impact of comprehensive cardiac rehabilitation on the physical activity of guch patients participating in a specially developed authorial program dedicated to this group of patients. its basic premise is that the low physical activity of guch patients increases their predisposition to cardiovascular diseases proportionally more in the case of those less active, similarly to the general population. the issue appeared especially interesting due to the potential for increasing the activity of adolescent patients with congenital heart diseases, which could consequently improve their general condition and reduce the risk of additional cardiovascular problems natural for this age in long - term observation. the aim of this study was to evaluate the influence of the ccr - guch program on the long - term physical activity of adolescent patients after chd surgery. fifty - seven patients with chds (30 women and 27 men) at the mean age of 23.7 4.1 years, who met the inclusion criteria and provided their free and informed consent, were invited to participate in the program of comprehensive cardiac rehabilitation designed at the chair and clinic of rehabilitation of the medical university of gdask in the years 2007 - 2009. all the patients had undergone cardiac surgery procedures in childhood (at the age of 6 on average) ; these included either the correction of ventricular septal defects (vsd) or the repair of atrial septal defects (asd). out of all patients invited to participate in the ccr - guch program, 31 patients (17 women and 14 men) were ultimately included in the program. the remaining 26 patients, who did not participate in the rehabilitation program due to logistic, economic, or social reasons, were provided information about the program as well as about the expected positive results of the involved controlled physical exertion and its importance for normal functioning. the chd patients constituting the study group were divided into two subgroups : those participating in rehabilitation (a) and non - participants (b). the control group consisted of 30 healthy students (15 women and 15 men) at the mean age of 24.4 1.97 years, free of any additional comorbidities, or physical / mental limitations, who provided their free and informed consent for their participation and the anonymous use of the obtained results for the purpose of the present study (group c). identical inclusion criteria were used for the examination of guch patients (groups a and b) : clinical diagnosis condition after the repair of shunt - related cardiac defects (asd or vsd) employing median sternotomy ; time from the cardiac surgery over 12 months. the inclusion criteria for all three groups (a, b, and c) were : age over 18 years as well as free and informed written consent for participation in the study. the exclusion criteria included : active inflammatory diseases, life - threatening cardiac dysrhythmias, impaired motor function preventing the patients from performing the tasks of the rehabilitation program, coexistence of other heart defects (congenital or acquired), significant deterioration of clinical condition within the past month, acute cardiac or neurological events within the past 6 months, positive results of the cardiac diagnostic test, lack of consent, or mental illnesses precluding patient cooperation. no statistically significant differences between groups a and b were observed in terms of the distribution of sex, height, body mass, or the basic functional indicators of the circulatory system. the mean age of the patients at the time of surgery was 5.18 2.8 years. the resting heart rate (hrrest) was 90 bpm on average ; right bundle branch block (rbbb) was revealed on resting electrocardiograms of 15 patients, premature ventricular contractions (pvc) were diagnosed in 4 patients, and premature supraventricular contractions (psvc) were diagnosed in 2 patients. no statistically significant differences were noted between groups a, b, and c in terms of selected initial anthropometric indices (table i). patient characteristics for the study groups (a undergoing rehabilitation, b no rehabilitation) and controls (c) bmi body mass index, sd standard deviation all patients (groups a, b, and c) underwent physical examination, and their medical histories were obtained. the patients underwent cardiopulmonary exercise testing on a cycloergometer using the metasoft studio software (cortex) ; the selected ramp protocol consisted of an initial load of 20 w increasing by 10 w per minute. limits of maximal fatigue were put in place along with the standard indications for test termination ; the following parameters were evaluated : hrrest, hrmax, exercise time, exercise load in watts, vo2 peak. the evaluation of physical activity was based on the stanford questionnaire, which served as a tool for the objective assessment of patients with different parameters. it was composed of questions concerning the patients current lifestyle and was divided into two parts, including questions about everyday exercise of low - activity (stanford i) and high - activity exercise (stanford ii). each test item was worth one point, and the final score reflected the current physical activity of the respondents. additionally, a written questionnaire was drawn up especially for the purposes of the ccr - guch program. the subjects from both study groups (a, b) and the controls (c) were asked to answer questions about their lifestyle (whether they considered themselves physically active and whether they were apprehensive of engaging in some forms of physical activity). they were also asked about their participation in physical education classes during their school years and about tobacco use. the guch patients (a, b) were also asked whether they had ever participated in cardiac rehabilitation and whether they saw the need for engaging in controlled physical training sessions. after 30 days following the initial examination and after completion of the ccr - guch program, the physical activity of patients in groups a and b was evaluated again, using the same methods as during the initial examination (stanford i and ii questionnaires, own questionnaire). the ccr - guch program was implemented during the course of four weeks and was based on recommendations concerning comprehensive cardiac rehabilitation in adults. the kinesiatric program involved a cycle of half - hour monitored cycloergometer training sessions and general fitness training with elements of aerobics and nordic walking. at the gym, the subjects engaged in exercises with and without equipment as well as resistance exercise. breathing exercises were introduced in order to strengthen the respiratory muscles and to teach proper breathing both during exertion and in situ ations requiring the improvement of ventilation, e.g. dyspnea or fatigue. resistance training was performed 2 - 3 times per week on fitness stations using weights up to 20 kg ; the sessions consisted of no more than 4 series of 15 repetitions. the psychological influence consisted of emotional support, the stimulation of self - confidence, and the improvement of self - esteem. during the educational part of the program, which included classes conducted by a cardiologist, a psychologist, and a dietician, the patients learned methods of coping with emotional stress and changing the habits adversely affecting their predisposition to cardiovascular diseases. the patients were instructed how to measure their pulse (monitoring the pulse and assessing potential cardiac dysrhythmias) and arterial pressure. moreover, the participants were instructed in the proper use of everyday exercise, including proper respiration during various forms of activity (walking, running, resistance training). they also received advice concerning the optimal selection, planning, and performance of exercises and were shown various forms of physical activity, including sports and recreational activities which exert favorable effects on the cardiovascular system and facilitate the achievement of good short - term and long - term results [8, 9 ]. the performed analysis of the initial stanford questionnaire results demonstrated that the guch patients (a and b) were less physically active than their healthy peers (mean scores : stanford i 1.9 vs. 2.9 points, p < 0.001 ; stanford ii preliminary results of the physical activity examination among the study groups guch patients (a and b) and the controls healthy students (c) before the start of the ccr - guch program guch grown - up congenital heart, ccr comprehensive cardiac rehabilitation, stanford i low - activity exercise, stanford ii high - activity exercise subsequently, the guch patients undergoing rehabilitation (a) were compared with the group that did not undergo the ccr - guch program (b). both groups were characterized by similar baseline physical activity according to the initial stanford questionnaire (table iii). results of the stanford questionnaire among the study groups (a undergoing rehabilitation and b no rehabilitation) before the start of the ccr - guch program ccr comprehensive cardiac rehabilitation, guch grown - up congenital heart, stanford i low - activity exercise, stanford ii high - activity exercise forty - seven guch patients (82%) did not participate, while all students (100%) did participate in physical education during their school years. apprehension concerning engaging in physical exertion in everyday life was reported by 35 guch respondents (61%), while all students declared that they felt no fear of exercise. twenty - six guch patients (45.6%) declared themselves to be physically active in everyday life ; all the students answered this question in the same manner. all the guch patients (100%) declared the need to improve their physical capacity and activity. similar answers were given to the question concerning the readiness to participate in controlled ccr - guch training sessions ; however, 26 patients (45.6%) did not ultimately participate in the program. three of the guch patients (5.2%) and 2 students (6.4%) reported tobacco use. after the intensive rehabilitation program conducted over the course of 1 month, the subjects were again asked to fill out the stanford questionnaire. seven patients who did not undergo rehabilitation (group b) did not answer the questions. the analysis of the remaining 50 questionnaires demonstrated that, after the period of 1 month, the patients who were not included in the ccr - guch program (group b) were characterized by significantly lower physical activity in comparison to the patients participating in the training sessions (group a) (mean scores : stanford i 1.8 vs. 3.2 points, p < 0.001 ; stanford ii 0.13 vs. 1.2 points, results of the stanford questionnaire among the study groups (a undergoing rehabilitation and b no rehabilitation) after the end of the one - month ccr - guch program ccr comprehensive cardiac rehabilitation, guch grown - up congenital heart, stanford i low - activity exercise, stanford ii high - activity exercise the analysis of both parts of the questionnaire also revealed that, after the completion of the ccr - guch program, the 31 patients from group a engaged in both low - activity (stanford i) and high - activity exercise (stanford ii) much more frequently (p < 0.001 and p < 0.001) (fig. the results of the stanford questionnaire in group a before the start and after the completion of the ccr - guch program are presented in table v. results of the stanford questionnaire among the study groups (a undergoing rehabilitation and b no rehabilitation) before the start and after the end of the one - month of the ccr - guch program ; a) stanford i ; b) stanford ii results of the stanford questionnaire in study group a (undergoing rehabilitation) before the start and after the end of the ccr - guch program (n = 31) ccr comprehensive cardiac rehabilitation, guch grown - up congenital heart, stanford i low - activity exercise, stanford ii high - activity exercise the unequivocally positive opinion of the effects of the ccr - guch program, expressed by 24% of the guch patients, should be underscored at this point. asked about the benefits that they believed were achieved by participating in the program, 20 group a patients (64.5%) responded that their subjective well - being was much better, 5 patients (16.1%) wrote that they tired less during everyday activities, 2 patients (6.4%) no longer feared increasing the load of physical exercise, and 3 patients (9.6%) felt safer during their everyday activity. their descriptive answers to the questionnaire handed out after the completion of the rehabilitation program were more likely to include information that they tended to use the stairs more often than the elevator, that they tended to walk rather than drive for short distances, and that they would more frequently go out for walks after dinner or supper. the questionnaire score reflecting improvements in everyday physical activity corresponds with the results of the stanford i questionnaire, assessing the same type of activity : the mean stanford i score changed from 1.97 points before ccr - guch to 3.2 after completion of the program. congenital heart defects surgery is typically performed during the patient 's childhood ; the age of patients undergoing these operations is increasingly lower, and increasingly younger children undergo successful cardiac surgical treatment using state of the art technology. the dominant group among adolescents and young adults with chds is formed by those who had undergone cardiac surgery, but, unfortunately, have predominantly sedentary lifestyles or undertake exercise that is not recommended for their health condition. this often results from the lack of precise information concerning the type and intensity of exercise that they could engage in. telling these patients : you can do everything, but in moderation or the basis for the selection of physical activity for individuals with chd is constituted by the standards of sports cardiology included in the recommendations of consecutive sports conferences in bethesda. the newest recommendations concerning the use of physical exercise by individuals with chd were listed in the european society of cardiology guidelines for the management of grown - up congenital heart disease (new version 2010). regrettably, many adolescents and young adults after the surgical treatment of simple shunt - related cardiac defects avoid intense physical activity [13, 14 ]. the bethesda recommendations permit any kind of exercise after the successful and uneventful repair of simple intracardiac shunts. the guidelines of the european society of cardiology (esc) also contain no contraindications for physical exercise in this patient group. the decided majority of patients operated on due to simple shunt - related heart defects (atrial and ventricular septal defects) may perform physical exercise of any kind and intensity starting from the 6th postoperative month, provided no complications, pulmonary hypertension, or dysrhythmias are observed. it should be stressed that information concerning the physical exercise recommended to this patient group is still lacking ; as a result, many patients are unsure what types of activities are truly safe. the patients are unfamiliar with their capacity or the potential risks and restrictions ; therefore, it appears necessary to create professional teams dedicated to managing patients after chd repair. physical activity may be objectively assessed using a questionnaire including questions concerning the use of various exercise types of different intensity. for the purposes of this study, i.e. determination of the current physical activity profiles of patients, the data obtained from the stanford i and ii questionnaires were immensely useful in evaluating guch patients, even though the test was originally designed for other purposes [7, 16 ]. the conducted analysis of the questionnaire 's results demonstrated that, after the completion of the rehabilitation program, the everyday physical activity of our patients increased. after the program ended, the subjects tended more often to report engaging in high - activity exercise (the average stanford ii scores increased from 0.13 before the rehabilitation to 0.63 after the rehabilitation). it is, therefore, to be expected that, owing to this active attitude, the patients will be able to maintain the improvement of physical capacity and exercise tolerance achieved during the comprehensive cardiac rehabilitation program for a longer period of time. it should be underscored that the ultimate examination with the stanford questionnaires demonstrated that the patients undergoing comprehensive rehabilitation had statistically better results than the patients who refused to participate in the rehabilitation program (mean scores : stanford i a : 3.2 vs. b : 1.8 ; stanford ii a : 1.2 vs. b : 0.13, p < 0.001). this may indicate that the elements of the program were properly selected and may confirm the program 's effectiveness, including in terms of the increase in awareness concerning the need for developing basic health - oriented habits and an active lifestyle. when analyzing the obtained responses, one should pay attention to the subjective opinions of patients concerning the benefits resulting from participating in the rehabilitation program. after completion of the training cycle, the patients declared that they felt much better, tired less, felt safer during physical activity, and did not fear engaging in types of activities that used to constitute an thus, it should be expected that, in the future, the group of patients who underwent the ccr - guch program will be more aware of their capabilities and will be better able to see their limits without overexerting themselves, which, unfortunately, tends to happen in the group of young adults with chds. the final results of the young adult guch patients were closer to the results of the controls, recruited (as in previous studies conducted by the authors) among young, healthy students. in accordance with the study 's methodology, cardiac rehabilitation conducted in the study group significantly improved the physical activity of over half of the participants. based on the obtained data, it can be surmised that the expected positive results of training and an improvement of the cardiovascular system 's adaptation to physical exercise were achieved during the program. the results confirm previously published literature data ; however, there are still few comprehensive studies devoted to the physical capacity of guch patients despite the growing number of such patients in the population [17, 18 ]. the ccr - guch rehabilitation in the study group was designed in accordance with the guidelines for conducting physical training, published by the american heart association and with the experience of the authors in providing treatment and conducting physical training sessions for cardiovascular patients [3, 14, 22 ]. one limitation of the study was the lack of randomization during group selection ; however, the authors deemed refraining to invite the patients to participate in the program unethical. the number of patients included in the program was also restricted by the financial limits of the study. the intensity of physical exercise was determined based on the initial cpet. in order to maintain safety, the patients were monitored with electrocardiography during cycloergometer training, while the intensity of general fitness training and nordic walking sessions was controlled only by pulse measurements. in accordance with the premises of the ccr - guch program, acquiring self - control reduced anxiety related to continuing the activities and physical training in home conditions, without the supervision of physical therapists or physicians. in conclusion, it should be underscored that the improvement of the initially lowered physical activity of guch patients undergoing rehabilitation documented in this study, contrasted with the results of patients who did not undergo rehabilitation, confirms the need to qualify and include guch patients in comprehensive programs of late rehabilitation [19, 20 ]. engaging in the comprehensive program of late cardiac rehabilitation improves the physical activity of patients after the surgical correction of congenital heart diseases. comprehensive cardiac rehabilitation reduces the fears concerning various forms of physical activity that are often encountered in this group of patients, and, in consequence, improves the patients quality of life. it appears justified to introduce the comprehensive rehabilitation program as a supplement to holistic care in the group of adolescent patients after congenital heart diseases. | introductionthe group of grown - up patients with congenital heart defects (grown - up congenital heart guch) complains of a number of specific medical and non - medical problems. the presented program of comprehensive cardiac rehabilitation (ccr - guch), dedicated to the above mentioned group, can potentially improve the physical activity of guch patients.aimthe aim of the study was to assess the effect of the comprehensive cardiac rehabilitation program on the physical activity of guch patients.material and methodsthe invitation to take part in the ccr - guch program was addressed to a group of 57 patients (mean age : 23.7 4.1 years) who had undergone the surgical correction of ventricular septal defects (vsd) or atrial septal defects (asd) at least 12 months earlier. the patients were divided into two groups : a patients undergoing rehabilitation, and b patients who did not participate in the program. the patients were initially examined using functional and stress tests, and the program of comprehensive cardiac rehabilitation was started in group a. after 30 days, the patients from both groups underwent further testing using the same methods as during the initial evaluation.resultsafter one month of rehabilitation, the physical activity parameters of patients participating in the ccr - guch program (group a) were significantly better than those observed among non - participants (group b).conclusionsthe introduction of the comprehensive rehabilitation program improves the physical activity and, consequently, the quality of life of guch patients. the ccr - guch program appears to be a justified supplement to holistic care in the late rehabilitation of patients after the surgical correction of congenital heart defects. |
over the past decade, we have learned much about the problems associated with acute brain dysfunction during critical illness ; currently, awareness of the ubiquitous presence of intensive care unit (icu) delirium is growing. the paper by pandharipande and colleagues in the previous issue of critical care adds insight into this complex area. in 2004, ely and colleagues published groundbreaking work that identified icu delirium as an event occurring in over 80% of mechanically ventilated patients ; those with icu delirium had a threefold higher independent mortality risk compared with those who never had icu delirium. over the last 10 years, this group of investigators has worked extensively in the development and validation of the confusion assessment method for the icu (cam - icu) to detect and better understand icu delirium. according to this tool, delirium is defined as an acute change or fluctuation in the course of mental status, plus inattention and either disorganized thinking or an altered level of consciousness. the cam - icu tool uses the richmond agitation - sedation scale (rass) to measure arousal. patients who are deeply unresponsive are categorized as comatose rather than delirious ; that is, they respond only to physical / painful stimulation by moving but do not open their eyes (rass score of -4) or have no response to verbal or physical stimulation (rass score of -5). although coma and delirium are different conditions, both can be placed in a category of acute brain dysfunction. delirium (like acute brain dysfunction, for that matter) is not a disease but a syndrome with a wide spectrum of possible etiologies. over the last few years, we have learned that icu delirium does not come as a ' one size fits all ' event. rather, it appears that the longer and more severe the delirium is, the worse the patient outcomes are. as reported in the previous issue of critical care, pandharipande and colleagues use data from the mends (maximizing efficacy of targeted sedation and reducing neurological dysfunction) trial, which compared dexmedetomidine with lorazepam for icu sedation in a randomized double - blinded fashion. sixty - one percent of patients (61/103) in the mends trial were admitted with sepsis. in this important post hoc analysis of these septic patients, dexmedetomidine - sedated patients had more delirium / coma - free days, deliriumfree days, and ventilator - free days and a lower 28-day mortality rate when compared with lorazepam - sedated patients. it is important to realize that the randomization scheme for the mends trial was to dexmedetomidine versus lorazepam, not septic versus non - septic. accordingly, the authors conclude (appropriately) that prospective clinical studies and further mechanistic preclinical studies are needed to confirm these preliminary observational results. the mechanisms by which such brain dysfunction occurs are not fully understood, but disturbances in inflammation and coagulation pathways leading to microvascular thrombosis are thought to be partly responsible. the commonplace administration of sedatives during mechanical ventilation of septic patients adds an additional layer of complexity to understanding acute brain dysfunction in these patients. as noted by pandharipande and colleagues, there is some evidence that benzodiazepines and alpha-2 adrenoceptor agonists exert opposing effects on the immune system. so it stands to reason that dexmedetomidine may be more efficacious than lorazepam with regard to acute brain dysfunction in patients with sepsis. as is the case in most well - designed trials, for example, in septic patients, how does one tease apart the impact of dexmedetomidine (compared with lorazepam) on sedation itself from the putative benefits of dexmedetomidine on immune modulation, apoptosis, and so on ? how does the timing of the cam - icu delirium assessment impact the findings in this study ? given the pharmacokinetic / dynamic properties of dexmedetomidine and lorazepam, whatever component of recovery from delirium or coma (or both) that is purely sedative - related is likely to occur over differing time intervals when these two drugs are compared (that is, slower recovery and longer delirium / coma with lorazepam). since the multicenter mends trial did not mandate one particular sedation algorithm, it may be that lingering effects of lorazepam may have affected the cam - icu delirium or coma assessments (or both) more in the dexmedetomidine group. the distinction between delirium and coma in the cam - icu tool is logical but arbitrary. as a person transitions from a rass score of -3 (opens the eyes or moves in response to voice but does not make eye contact) to -4 (responds only to physical / painful stimulation by moving but does not open the eyes), the term coma, rather than delirium, is used. with regard to acute brain dysfunction, is the delirium - to - coma transition merely a continuum of progressively lesser degrees of arousal, or is there a fundamental change in the pathophysiology of the acute brain dysfunction with this transition ? the paper by pandharipande and colleagues is an important advance in our understanding of the complex interconnections between acute brain dysfunction, sedation, and sepsis. however, we need further progress in our understanding of the complex pathophysiology of acute brain dysfunction in critically ill patients who require mechanical ventilation. this hypothesis - generating study lays important groundwork for future investigations of sepsis and sedation in this area. cam - icu : confusion assessment method for the intensive care unit ; icu : intensive care unit ; mends : maximizing efficacy of targeted sedation and reducing neurological dysfunction ; rass : richmond agitation - sedation scale. jpk has been the recipient of an unrestricted grant from hospira (lake forest, il, usa) and is on their speakers ' bureau. | critically ill patients requiring mechanical ventilation frequently suffer from intensive care unit delirium, a syndrome associated with numerous poor measured outcomes. the relationship between delirium, sepsis, and sedation is complex. a discussion of the recent study (' effect of dexmedetomidine versus lorazepam on outcome in patients with sepsis : an a priori - designed analysis of the mends [maximizing efficacy of targeted sedation and reducing neurological dysfunction ] randomized controlled trial ') by pandharipande and colleagues is presented in this commentary. |
mast cells are increasingly recognised as playing an important pathogenic role in a variety of allergic and autoimmune diseases [14 ]. however, there is also developing evidence of their participation in tissue repair and resolution of inflammation [57 ], as well as for their exhibiting pathogenic and protective roles in cancer [8, 9 ]. such contradictory evidence relating to mast cell function most likely reflects that mast cells, which are haematopoietic cells found in all vascularised organs [1013 ], constitute a heterogeneous cell population varying in morphology, function, and location with subpopulations being characterised by their differential protease, eicosanoid, and proteoglycan content [10, 1317 ]. such heterogeneity arises because bone marrow - derived mast cell progenitors [18, 19 ] arrive in tissue before they are fully matured allowing the different cytokines, hormones, and reactive oxygen and/or nitrogen species produced by various microenvironments to essentially create moreover, the functional response of mast cells depends on the stimuli received ; for example, following classical activation via the ige receptor, fcr1, mast cells degranulate rapidly (within minutes) to exocytose prostaglandins and leukotrienes as well as preformed cytokines, tryptase, histamine, heparin, and platelet activating factor (paf) whilst de novo synthesised cytokines exhibit a more delayed (hours) release [8, 21 ]. however, mast cells can also be activated independently of fcri and this can be initiated by cytokines or other proinflammatory mediators reflecting a direct interaction with triggering factors such as lps, parasite molecules, or allergic stimuli in the skin or the mucosa. hence, mast cells are frequently the first cell type to respond during inflammation, as evidenced by the important roles played by mast cells in bacterial and parasitic infections [2225 ]. however, mast cells are also able to influence disease progress subsequently ; both directly via the release of proinflammatory mediators, and indirectly via their effects on other immune cells, including dendritic cells (dc), t and b cells, and macrophages. isolation of in vivo differentiated mast cell subsets is difficult due to their limited numbers in tissue [5, 26 ] and this has led to the development of in vitro culture protocols to generate human and mouse mast cells, as defined by their expression of cd117, fcr1 and the il-1 receptor family member, st2 (suppression of tumorigenicity 2), that can be subclassified as distinct phenotypes due to their differential granular phenotypic and functional responses. thus, although mast cells make up 95% mast cells was routinely obtained as evidenced by the surface expression of cd117, fcr1 and st2 and viability was determined by trypan blue staining. g / ml murine anti - dnp ige for 18 h prior to stimulation. in experiments investigating immunomodulation by es-62, mast cells were incubated with es-62 (2 g / ml) simultaneously with ige during the sensitisation period. cells were then stimulated (1 10 cells / ml except where indicated) by addition of medium, 0.5 g / ml dnp - hsa to cross - link fcr1, 0.5 g / ml lps (salmonella minnesota) or pma (phorbol myristate acetate ; 1 m) plus ionomycin (1 m). reactions were terminated after the desired culture period by centrifugation at 400 g and supernatants aspirated for determination of mediator release whilst the cell pellets were stored at 20c until subjected to western blot analysis. es-62 was purified to homogeneity from spent culture medium of adult acanthocheilonema viteae using endotoxin - free reagents as described previously. the purity and identity of each batch were confirmed by sds - page and the level of endotoxin in the es-62 sample was determined using a limulus amebocyte lysate (lal) qcl-1000 kit (lonza biologics). es-62 is used at a working concentration that has an endotoxin reading of < 0.003 endotoxin units / ml. to identify mast cells by positive staining of heparin with toluidine blue, cells (0.01 10) were cytofuged using a shandon cytospin3 (thermo shandon) at 500 rpm for 5 min. briefly, cells were pre - incubated with 50 l fc receptor (fcr) blocking buffer (anti - cd16/32, clone 2.4g2, hybridoma supernatant, 10% mouse serum, and 0.1% sodium azide) for 20 min at 4c prior to incubation with the appropriate flurochrome - conjugated or biotinylated antibodies (2 g / ml ; suspended in 50 l fc block ; cd117, ebioscience ; fcri, ebioscience ; st2, md bioproducts ; and tlr4/md2, ebioscience) for 30 min, 4c. following washing, for biotinylated primary antibodies, after labelling, cells were washed twice with 3 ml facs buffer (pbs containing 2% fbs and 2 mm edta) at 1500 rpm for 6 min, 4c and then resuspended in facs buffer. to enable exclusion of dead cells from the analyses, cells were either stained with live / dead viability / cytotoxicity kit (invitrogen) before commencement of staining or by the addition of 1 l 7-aad (7-amino actinomycin d ; ebioscience) immediately prior to data acquisition. cellular fluorescence data were acquired using a becton dickinson lsr ii or facscalibur flow cytometer and analysed using flowjo software (tree star inc). cells were loaded with the fluorescent calcium - sensing dye fura-2/am (5 m ; invitrogen) in hbss (145 mm nacl, 5 mm kcl, 1 mm mgso4, 1 mm cacl2, and 10 mm hepes) supplemented with 0.18% (w / v) d - glucose and 0.2% (w / v) bsa for 30 min at 37c in the dark. for measurement of intracellular calcium mobilisation in the absence of extracellular calcium, calcium - free hbss supplemented with 100 m egta (ethylene glycol tetraacetic acid) to chelate any remaining free calcium, was used. cells (10) were added to a stirred glass cuvette in a hitachi f-700 fluorescence spectrophotometer at 37c and stimulated as indicated at t = 50 s and measurements acquired for a total of 180 s. calcium levels were detected every 500 ms using excitation - emission ratios of 340/380 nm. following each experiment rmax and rmin values were determined by the addition of 1% triton - x100 and subsequent addition of 20 mm egta ph 7.4, respectively. elisas for il-6, il-13, mcp-1, and tnf (limits of detection 4 pg / ml, 4 pg / ml, 15 pg / ml, and 8 pg / ml resp. ; ebioscience) and pgd2 (cayman chemicals) were performed on triplicate samples according to the suppliers ' recommendations and developed using tmb substrate and absorbances were determined using a tecan sunrise microplate reader. the level of degranulation was determined using a modified colorimetric assay to assess the release of -hexosaminidase. mast cells (0.2 10) were suspended in 200 l tyrode 's buffer supplemented with 1% fcs and stimuli were added for 30 min at 37c. reactions were terminated by centrifugation (400 g) and 50 l aliquots of supernatants assayed for release of -hexosaminidase and normalised to total cellular -hexosaminidase following cell lysis by the addition of 1% triton - x 100 and by incubation with 1 mm p - nitrophenyl - n - acetyl--d - glucosamine (nag) in 200 l 0.05 m citrate buffer, ph 4.5. after incubation in the dark at 37c for 1 h the reaction was quenched by the removal of 62.5 l of the reaction mix into a clean well and the addition of 125 l / well 0.1 m sodium bicarbonate buffer and optical density determined by a tecan sunrise microplate reader at 405 nm. mast cells (2 10/ml) were stimulated as indicated and reactions terminated by the addition of ice - cold pbs and centrifugation at 400 g at 4c for 5 min. lysis was performed by the addition of 50 l ice - cold, modified ripa lysis buffer (50 mm tris buffer, ph 7.4 containing 150 mm sodium chloride, 2% (v / v) np40, 0.25% (w / v) sodium deoxycholate, 1 mm egta, 10 mm sodium orthovanadate, 0.5 mm phenylmethylsulfonylfluoride, chymostatin (10 g / ml), leupeptin (10 g / ml), antipain (10 g / ml), and pepstatin a (10 g / ml)). after vortexing, the cells were incubated on ice for 30 min before microcentrifugation of lysates at 12000 rpm for 15 min. equal protein loadings of cell lysates (3040 g protein per lane), determined by bca protein assay (bicinchoninic acid assay ; thermo pierce), were resolved using the xcell surelock mini - cell kit with nupage novex high - performance precast bis - tris gels and nupage buffers and reagents (invitrogen). proteins were then transferred to nitrocellulose (amersham) or pvdf membranes and protein loading was validated by ponceau red staining. membranes were washed in tris - buffered saline (tbs) (0.5 m nacl and 20 mm tris ph 7.5) with 0.1% (v / v) tween-20 (tbs / tween) and blocked for 1 h in tbs / tween with 5% nonfat milk (marvel). all antibodies were diluted in tbs / tween with either 5% nonfat milk or 5% bsa. following incubation with primary antibody the membranes were washed with tbs / tween and incubated in the appropriate horseradish peroxidase (hrp)-conjugated secondary antibody for 2 h at room temperature before visualisation using the ecl detection system and kodak x - ray film. statistical analysis was by unpaired t - test or one - way anova with tukey 's post - test and p is significant at p < 0.05, p < 0.01, p < following expansion of pdmc and derivation of mucosal - type bmmc and ctmc for 46 weeks in vitro, these mast cell populations were phenotyped for mast cell lineage markers (cd117, fcri, and st2) and also for tlr4 (toll - like receptor-4) and proteoglycan (heparin) expression (figure 1). this analysis revealed all three mouse subsets to be of similar size, although the ctmc appeared rather less granular (figure 1(a)), with each subset comprising a homogeneous population of cd117fcri mast cells (figure 1(b)). moreover, all 3 populations expressed tlr4 (figure 1(c)) and st2 (figure 1(d)), with bmmc typically expressing the highest levels of tlr4 and ctmc showing most st2 expression. similarly, pdmc, mucosal - type bmmc, and ctmc all contained heparin - containing granules as indicated by toluidine blue staining (figure 1(e)), although there was some heterogeneity in the ctmc, and to a lesser extent, the mucosal - type bmmc populations, perhaps reflecting their differences in granularity (figure 1(a)). mucosal - type mast cells from mice have generally been considered to express little or no heparin, but in agreement with our results, it has been reported that heparin expression can be upregulated in response to scf in these cells : this functional plasticity is consistent with the ability of mouse bmmc to repopulate both serosal and mucosal compartments of mast cells in vivo. it has previously been reported that serosal- and mucosal - type mast cells exhibit differential functional responses with pdmc displaying strong degranulation responses whilst bmmc preferentially produce chemokines and cytokines, and that these responses can be further fine - tuned selectively in response to inflammatory stimuli and microenvironment. we therefore characterised the differential responses of pdmc, mucosal - type bmmc, and ctmc in response to ag - mediated crosslinking of fcri, lps / tlr4 signalling and also the pharmacological stimulus pma plus ionomycin (p / i) in terms of degranulation (-hexosaminidase), eicosanoid (pgd2), chemokine (mcp-1), and cytokine (il-6, il-13 and tnf) release (table 1). these data confirmed that pdmc were the subtype that degranulated most strongly in response to fcri and pma plus ionomycin and demonstrated that all of these subtypes generated in vitro exhibited little or no degranulation in response to lps. all of the mast cell populations constitutively secreted high levels of pgd2 (lowest in pdmc) ; however, whilst pdmc did not produce any more pgd2 in response to fcri crosslinking, both mucosal - type bmmc and ctmc responded further to this stimulus and also to lps. further differential responses were observed in terms of chemokine release as whilst lps stimulated release of mcp-1 in all subtypes, fcri crosslinking induced little or no release of mcp-1 over the basal levels in pdmc, yet strongly stimulated release of this chemokine from mucosal - type bmmc and ctmc. only pma plus ionomycin were able to induce substantial secretion of il-6 and il-13 by pdmc, and none of the stimuli were routinely able to trigger tnf release by these cells. similarly, whilst fcri- and lps / tlr4-signalling induced little or no il-13 or tnf production by ctmc, lps, but not fcri crosslinking, triggered strong secretion of il-6. by contrast, fcri and lps / tlr4 signalling both induced the production of all three cytokines (il-6, il-13 and tnf) by mucosal - type bmmc. it has been reported that bmmc and freshly isolated pdmc derived from c57bl/6 mice exhibited higher levels of degranulation (-hexosaminidase) and generated lower levels of cytokine and prostaglandin production than those derived from balb / c mice but we did not find this to be a significantly reproducible trend in this study (data are not shown). given the differential functional responses of the mast cell subtypes, we next investigated whether the filarial nematode product showed selectivity in its desensitisation of mast cell responses, both in terms of the mast cell subtype targeted and also with respect to their differential responses to the individual proinflammatory stimuli. these studies showed that exposure to es-62 significantly inhibited the degranulation of pdmc and the mucosal - type bmmc, but not ctmc, in response to fcri signalling (figure 2(a)). whilst the responses to lps were typically too low (table 1) to show significant effects of es-62 (data not shown), degranulation in response to pma plus ionomycin was also significantly inhibited in pdmc (figure 2(b)) and likewise observed in ctmc (2/2 experiments) and the mucosal - type bmmc (3/5 experiments). although pdmc produced little or no chemokines / cytokines in response to either fcri crosslinking or lps - stimulation (table 1), generally, the very low levels of mcp-1, il-6, and il-13 observed were inhibited by es-62 (data not shown). with respect to mucosal - type bmmc and ctmc, whilst es-62 was only able to significantly inhibit mcp-1 production by fcri - stimulated mucosal - type bmmc (figures 3(a) and 3(b)), it inhibited lps - stimulated il-6 production by both subtypes as well as that seen in mucosal - type bmmc in response to crosslinking of fcri (figures 3(c) and 3(d)). by contrast, the il-13 response to fcri crosslinking was suppressed by es-62 in both subtypes (figures 3(e) and 3(f)) whilst the fcri - mediated tnf response only seen in mucosal - type bmmc was also inhibited by es-62 (data not shown). collectively, therefore, we have shown that es-62 can target both serosal- and connective - tissue phenotypes to render these cells hyporesponsive to proinflammatory stimuli. that es-62 can modulate the responses of both mature and immature cells is consistent with our previous studies showing that the parasite product can target immature bone marrow progenitors of macrophages and dendritic cells to generate a more anti - inflammatory environment in vivo [37, 39 ]. the finding that es-62 did not modulate expression of fcri, cd117, or st2 on resting or sensitized mast cells (figures 2(c)2(e) and data not shown), however, suggested that it was not affecting their phenotypic status but rather targeting their functional plasticity. the observed differential targeting of particular responses may indicate selective actions in particular microenvironments and consequently, recruitment of other innate cells such as neutrophils to the site of inflammation as well as mast cell promotion of the polarisation of particular immune responses [4, 14 ], dependent on the site and type of inflammation (e.g., protective inflammation to fight infection versus aberrant autoimmune or allergic hyperinflammation). to address how es-62 may be differentially targeting the functional responses of pdmc, the mucosal - type bmmc, and ctmc, we investigated the effects of the parasite product on calcium mobilisation and expression of pkc, as we had previously shown modulation of these two key signals in degranulation and cytokine signalling [4042 ] to be crucial to the desensitisation of fcri - mediated human mast cell responses [35, 36 ]. moreover, es-62 exerts its effects via subversion of tlr4 signalling whilst the canonical tlr4 ligand lps typically acts to enhance fcri functional responses [43, 44 ], the latter accounting at least in part for the widely established finding that lps exacerbates airway hyperresponsiveness. lps has been reported to do this by increasing fcri - driven calcium mobilisation by upregulating the orai1 and stim1 subunits of the store - operated calcium (soc) channel and hence stimulating calcium influx. in addition, pkc signalling, including that of pkc, has been shown to be important to lps / tlr4 responses in a variety of innate cells. as a first step, we investigated whether fcri- and lps / tlr - signalling induced calcium mobilisation in each of pdmc, mucosal - type bmmc and ctmc (figure 4). interestingly we found that not only as expected, ige - sensitisation of mast cells was essential for fcri - mediated calcium mobilisation, but also that it enhanced that seen in response to lps (pdmc ; figures 4(a) and 4(b)). moreover, it was clear that whilst the fcri signal reflected a mix of mobilisation of intracellular calcium and calcium influx, as indicated by the observed transient spike in the absence of extracellular calcium (egta), the calcium response to lps in all mast cell subtypes predominantly reflected calcium influx (figures 4(c)4(h)). consistent with lps inducing calcium influx, we also found as reported previously that lps enhanced fcri - mediated calcium mobilisation (data not shown). by contrast, although preexposure to es-62 did not modulate the baseline calcium levels, the parasite product suppressed the subsequent calcium mobilisation in response to both fcri - crosslinking and lps / tlr4 signalling in pdmc (figures 5(a) and 5(b)). in addition, es-62 was found to downregulate pkc expression in pdmc, mucosal - type bmmc and ctmc (figures 5(c) and 5(d)), suggesting, that as with human mast cells, es-62 was targeting this signal in pdmc, mucosal - type bmmc and ctmc to suppress degranulation and cytokine responses. pkc has been shown to be degraded via both proteosomal and caveolae / lipid raft, lysosomal routes [46, 47 ] and our preliminary studies in human mast cells showed that the inhibitor of caveolae / lipid raft trafficking, nystatin could protect against such downregulation following exposure to es-62 for 24 h. furthermore, our earlier studies had shown that es-62-mediated downregulation of pkc expression in b cells could be prevented by treatment with the cysteine protease inhibitor leupeptin ; findings also consistent with a lysosomal mechanism [49, 50 ] of degradation of this key signalling element. we have therefore further investigated the (differential) mechanisms involved in es-62-driven degradation of pkc in mast cell subtypes. our data in ctmc are consistent with that of our previous study on human mast cells as they showed that nystatin but not lactacystin protected against pkc degradation and we have further confirmed the role of an endosomal route by showing that protection is also afforded by the combination of the lysosomal inhibitors, e64d plus pepstatin a (figure 5(d)). however, our comprehensive analysis of mechanism in pdmc and the mucosal - type bmmc has revealed a more complicated scenario in which both nystatin and lactacystin can offer some protection at differential time points (data not shown). interestingly, these findings are consistent with reports that both mechanisms can coexist in cells not only in a temporal and spatially distinct manner but can also be triggered to regulate pkc expression in response to a single agonist. hence, the differential recruitment of one or more of these degradative pathways may provide a rationale for fine tuning the level of pkc desensitisation required to downregulate hyperinflammatory responses ; an attractive proposal gave that ctmc exhibit the least degranulation potential and are not as effective at producing cytokines as the mucosal - type bmmc. overall, as we have also found that the inhibitors alone can modulate pkc expression in pdmc and mucosal - type bmmc, these findings collectively indicate that regulation of this key signalling element in mast cells is tightly controlled by a complex and dynamic system involving both proteosomal and lysosomal routes of degradation. finally, whilst we have shown that es-62 can suppress the cytokine responses of both pdmc and the mucosal - type bmmc, it is clear that the levels of cytokines produced by pdmc in response to fcri- and lps / tlr4 signalling are very low compared to those secreted by mucosal - type bmmc. we have therefore addressed identifying which signals may be contributing to such higher levels of cytokine production by also determining the effects of es-62 on pkc expression as this signalling element has not only been shown to be important for functional responses to fcri- and lps / tlr4 signalling [40, 45, 5153 ] but also to be a target for downregulation by the parasite product in human mast cells and b cells [35, 48 ]. in addition, we have also examined the effect of es-62 on myd88, a pivotal signal transducer of tlr4 [45, 51, 54 ] as we have shown it to be a target of es-62 in countering th17 pathology and only the myd88-, and not the trif - dependent pathway of tlr4 signalling, appears to be active in bmmc. consistent with the hypothesis that additional signals such as myd88 and pkc are required for the augmented cytokine responses observed in bmmc relative to pdmc, these studies show that whilst myd88 expression in pdmc is unchanged by exposure to es-62, culture with the parasite product results in downregulation of both myd88 and pkc in mucosal - type bmmc (figure 5(e)). pdmc, mucosal - type bmmc, and ctmc mast cell populations display differential functional responses with mature serosal mast cells predominantly acting like cells that perform a specialised degranulation function. by contrast, bmmc, which have been reported to possess an immature mucosal - like phenotype that can further differentiate into mucosal or serosal mast cells display reduced degranulation and increased cytokine responses. consistent with the idea that bmmc are plastic and can differentiate into either mucosal or serosal mast cells, ctmc display a comparable degranulation potential to that of mucosal - type bmmc and a cytokine profile intermediate of mucosal - type bmmc and mature serosal / connective tissue pdmc. all three mast cell populations can be rendered hyporesponsive by es-62 but the selective nature of these effects suggests that es-62 may be targeting functions of the individual subtypes that are specific to the particular inflammatory microenvironment and phenotype. the mechanisms underlying such desensitisation have not been fully delineated but our working model (figure 6) is that reduced degranulation and low level cytokine secretion reflect desensitisation of fcri- and lps / tlr4-mediated calcium mobilisation and pkc signalling whilst suppression of the high levels of cytokine production by mucosal - type bmmc in response to these signals requires downregulation of additional signals such as myd88 and pkc. such a rheostat effect allowing differential signal strength - dependent desensitisation of receptor signalling would allow es-62 to provide an appropriate level of hyporesponsiveness that would prevent development of aberrant autoimmune and allergic inflammatory disorders whilst allowing appropriate levels of inflammation to generate protective immune responses to pathogenic infection. | es-62, an immunomodulator secreted by filarial nematodes, exhibits therapeutic potential in mouse models of allergic inflammation, at least in part by inducing the desensitisation of fcri - mediated mast cell responses. however, in addition to their pathogenic roles in allergic and autoimmune diseases, mast cells are important in fighting infection, wound healing, and resolving inflammation, reflecting that mast cells exhibit a phenotypic and functional plasticity. we have therefore characterised the differential functional responses to antigen (via fcri) and lps and their modulation by es-62 of the mature peritoneal - derived mast cells (pdmc ; serosal) and those of the connective tissue - like mast cells (ctmc) and the mucosal - like mast cells derived from bone marrow progenitors (bmmc) as a first step to produce disease tissue - targeted therapeutics based on es-62 action. all three mast cell populations were rendered hyporesponsive by es-62 and whilst the mechanisms underlying such desensitisation have not been fully delineated, they reflect a downregulation of calcium and pkc signalling. es-62 also downregulated myd88 and pkc in mucosal - type bmmc but not pdmc, the additional signals targeted in mucosal - type bmmc likely reflecting that these cells respond to antigen and lps by degranulation and cytokine secretion whereas pdmc predominantly respond in a degranulation - based manner. |
it is classified as primary or secondary on the basis of the presence or absence of underlying lung disease predisposing to the event. its clinical picture includes retrosternal chest pain (most common symptom), subcutaneous emphysema, dyspnea, dysphagia, dysphonia, asthenia, and the classical sign of hamman 's crunch (a crepitant sound that varies with the heartbeat on auscultation of the precordium). in most cases, it is possible to identify a triggering event, such as acute exacerbations of asthma. other known triggers include those related to the valsalva maneuver, such as strenuous exercise, weight lifting, inhalation of illicit drugs, cough, forced evacuation, and labor, as well as vomiting, respiratory infections, foreign body aspiration, and barotrauma. treatment is supportive, consisting of using painkillers, resting, and avoiding maneuvers that generate an increase in transpulmonary pressure, such as the valsalva maneuver and spirometry. a 50-year - old male patient underwent orchiectomy for testicular swelling and an increased alpha - fetoprotein level. the patient was started on chemotherapy with cisplatin, etoposide, and bleomycin (cumulative dose at the end of the course, 300 iu). during treatment, the patient had an episode of febrile neutropenia and received granulocyte colony - stimulating factor (g - csf) at that time. three months after the start of chemotherapy, the patient had a medical visit in which he complained of rapidly progressive dyspnea, presenting with hypoxemia (spo2 = 87%) and crackles at both lung bases. an hrct scan (figure 1) showed diffuse, relatively symmetric, reticular lung opacities in a peripheral and sometimes peribronchovascular distribution, predominantly at the lung bases, associated with irregular interlobular septal thickening, in addition to some foci of consolidation and areas of ground - glass opacity, bilaterally distributed, predominantly in the subpleural regions. a presumptive diagnosis of bleomycin - induced interstitial pneumonitis was made, and corticosteroid treatment was started. the patient underwent pulmonary function testing (pft), which revealed severe restrictive lung disease and markedly reduced dlco. figure 1 in a, axial hrct scan showing reticular opacities (dashed arrow) associated with irregular interlobular septal thickening (solid arrow), as well as foci of consolidation and areas of ground - glass opacity (arrowhead), bilaterally distributed, predominantly in the subpleural regions. in b, coronal hrct scan showing the distribution of those changes along the longitudinal axis of both lungs, with a slight basal predominance. two days after undergoing pft, the patient presented to the emergency room with significantly worsened dyspnea. physical examination revealed tachycardia (hr, 110 bpm), tachypnea (rr, 28 breaths / min), and hypoxemia (spo2 = 75%), as well as subcutaneous emphysema in the right cervical region. lung auscultation revealed decreased breath sounds in the lower thirds and crackles at both lung bases. laboratory tests revealed leukocytosis (17,040 cells / mm with a shift to myelocytes, thrombocytopenia, an elevated level of nitrogenous compounds, and an increased level of c - reactive protein (43.9 mg / dl). a chest x - ray (figure 2) allowed the visualization of pneumomediastinum and subcutaneous emphysema, with a small pneumothorax and an increase in ground - glass opacities. the same changes were seen on hrct (figure 3), which showed extensive pneumomediastinum and subcutaneous emphysema, as well as a significant increase in reticular opacities and areas of ground - glass opacity, compared with the initial examination. the patient was started on treatment for an infectious etiology (piperacillin / tazobactam) and progression of interstitial disease (methylprednisolone, 1 mg / kg). the patient continued to have fever despite further antibiotic therapy (vancomycin and sulfamethoxazole / trimethoprim were added) and developed respiratory failure requiring orotracheal intubation, mechanical ventilation, and chest tube drainage. figure 2 (front view) chest x - ray showing an air - density band around the mediastinum (arrows), characterizing pneumomediastinum, which extends to the cervical region and chest wall, dissecting along the fibers of the pectoral muscles (dashed arrow). note the extensive involvement of the lungs by areas of consolidation and reticular opacities, distributed in the lung periphery, especially on the right side, where one can also see a small pneumothorax (arrowheads). figure 3 in a, oblique sagittal reformatted hrct scan showing air dissecting within the lung interstitium along the right inferior pulmonary vein (arrow) and entering the mediastinum. in b, curved coronal reformatted hrct scan showing the upward path of the air around the right inferior pulmonary vein (arrow) into the pneumomediastinum (arrowheads). note the integration between the pneumomediastinum and the cervical emphysema (dashed arrows). in c, curved coronal reformatted hrct scan showing the large amount of air that dissects along the mediastinal fat planes and pneumomediastinum (arrowheads). in d, axial hrct scan showing pneumomediastinum (arrowheads) associated with chest wall emphysema (asterisk, also found in a). note also a small right pneumothorax (arrow), as well as reticular lung opacities and areas of groundglass opacity (dashed arrow). the pathophysiological mechanism involved in the genesis of pneumomediastinum is the emergence of a pressure gradient between the alveoli and surrounding structures that, upon reaching a critical level, causes alveolar rupture with an air leak into the interstitium, causing interstitial emphysema. the air dissects along the bronchovascular bundle (figures 3a and 3b) and, since the pressure is always lower in the mediastinum than in the lung parenchyma, the air tends to move toward the hilum and spread through the mediastinum (figure 3c). because of the contiguity between the fasciae, the air can reach the subcutaneous cellular tissue (figures 3a and 3d) and the peritoneum. when mediastinal pressure rises abruptly and decompression by alternative pathways is insufficient to relieve pressure, there can be rupture of the mediastinal pleura and the development of pneumothorax (figure 3d). the brazilian journal of pulmonology has recently published a letter to the editor describing a case of spontaneous pneumomediastinum and reporting, among other findings, pneumorrhachis, which is extremely rare. however, in that case, no imaging findings of dissection of air along the bronchovascular bundle were found. in a case report of pneumomediastinum in a bone marrow transplant recipient, some hrct findings were similar to those of the present case, although they were of lesser magnitude and there was no pneumothorax. those authors demonstrated that most air leak syndromes did not result from rupture of subpleural bullae, but rather from alveolar rupture. in addition, they introduced the concept that the presence of positive pressure within the alveoli is not necessary for rupture to occur. what is essential is the pressure gradient between the alveolus and the perivascular sheath of the adjacent septum. a very negative pressure in the interstitial space could therefore lead to alveolar rupture even without the occurrence of extreme positive alveolar pressures. for this reason, intense respiratory efforts, adjacent atelectasis, and low intravascular pressure could be involved in the development of these syndromes. the first of such reports described a previously healthy 23-year - old medical student who developed extensive pneumomediastinum and subcutaneous emphysema after undergoing pft. in spirometry, the patient is instructed to inhale up to total lung capacity, hold his or her breath, and exhale vigorously. when the patient inhales, there is a decrease in intrathoracic pressure, an increase in alveolar air volume, and an increase in venous return, resulting in increased pulmonary vein diameter. therefore, there is no pressure gradient in the interstitial space, since all compartments elongate and increase in diameter symmetrically. however, when the patient " holds his or her breath ", there is venous stasis, resulting in decreased pulmonary venous filling and vessel lumen reduction, allowing the emergence of the pressure gradient necessary to cause alveolar rupture. subsequent reports have documented pneumomediastinum as a complication of pft associated with extremely different underlying lung diseases, such as rheumatic diseases with interstitial lung involvement. in these situations, there is a summation of the aforementioned pressure changes induced during the test, the existence of inflammation combined with increased elastic recoil as seen with fibrosis, and a collapse of adjacent regions, making the lung vulnerable to segmental hyperdistension and to the emergence of a pressure gradient. when reporting the case of a healthy medical student who developed pneumomediastinum after use of marijuana, one group of authors understood that the event was caused by the fact that the young man performed various valsalva - like maneuvers (holding his breath at maximal inhalation against a partially closed or fully closed glottis) to prolong the effect of the drug. the valsalva maneuver is a factor classically related to air leak syndromes because it generates increased intrapulmonary pressure. therefore, labor and forced evacuation are associated with reports of pneumomediastinum. as mentioned previously however, this apparent benignity should be regarded with caution, since, in patients with underlying lung diseases, the existence of air leak syndromes is associated with greater severity. it is known that this syndrome can be associated with the spread of infections and the release of inflammatory mediators. in addition, it can result in the feared air - block syndrome (a condition in which the presence of air in the interstitium and mediastinum does not find a way out, culminating in the generation of large pressures on the mediastinum, which affects circulation by compressing the vessels and the heart and prevents lung inflation and deflation). in the present case, it is possible that the pneumomediastinum acted as a contributing factor to the fatal outcome because it facilitated the spread of infection and the perpetuation of inflammation, but without acting as a determinant of death, which was related to the interstitial lung disease and the severe, superimposed infectious process. in addition, it is of note that, in the present case, there was no indication for specific treatment for pneumomediastinum, such as mediastinotomy. this type of procedure would only be beneficial in cases of hypertensive pneumomediastinum, which are rare. because pft is often performed in patients with acute chronic respiratory disease, its association with air leak syndromes should be studied and reported. the most common form of pulmonary toxicity associated with bleomycin is subacute progressive pulmonary fibrosis. other less common lesions include organizing pneumonia, hypersensitivity pneumonia, and acute chest pain syndrome. it occurs more frequently in elderly subjects, with higher cumulative doses of the medication, renal failure, use of oxygen (especially when high fio2 is used), combination of other chemotherapeutic agents (such as cisplatin and gemcitabine), thoracic radiation therapy, and use of g - csf. bleomycin - induced interstitial pneumonitis is understood as a causative factor for the occurrence of pneumomediastinum in the current literature, on the basis of two studies that reported three cases in which this association occurred. curiously, in at least two of those cases, pft was performed before the development of pneumomediastinum ; however, those authors did not infer a causal relationship between pft and the occurrence of the complication. it is evident that interstitial pneumonitis includes previously mentioned factors, such as local inflammation and alveolar collapse, that facilitate the development of pneumomediastinum. under these conditions, there can be hyperinflation of adjacent regions, increased elastic recoil, and reduced lung compliance, predisposing to the emergence of an increased pressure gradient, but it seems pertinent to investigate what role pft might also have played in those reports. in the present case, because of the strong temporal correlation between the patient having undergone pft and the occurrence of pneumomediastinum, in addition to the pathophysiological rationale, we consider that the " trigger " for the occurrence of air leak was the patient having undergone the test, although it is undeniable that the presence of bleomycin - induced interstitial pneumonitis created a predisposition to the event. the present report and a review of cases in the literature suggest the need for caution and proper orientation of patients with bleomycin - induced interstitial pneumonitis who perform pft maneuvers. o pneumomediastino espontneo um evento incomum que acomete preferencialmente jovens do sexo masculino. classificado como primrio ou secundrio de acordo com a ausncia ou presena de doena pulmonar de base predispondo ao evento. o quadro clnico inclui dor pleurtica retroesternal (sintoma mais frequente), enfisema subcutneo, dispneia, disfagia, disfonia, astenia e o clssico sinal de hamman (crepitao sncrona com os batimentos cardacos, audvel no precrdio). na maioria dos casos, possvel identificar um evento desencadeador, como a exacerbao aguda de asma. outros desencadeantes conhecidos so aqueles relacionados realizao da manobra de valsalva, como exerccio fsico extenuante, levantamento de peso, inalao de drogas ilcitas, tosse, esforo vigoroso de evacuao e trabalho de parto, alm de vmitos, infeces respiratrias, aspirao de corpo estranho e barotrauma. a evoluo geralmente benigna e autolimitada. o tratamento de suporte, tendo por base o uso de analgsicos e o repouso, alm de evitar manobras que aumentam a presso transpulmonar, como, por exemplo, a manobra de valsalva e a espirometria. paciente masculino de 50 anos, submetido orquiectomia por aumento de volume testicular e elevao de alfa - fetoprotena. foi iniciada quimioterapia com cisplatina, etoposdeo e bleomicina (dose acumulada ao final dos ciclos, 300 ui). durante o tratamento, o paciente apresentou um episdio de neutropenia febril e fez uso de granulocyte colony - stimulating factor (g - csf, fator estimulador de crescimento de colnias de granulcitos) nessa ocasio. trs meses aps o incio da quimioterapia, o paciente compareceu a consulta mdica queixando - se de dispneia rapidamente progressiva, apresentando hipoxemia (spo2 = 87%) e com estertores crepitantes bibasais. a tcar (figura 1) evidenciou opacidades pulmonares reticulares difusas, relativamente simtricas, com distribuio perifrica e, por vezes, peribroncovascular, com predominncia nas regies basais, associadas a um espessamento irregular dos septos interlobulares, alm de alguns focos de consolidao e reas de vidro fosco, distribudos bilateralmente, predominando nas regies subpleurais. foi feita a hiptese diagnstica de pneumopatia intersticial por bleomicina e foi iniciado o tratamento com corticosteroides. foi realizada a prova de funo pulmonar (pfp), que mostrou distrbio ventilatrio restritivo acentuado e reduo acentuada na dlco. figura 1 em a, imagem axial de tcar demonstrando opacidades reticulares (seta descontnua), associadas a espessamento irregular dos septos interlobulares (seta contnua), alm de alguns focos de consolidao e reas de vidro fosco (cabea de seta), distribudas bilateralmente, predominando nas regies subpleurais. em b, imagem coronal de tcar demonstrando a distribuio dessas alteraes ao longo do eixo longitudinal de ambos os pulmes, com leve predomnio nas bases. dois dias aps a realizao da pfp, o paciente procurou o pronto - socorro por piora intensa da dispneia. ao exame fsico, encontrava - se taquicrdico (fc, 110 bpm), taquidispneico (fr, 28 ciclos / min) e hipoxmico (spo2 = 75%), com presena de enfisema subcutneo em regio cervical direita e ausculta pulmonar com murmrio vesicular reduzido em teros inferiores e estertores crepitantes bibasais. os exames laboratoriais mostraram leucocitose (17.040 clulas / mm com desvio at mielcitos, plaquetopenia, elevao de escrias nitrogenadas e aumento de protena c reativa (43,9 mg / dl). a radiografia de trax (figura 2) permitiu a visualizao de pneumomediastino e enfisema subcutneo, com pequeno pneumotrax e aumento de opacidades em vidro fosco. as mesmas alteraes foram observadas tcar (figura 3), que evidenciou grande extenso de pneumomediastino e enfisema subcutneo, bem como um aumento significativo das opacidades reticulares e reas de vidro fosco, comparativamente ao exame inicial. foi institudo um tratamento contemplando uma etiologia infecciosa (piperacilina / tazobactam) e progresso de doena intersticial (metilprednisolona, 1 mg / kg). o paciente continuou apresentando febre a despeito do escalonamento da terapia antibitica (foram acrescentados vancomicina e sulfametoxazol / trimetoprima) e evoluiu com insuficincia respiratria, sendo submetido a intubao orotraqueal, ventilao mecnica e drenagem torcica. o paciente evoluiu a bito por choque sptico sete dias aps a admisso. figura 2 radiografia de trax (frente) demonstrando faixa com densidade de ar contornando o mediastino (setas), configurando pneumomediastino, que se estende para a regio cervical e para a parede torcica, dissecando as fibras musculares peitorais (setas descontnuas). notar o extenso acometimento pulmonar por reas de consolidao e opacidades reticulares, distribudas na periferia pulmonar, especialmente direita, onde tambm se delimita um pequeno pneumotrax (cabeas de seta). figura 3 em a, reformatao sagital oblqua de tcar, que demonstra ar dissecando o interstcio pulmonar ao longo da veia pulmonar inferior direita (seta), at atingir o mediastino. em b, reformatao coronal curva de tcar mostrando o trajeto ascendente do ar ao redor da veia pulmonar inferior direita (seta), continuando - se com o pneumomediastino (cabeas de seta). notar a continuidade do pneumomediastino com o enfisema cervical (setas descontnuas). em c, reformatao coronal curva de tcar demonstrando a grande quantidade de ar que disseca os planos gordurosos mediastinais e pneumomediastino (cabeas de seta). em d, imagem axial de tcar demonstrando o pneumomediastino (cabeas de seta) associado ao enfisema da parede torcica (asterisco, tambm observado em a). notar tambm um pequeno pneumotrax direita (seta) e as opacidades pulmonares reticulares e reas de vidro fosco (seta descontnua). o mecanismo fisiopatolgico implicado na gnese do pneumomediastino o surgimento de uma diferena de presso entre os alvolos e estruturas adjacentes que, ao atingir um nvel crtico, ocasiona ruptura alveolar, com extravasamento de ar para o interstcio, ocasionando enfisema intersticial. o ar disseca o feixe broncovascular (figuras 3a e 3b) e, como a presso no mediastino sempre menor do que no parnquima pulmonar, o ar tende a se mover na direo do hilo e se espalhar pelo mediastino (figura 3c). devido contiguidade entre as fscias, o ar pode atingir o tecido celular subcutneo (figuras 3a e 3d) e o peritnio. quando a presso mediastinal se eleva abruptamente e a descompresso por vias alternativas no suficiente para aliviar a presso, pode haver ruptura da pleura mediastinal e o surgimento de pneumotrax (figura 3d). recentemente, foi publicada no jornal brasileiro de pneumologia uma carta ao editor descrevendo um caso de pneumomediastino espontneo e mostrando, entre outros achados, o rarssimo pneumorraque ; porm, naquele caso, a imagem de disseco do feixe broncovascular no foi caracterizada. em um relato de pneumomediastino em um paciente receptor de transplante de medula ssea, foi observada uma imagem tomogrfica semelhante do presente caso ; porm, de menor magnitude e sem a presena de pneumotrax. a descrio dessa cadeia de eventos foi feita por macklin & macklin na dcada de 40. a maioria das sndromes de vazamento de ar no era resultante da ruptura de bolhas subpleurais, mas decorrente de ruptura alveolar. alm disso, apresentaram o conceito de que no necessria a presena de presso positiva dentro dos alvolos para a ocorrncia da ruptura. o fundamental a diferena de presso entre o alvolo e a bainha perivascular do septo adjacente. uma presso muito negativa no espao intersticial poderia, portanto, levar a rotura alveolar mesmo sem a ocorrncia de presses positivas extremas no alvolo. dessa maneira, esforos inspiratrios intensos, atelectasias adjacentes e baixa presso intravascular poderiam estar implicados no surgimento dessas sndromes. em 1973, o primeiro relato dessa natureza descreveu o caso de um estudante de medicina de 23 anos, previamente hgido, que evoluiu com extenso pneumomediastino e enfisema subcutneo aps realizar uma pfp. em uma espirometria, o paciente instrudo a realizar uma inspirao mxima at a capacidade pulmonar total, prender a respirao e expirar vigorosamente. no momento em que o indivduo inspira, ocorre uma reduo da presso intratorcica, aumento do volume de ar nos alvolos e aumento do retorno venoso, com consequente aumento do dimetro das veias pulmonares. por isso, no ocorre diferena de presso no espao intersticial, j que todos os compartimentos se alongam e aumentam de dimetro de maneira simtrica. no entanto, quando o indivduo " prende a respirao ", h estase venosa, com consequente reduo do enchimento venoso pulmonar e reduo do lmen dos vasos, possibilitando o surgimento da diferena de presso necessria ruptura alveolar. relatos que se seguiram notaram o pneumomediastino como uma complicao da pfp associada a doenas pulmonares subjacentes extremamente diversas, como, por exemplo, doenas reumatolgicas com acometimento intersticial pulmonar. nessas situaes, somam - se as j citadas alteraes de presso induzidas na realizao do exame, a existncia de inflamao combinada com aumento do recolhimento elstico da fibrose e o colapso de reas adjacentes que tornam o pulmo vulnervel a hiperdistenso segmentar e ao surgimento de gradiente de presso. esse o mecanismo que acreditamos aplicar - se tambm no presente caso. ao relatar o caso de um estudante saudvel que desenvolveu pneumomediastino aps o uso de maconha, um grupo de autores entendeu que o mesmo foi causado pelo fato de o jovem ter realizado diversas manobras do tipo valsalva (prendendo a respirao aps inspirao mxima contra a glote parcial ou totalmente fechada) com o intuito de prolongar o efeito da droga. a manobra de valsalva um fator clssico relacionado s sndromes de vazamento de ar por gerar um aumento de presso intrapulmonar. por esse motivo, o trabalho de parto e esforos de evacuao vigorosos esto associados a relatos de pneumomediastino. conforme j anteriormente citado, o pneumomediastino costuma ter uma evoluo benigna e autolimitada. entretanto, devemos olhar com cautela essa aparente benignidade, j que, em pacientes com doenas pulmonares subjacentes, a existncia de sndromes de vazamento de ar est associada a maior gravidade. sabemos que essa sndrome pode associar - se a disseminao de infeces e liberao de mediadores inflamatrios. alm disso, pode resultar na temida sndrome airblock (condio em que a presena de ar no interstcio e no mediastino no encontra uma rota de sada, culminando na gerao de grandes presses sobre o mediastino, que interfere na circulao por compresso dos vasos e do corao e impede a insuflao e desinsuflao pulmonar). no presente caso, possvel que o pneumomediastino tenha atuado como um fator contribuinte para o desfecho fatal por facilitar a disseminao da infeco e a perpetuao de inflamao ; porm, sem atuar como o determinante do bito, sendo esse relacionado intersticiopatia e ao grave processo infeccioso sobreposto. vale ainda ressaltar que, no presente caso, no existia a indicao de tratamento especfico para o pneumomediastino como, por exemplo, mediastinotomia. esse tipo de procedimento s seria benfico nos raros casos de pneumomediastino hipertensivo. como as pfp so frequentemente realizadas em portadores de doena respiratria aguda e crnica, sua associao com sndromes de vazamento de ar deve ser estudada e relatada. a principal forma de outras leses menos comuns so a pneumonia em organizao, a pneumonia de hipersensibilidade e a sndrome da dor torcica aguda. ocorre mais frequentemente em idosos, com maior dose acumulada da medicao, insuficincia renal, uso de oxignio (principalmente quando em altas fraes), associao de outros quimioterpicos, como cisplatina e gemcitabina, radioterapia torcica e uso de g - csf. a pneumopatia intersticial induzida por bleomicina entendida na literatura atual como um fator causal para a ocorrncia de pneumomediastino baseado em dois estudos que relataram trs casos nos quais essa associao ocorreu. curiosamente, em pelo menos dois desses casos, a pfp foi realizada previamente ao surgimento do pneumomediastino ; porm, os autores no inferiram nexo causal entre a pfp e a ocorrncia da complicao. evidentemente que a pneumopatia intersticial contempla fatores j mencionados, como inflamao local e colapso alveolar, que facilitam o surgimento de pneumomediastino. nessas condies, pode ocorrer hiperinsuflao de reas adjacentes, aumento do recolhimento elstico e reduo da complacncia pulmonar, predispondo ao surgimento de gradiente de presso aumentado, mas nos parece pertinente investigar qual papel a pfp pode ter desempenhado tambm naqueles relatos. no presente caso, devido forte correlao temporal entre a realizao da pfp e a ocorrncia do pneumomediastino, alm do racional fisiopatolgico, consideramos que o " gatilho " para a ocorrncia do vazamento de ar foi a realizao do exame, embora seja inegvel a predisposio para o evento gerada pela presena de pneumopatia intersticial induzida por bleomicina. o presente relato e a reviso de casos na literatura sugerem a necessidade de cautela e orientao adequada de pacientes com pneumopatia intersticial induzida por bleomicina que realizam manobras de pfp. | spontaneous pneumomediastinum is an uncommon event, the clinical picture of which includes retrosternal chest pain, subcutaneous emphysema, dyspnea, and dysphonia. the pathophysiological mechanism involved is the emergence of a pressure gradient between the alveoli and surrounding structures, causing alveolar rupture with subsequent dissection of the peribronchovascular sheath and infiltration of the mediastinum and subcutaneous tissue with air. known triggers include acute exacerbations of asthma and situations that require the valsalva maneuver. we described and documented with hrct scans the occurrence of pneumomediastinum after a patient with bleomycin - induced interstitial lung disease underwent pulmonary function testing. although uncommon, the association between pulmonary function testing and air leak syndromes has been increasingly reported in the literature, and lung diseases, such as interstitial lung diseases, include structural changes that facilitate the occurrence of this complication. |
huntington 's disease (hd) is a neurodegenerative disorder characterized by selective loss of neurons in the striatum and cortex, which leads to progressive motor dysfunction, cognitive decline, and psychiatric disorders. hd is a genetic disorder caused by a trinucleotide (cag) repeat expansion in the gene encoding for huntingtin (htt) on chromosome 4p16.3. the normal htt presents less than 36 cag repeats. mutated htt with 40 or more cag repeats results in hd with complete penetrance. expansions varying from 36 to 39 cag repeats may also result in hd, but with incomplete penetrance [2, 3 ]. the cag repeat expansion in htt encodes an expanded polyglutamine tract in the huntingtin (htt) protein resulting in a mutant protein. the physiological functions of htt are still a matter of debate. a range of evidence points to htt 's central role in embryonic development. htt has also been implicated in controlling brain neurotrophic factor (bdnf) production, neuronal gene transcription, and synaptic transmission. the neuronal death observed in hd might result from loss of function and/or gain of toxicity of mutant htt, but a range of evidence suggests that hd arises mainly from gain of toxicity from an abnormal conformation of mutant htt. although the cause of hd is well known and the mutant htt is pivotal to hd pathophysiology, the ultimate cause of neuronal death is still uncertain. apart from impairment in systems for handling abnormal proteins, other metabolic pathways and mechanisms might contribute to neurodegeneration and progression of hd. among these, inflammation seems to play a role in hd pathogenesis (reviewed in). at first, neuroinflammation can be regarded as beneficial for neuronal tissue since it promotes clearance of cell debris, but scenario is definitely more complex. for instance, apart from modulating immune cell functions, inflammatory mediators also act on neurons, possibly contributing to neuronal death. neuronal death further activates inflammatory mechanisms, resulting in a vicious cycle of inflammation and neurodegeneration. besides inflammatory processes occurring in the central nervous system (cns) the objective of the current review is to summarize the available evidence about inflammatory / immune changes in hd. herein, we showed that hd is associated with increased inflammatory mediators in both cns and periphery. in addition, we briefly presented the studies targeting immune disruption as an attempt to slow disease progression. robust evidence regarding neuroinflammation in hd comes from postmortem studies with brain tissue from patients with hd. the term neuroinflammation was initially used to describe the infiltration of peripheral immune cells in the cns in response to infectious agents or trauma. the pathological hallmark of hd is prominent degeneration of the caudate and putamen (collectively known as striatum), along with accumulation of nuclear and cytoplasmic inclusions of mutant huntingtin in neurons. postmortem studies also showed significant microglial activation in brain regions implicated in hd pathogenesis, that is, striatum, globus pallidus, and cortex [9, 10 ]. interestingly, the number of activated microglia in the striatum and cortex correlated with neuronal loss, corroborating the view that neuroinflammatory changes may be elicited by degenerating neurons. indeed, infiltration of peripheral inflammatory cells is not usually seen in hd [911 ]. these results regarding microglial activation in postmortem human brains can be recapitulated in mouse models of hd. it has been shown that mutant htt was expressed in murine microglial cells, facilitating autonomous microglial activation, which was sufficient to trigger neurodegeneration. as observed in patients, increased microglial activation was detected in the striatum of presymptomatic r6/2 mice (mouse model expressing human mutant htt exon 1 containing 150 cag repeats). in addition, macrophages and microglia derived from r6/2 and yac128 (expressing the full - length human htt gene containing 128 cag repeats) were also more active in response to stimulation [8, 14 ]. notably, changes in striatal and microglial morphology were shown to be age - dependent in yac128 mice. in parallel with microglial activation, increased number of astrocytes has also been described in postmortem brains of patients with hd [9, 11 ]. the onset of microglial activation and astrocytosis might differ depending on the neuropathological severity grade (based on the severity of atrophy and neuronal loss). no reactive astrocytes were observed in grade 0 (no abnormality on conventional neuropathological evaluation) with increase in other stages. in contrast, the increase in the density of microglial cells was already observed in grade 0, with modest increase from grade 0 to 3 and significant increase in grade 4 over controls. while the increase in microglial activation seemed to be a very early event, being present even before neuropathological changes, reactive astrocytosis was observed only after neurodegeneration. interleukin- (il-) 6, il-8, tumor necrosis factor- (tnf-), monocyte chemoattractant protein- (mcp-) 1/ccl2, and il-10 mrna levels were markedly increased in the striatum of patients with hd in comparison with controls [8, 16 ]. in addition, upregulated expression of il-6, il-8, and matrix metalloproteinase- (mmp-) 9 was also described in the cortex and cerebellum of patients with hd. given the important role of astrocytes in supporting neurons, studies have speculated whether astrocytic dysfunction might be involved in neurodegeneration in hd. marked accumulation of the chemokine regulated on activation normal t cell expressed and secreted (rantes / ccl5) was found in astrocytes in the frontal cortex, substantia nigra, and caudate of patients with hd, but not in astrocytes of matched controls. moreover, the expression of the amino - terminal mutant htt (160q) exclusively in astrocytes was sufficient to induce neurological symptoms in mice. intracellular signaling pathways regulate the expression of genes influencing a broad range of biological processes, including innate and adaptive immunity, inflammation, and stress responses. the nuclear factor kappa b (nf-b) is a major downstream transcription factor that is responsible for promoting the transcription of inflammatory mediators upon stimulus. the nuclear localization of p65 (a subunit of nf-b) was found in gfap - positive astrocytes in the caudate nucleus of patients while no nuclear localization of p65 was observed in matched controls. nf-b signaling cascade acts in parallel with other pathways, including the signaling pathways initiated by phosphatidylinositol 3-kinase (pi3k) and protein kinase b (pkb, also known as akt). caspase-3-generated akt product (akt 49 kda) was found in cerebellum and cortex from patients, but not from controls. the same work group showed later that akt is decreased by caspase-3 cleavage in the brains of patients with hd. an interest in evaluating microglial activation in vivo has been raised mainly due to the results obtained from postmortem studies. a growing body of evidence has shown that microglial activation as assessed in vivo using positron emission tomography (pet) is associated with hd progression. the peripheral benzodiazepine binding sites for the isoquinoline c - pk11195 are expressed by the mitochondrial membranes of activated but not resting microglia / brain macrophages. c - raclopride pet is a marker of dopamine d2 receptor binding and hence striatal gabaergic cell function. a significant increase in striatal c - pk11195 binding was found in patients with hd compared with controls. c - pk11195 binding significantly correlated with disease severity as assessed by striatal reduction in c - raclopride binding, the unified huntington 's disease rating scale (uhdrs) score, and the number of patients ' cag repetitions. presymptomatic hd gene carriers also presented increased striatal c - pk11195 binding which was associated with the severity of striatal neuronal dysfunction evaluated by c - raclopride pet. these data were corroborated by a later study that combined magnetic resonance imaging (mri) and pet analyses. worsening in atrophy evaluated by mri was accompanied by a reduction in c - raclopride and an increase in c - pk11195 bindings in patients with hd. in premanifest hd, increased level of microglial activation in the associative striatum and in the regional network associated with cognition correlated with 5-year probability of hd onset. based on the alterations in metabolism, sleep, and circadian rhythms as well as in the hypothalamic - pituitary axis described in hd, one study aimed to evaluate in vivo d2 receptor 's loss / dysfunction and changes in microglial activation in the hypothalamus of patients. a significant decrease in mean hypothalamic c - raclopride binding and a significant increase in c - pk11195 binding were reported in patients with hd in comparison with controls. these pathological changes occur very early in the course of the disease since they were observed in both presymptomatic and symptomatic patients. more recently, increased microglial activation in somatosensory cortex (as evaluated through c - pk11195 binding) was associated with higher plasma levels of il-1, il-6, il-8, and tnf- in premanifest hd gene carriers. another way to investigate inflammatory changes in the cns in living patients is using cerebrospinal fluid (csf) samples. the complement factors c1qc, c2, and c3 and other proteins associated with inflammatory pathways, namely, peptidoglycan recognition protein 2 (pglyrp2) and apolipoprotein a4 (apoa4), were found to be increased in csf from patients with hd in comparison with controls. csf levels of these proteins followed the trend consistent with elevating as disease progressed (control < hd - early stage < hd - mid stage). clusterin is a molecular chaperone associated with the clearance of cellular debris and apoptosis and is intimately associated with the regulation of complement - mediated membrane attack. it is possible that the elevated level of these complement related proteins could contribute to disease progression. however, r6/2 mice crossed with complement c3 deficient mice exhibited no alteration in multiple behavioral assays, weight, and survival. glia cells produce matrix metalloproteinases (mmps) and free radicals in response to proinflammatory stimuli. -mmps and their regulators, tissue inhibitors of metalloproteinases (timps), play important roles in inflammation and wound healing. mmp-3 and mmp-9 levels are increased in csf from patients with hd in comparison with controls. moreover, mmp-3, mmp-9, and timp-1 levels were associated with disease severity in hd as assessed by motor scores. mmp-3 is an endogenous neuronal activator of microglia and also induced increased cytokine release by microglia in yac128 mice, that is, mice expressing full - length mutant human huntingtin with 128 polyglutamine repeats. however, not all studies showed altered levels of inflammatory / immune markers in the csf of patients with hd. for instance, there was no difference in csf levels of ykl-40 between patients and controls. ykl-40 (chi3l1) is a protein usually upregulated in inflamed tissues in several inflammatory diseases. astrocytes and microglia cells express ykl-40 in the brain, and its level is significantly elevated in different acute and chronic neuroinflammatory diseases. the meaning of this study may be twofold : csf level of ykl-40 is not suitable as hd marker, and ykl-40 may be altered only in overt inflammatory conditions. in order to investigate the relationship between central and peripheral inflammatory processes, il-6 and il-8 levels were measured in matched plasma and csf samples from patients with hd and controls. csf and plasma levels of il-6 and il-8 correlated closely in both hd and controls ' samples. unfortunately, authors did not compare csf levels of these molecules between samples from hd and controls subjects. the evidence of microglial activation and changes in csf inflammatory - related proteins levels pointed to immune changes in hd. given the cns and immune system cross - talk, it is reasonable to hypothesize that patients with hd present changes in peripheral inflammatory / immune parameters. the search for changes in the peripheral immune system is important for helping not only to elucidate hd pathophysiology but also to identify biomarkers useful for noninvasive monitoring of disease progression and/or response to treatment. nearly one decade before the discovery of the gene that causes hd, one study had speculated that gene defects could be expressed in nonneural as well as neural cells. attempting to prove this hypothesis, gollin. evaluated abnormalities in peripheral blood lymphocytes. using flow cytometry combined to the fluorescent membrane probe 8-anilino - l - naphthalene sulfonate (ans), they observed an increase in ans fluorescence intensity in lymphocytes harvested from patients with hd in comparison with controls. decades later, studies confirmed this hypothesis, showing that mutant htt can be found in several cells types, including lymphocytes. although htt is an ubiquitously expressed constitutive protein, htt mrna expression is higher in immune cells in comparison with other cell types [6, 34 ]. mutant htt was detected in monocytes, t cells, and b cells of patients with hd. in addition, mean mutant htt levels increased steadily with disease progression, with significant differences between premanifest patients (hd gene carriers) and patients with hd (manifest hd patients). monocyte and t cell mutant htt levels were significantly associated with disease burden scores and caudate atrophy rates in patients with hd. abnormal levels of mutant htt in peripheral immune cells might result in changes in these cells functions. for instance, monocytes from hd mutation carriers (premanifest subjects) stimulated with lipopolysaccharide (lps) displayed excess il-6 production compared with cells from control subjects. in addition to il-6, monocytes from patients with hd produced higher levels of tnf- and il-1 after lps stimulation when compared with controls. these data show that myeloid cells isolated from patients with hd are hyperreactive, producing elevated levels of several key proinflammatory cytokines following stimulation. this finding corroborates the view that abnormal levels of mutant htt impact on monocyte function. peripheral lymphocytes harvested from patients with hd produced a migration inhibitor factor when exposed to brain tissue from hd. the same did not occur when lymphocytes from control subjects were exposed to the same tissue. hd lymphocytes also responded to presence of multiple sclerosis brain tissue with a migration inhibition factor production. further in vitro and in vivo evidence show that mutant htt impairs immune cell migration. using well - characterized mouse models of hd (i.e., yac128 and bachd mice that express full - length htt and present many behavioral and pathological features of the disease), kwan. (2012) found that primary microglia from early postnatal hd mice were profoundly impaired in their migration to chemotactic stimuli. the expression of a mutant htt fragment in microglial cell lines was sufficient to reproduce the impairment in migration. leukocyte recruitment was impaired after induction of an inflammatory stimulus (i.e., peritonitis) in hd mice. in line with these experimental results, migration of monocytes harvested from premanifest hd subjects upon chemotactic stimuli was also severely impaired. 2015) evaluated whether patients with hd and controls exhibit differences regarding the frequency of blood lymphocytes. the activation levels of cd4 + and cd8 + t lymphocytes were also assessed by cd62l expression. the ability of lymphocytes to rapidly proliferate in response to a stimulus is a key feature of the adaptive immune system. the proliferative response of t lymphocyte populations (cd3 +, cd3+cd4 +, and cd3+cd8 +) was not impaired in hd in comparison with controls in response to stimuli (either anti - cd3 and anti - cd28 antibodies or pha - p). cd4 + lymphocytes from patients with hd and controls did not differ regarding the production of cytokines [il-2, il4, il-6, il-8, il-5, il-13, interferon- (ifn-), il-10, il-1b, il-12p70, and tnf- ] upon anti - cd3/cd-28 antibodies stimulation. the authors concluded that peripheral immune dysfunction in hd is likely to be mediated primarily by the innate rather than the adaptive immune system. however, the conclusions based on this work are limited due to the small sample size (varying from 5 to 12 subjects per group). studies comprising larger samples are needed in this regard. corroborating the hypothesis of normal adaptive immune response in hd, plasma levels of immunoglobulin g (igg), iga, and igm conversely, one study found that patients with hd presented higher serum levels of iga than controls. in addition, patients who died within one year after laboratory measurement presented higher levels of igm, circulating immune complexes and neopterin compared to survivors. nevertheless, as there is an intimate interaction between peripheral and cns immune system, changes in one of them may influence the other and vice versa. based on this assumption, abnormalities in circulating (i.e., blood) levels of inflammatory molecules inflammatory changes can occur in early stages of the disease, even in preclinical stages. for instance, c - reactive protein (crp) levels were increased in premanifest hd subjects compared to controls. the majority of studies found that inflammatory molecules levels increase as the disease advances. plasma levels of il-6 were increased in moderate hd in comparison with both controls and early hd patients. not only il-6, but also il-8 levels increased across groups from controls to different stages of the disease. il-6 levels were increased in hd gene carriers with a mean of 16 years before the predicted onset of clinical symptoms. tnf-, il-10, and il-4 were increased only in moderate stage hd patients in comparison with controls. in addition, plasma levels of il-8 and tnf- correlated with clinical symptoms, as evaluated by the uhdrs. cytokines involved in the innate immune response (il-6 and il-8) were increased in the earlier stages of disease (even 16 years before the predicted onset of clinical symptoms), while anti - inflammatory cytokines il-10 and il-4 increased only in moderate stage of disease ; therefore, the authors proposed that early innate immune activation could be a target in the development of disease - modifying therapies. plasma levels of the chemokines ccl26/eotaxin-3, ccl4/macrophage inflammatory protein- (mip-) 1, ccl11/eotaxin, ccl2/mcp-1, and ccl13/mcp-4 were statistically elevated above control levels in hd, with the first three increasing linearly with disease progression. agreeing with these results, one study described that plasma levels of proteins involved in regulation of the innate immune system, namely, alpha-2-macroglobulin (a2 m), c7, c9, and clusterin, are increased in patients with hd in comparison with controls. data obtained in mouse models were similar to those seen in patients with hd, as plasma levels of several inflammatory molecules, including il-6, il-8, tnf-, il-10, il-1, and il-12p70, were elevated in the hd mouse models r6/2, hdhq150 (murine model expressing 150 cag repeats inserted in the mouse endogenous htt gene first exon), and yac128 [8, 12, 42 ]. il-6 is particularly important in hd as the administration of an antibody that neutralizes il-6 into r6/2 mice decreased weight loss at late stages and partially rescued motor deficits. for instance, the levels of adipokines, namely, leptin and adiponectin, were not changed in hd. accordingly, plasma levels and diurnal rhythmicity of leptin, adiponectin, and resistin were not significantly different between patients and controls. however, when corrected for fat mass, mean plasma leptin concentration as well as basal, pulsatile, and total secretion rates increased with the size of the cag repeat mutation. both higher pulsatile leptin secretion and higher mean adiponectin levels were associated with a greater degree of motor and functional impairment in patients with hd. cag repeat size - dependent interference of the hd mutation with adipose tissue function may contribute to weight loss in patients with hd. (2014) did not find any difference in circulating levels of tnf- between patients with hd and controls. it is worth mentioning that tnf- does not seem to be a stable molecule, and its soluble receptors, which are released in response to changing levels of tnf-, seem to be more reliable markers of tnf- activity than tnf- itself. in this sense, serum levels of soluble tnf receptor type i (stnfri) were increased in patients with hd. in sum, we found that patients with hd exhibit increased circulating levels of inflammatory molecules, mainly related to innate immunity. this increase is already observed in early stages of the disease, that is, years before the onset of motor symptoms. it remains to be further investigated whether inflammatory / immune mediators can be used as biomarkers for disease progression and treatment response. this is a special need raised by clinical studies investigating the efficacy of disease - modifying drugs in hd. the most commonly activated signaling pathway downstream of cytokine receptors is the janus kinase / signal transducer and activator of transcription (jak / stat) pathway, coordinating cytokine - mediated gene expression and repression. using flow cytometry, trger. (2013) investigated the jak / stat pathway in monocytes from patients with hd. at baseline, while both phosphorylated (p)stat1 and pstat3 levels were unchanged, pstat5 levels were significantly elevated in hd gene carriers ' monocytes compared with control cells. targeted activation of jak / stat signaling molecules using specific stat1, stat3, and stat5 activators (ifn-, il-6, and granulocyte - macrophage colony - stimulating factor (gm - csf), resp.) found the same level of pathway activation in control and hd monocytes, indicating normal function of the signaling cascade in hd. it has been demonstrated that the ib kinase / nf-b signaling pathway, which promotes cytokines and chemokines release, was upregulated by mutant htt contributing to neurotoxicity. htt bound i kappa b kinase (ikk) complex in a cag repeat length dependent manner. activation of the ikk complex leads to the phosphorylation and degradation of ib, the endogenous inhibitor of nfb. ib was degraded more rapidly and over a prolonged period of time in monocytes from patients with hd when compared to controls, as a result of ikk activation. in addition, levels of phosphorylated ib were increased in monocytes isolated from patients with hd compared with control subjects. one study demonstrated a progressive but marked alteration of a prosurvival pathway in hd and further implicated it as a key transduction pathway regulating the toxicity of htt. levels of akt levels were twice higher in immortalized lymphoblasts from patients with hd than controls, but the amount of activated akt (pakt s473) was not different. accordingly, the active akt / total akt ratio in patients was significantly lower than that in controls. the same pattern of akt and pakt observed in immortalized lymphoblasts was found in primary lymphocytes. a schematic summary of inflammatory / immune changes evidence from human studies is presented in figure 1. based on data about immune / inflammatory changes in hd, some research groups have attempted to show the potential effects of immunomodulatory- and/or anti - inflammatory - based therapies in hd. the majority of studies are still preclinical ; that is, they used in vitro cell cultures and animal models of hd for testing these strategies. for instance, chronic treatment with the selective cyclooxygenase- (cox-) 2 inhibitors celecoxib and meloxicam attenuated behavioral and biochemical changes in a quinolinic acid - induced rat model of hd. another study, however, failed to demonstrate positive effects of the nonselective cox inhibitor acetylsalicylate or the selective cox-2 inhibitor rofecoxib in n171 - 82q and r6/2 hd transgenic mice. although not belonging to the classical definition of anti - inflammatory drugs, they are known to present anti - inflammatory properties. minocycline is a second - generation tetracycline that has been in therapeutic use for over 30 years. in addition to its antibiotic properties, minocycline can exert a variety of biological actions, including anti - inflammatory and antiapoptotic activities. a meta - analysis conclude that minocycline exerts neuroprotective effects in rodent models of neurodegenerative diseases, including hd. this effect is explained, at least in part, by minocycline anti - inflammatory, antiapoptotic, and antioxidant activities. the evidence from preclinical studies in hd supported further testing in patients with hd. a clinical study (nct00277355, table 1, results published in) showed that minocycline at 100 and 200 mg / day was well tolerated during 8-week treatment. the study involved 60 patients who were randomly assigned to receive placebo (n = 23), minocycline 100 mg / day (n = 18), or minocycline 200 mg / day (n = 19). corroborating these results, minocycline 200 mg / day was considered safe and well tolerated in a 6-month treatment protocol. here again, no noticeable changes were reported in cognitive and motor symptoms as evaluated by the mini - mental state examination, uhdrs, and abnormal involuntary movement scale. due to their anti - inflammatory properties, cannabinoids have been studied as a potential therapeutic approach in neuroinflammatory diseases. a case report described a great improvement in chorea and behavioral symptoms, mainly irritability, with nabilone ; however, this was not confirmed by a clinical trial. a crossover study involving 44 patients with hd evaluated the benefits of nabilone over placebo on uhdrs measures and the neuropsychiatry inventory. although nabilone was considered safe and well tolerated, there was no significant difference on the evaluated outcomes. the use of cannabidiol was considered safe and well tolerated in a clinical trial conducted for 6 weeks with 15 patients with hd. whereas these two clinical trials indicate lack of benefit, both were underpowered to detect differences, and thus no definite conclusions can be drawn. based on the evidence of increased levels of tnf- in hd, one study investigated the therapeutic potential of xpro1595, a dominant negative inhibitor of soluble tnf-. xpro1595 suppressed the inflammatory responses in two in vitro models : (i) primary astrocytes - enriched culture isolated from a transgenic mouse model (r6/2) exposed to lps and (ii) human astrocytes - enriched culture derived from induced pluripotent stem cells (ipscs) of patients with hd stimulated with cytokines. moreover, xpro1595 protected the cytokine - induced toxicity in both in vitro models. in vivo, xpro1595 decreased tnf- levels in the cortex and striatum, improved motor function, reduced caspase activation, diminished the amount of mutant htt aggregates, increased neuronal density, and decreased gliosis in the brain of r6/2 mice. laquinimod is an immunomodulator that downregulates both proinflammatory cytokine production in peripheral blood mononuclear cells and astrocytic and microglial activation in the brain. laquinimod was able to dampen hyperreactive production of cytokines from monocytes exposed to lps harvested from both premanifest and symptomatic hd mutant human carriers. a clinical trial is currently recruiting patients with hd in order to investigate the safety and efficacy of laquinimod (nct02215616, table 1). other drugs with potential anti - inflammatory / immunomodulatory effects have been tested in hd. vx15/2503 is a monoclonal antibody that blocks the activity of semaphorin 4d, a protein known to contribute to neuroinflammation. however, the role of cns and peripheral inflammatory changes in hd is still poorly understood. it is not clear whether inflammatory changes result from neurodegeneration and/or represent an independent pathological mechanism in hd. it is important to refine the understanding of the more specific immune / inflammatory mechanisms that are involved in hd. in addition, it remains to be investigated whether peripheral alterations mirror cns changes and the putative routes of these interactions. although there is no evidence of peripheral immune cell infiltration into the brain of patients with hd, there are consistent results showing other neuroinflammatory processes in hd. second, the levels of inflammatory mediators are increased in the brain and csf of patients. third, the levels of inflammatory mediators are also increased in peripheral blood from patients. inflammation is likely an early event in the pathological process given that immune activation is shown to be present up to 15 years before symptom onset. it remains a matter of debate whether the inflammatory response is an active or a reactive (or both) mechanism in hd pathophysiology. further studies are needed, mainly to help to study inflammation as a valid target for new therapeutic interventions to halt the progression of hd. | huntington 's disease (hd) is a neurodegenerative disorder characterized by selective loss of neurons in the striatum and cortex, which leads to progressive motor dysfunction, cognitive decline, and psychiatric disorders. although the cause of hd is well described hd is a genetic disorder caused by a trinucleotide (cag) repeat expansion in the gene encoding for huntingtin (htt) on chromosome 4p16.3the ultimate cause of neuronal death is still uncertain. apart from impairment in systems for handling abnormal proteins, other metabolic pathways and mechanisms might contribute to neurodegeneration and progression of hd. among these, inflammation seems to play a role in hd pathogenesis. the current review summarizes the available evidence about immune and/or inflammatory changes in hd. hd is associated with increased inflammatory mediators in both the central nervous system and periphery. accordingly, there have been some attempts to slow hd progression targeting the immune system. |
one of the first observable responses to dna damage is activation of dna - pk family kinases and resulting phosphorylation of the histone variant h2ax on s139. due to the availability of excellent antibodies to s139-phosphorylated h2ax (a form of the protein called -h2ax), this modification is a widely recognized early marker of both genotoxic stress and normal dna replication. the tail region of h2ax includes a conserved sq motif (s139 q140) that is recognized as the core target motif of dna - pk family serine / threonine kinases (atm, atr, and dna - pk [3, 5 ]). within minutes after dna damage the foci actually include large areas of chromatin flanking points of dna damage. after dna damage, these include the breast cancer susceptibility gene brca1, rad51, the nbs1/rad50/mre11 complex [1, 8 ], 53bp1 [9, 10 ], and mdc1 [11, 12 ]. recruitment of most proteins to radiation - induced foci is dependent on atm / atr activity and formation of -h2ax, indicating that h2ax phosphorylation plays a key role in maintenance of irradiation - induced foci. atm is the major h2ax kinase in response to -irradiation while atr plays a larger role during dna synthesis. s139 phosphorylation of h2ax is greatly reduced in atm / atr knockout cells and is completely blocked by treatment with wortmannin, an inhibitor of dna - pk kinases. genetic and biochemical experiments support roles for brca1 in homologous recombination [7, 14 ], nonhomologous end joining [15, 16 ], and transcription - coupled repair. brca1 null cells are extremely sensitive to -irradiation and other types of genotoxic stress [18, 19 ]. although the role of brca1 in dna repair is not known, the n - terminal ring finger of brca1 interacts with the ring finger of bard1, and the complex has been shown to possess e3 ubiquitin ligase activity in vitro. the e2 ubiquitin - conjugating enzyme (ubch5c) has been shown to associate with this complex [21, 22 ], and several in vitro substrates have been identified, including monoubiquitinated histones h2ax, h2a, h2b, h3, and h4 (but not h1). brca1 association with chromatin is properly considered an intermediate or late event in chromatin repair [1, 23 ]. here we demonstrate by differential fractionation of chromatin bound brca1 complexes that brca1 and -h2ax form a biochemical complex in the chromatin fraction of cells as a late event following dna damage. the complex is resistant to nonionic detergent extraction and is dependent on wortmannin - sensitive kinases, features that are distinct from brca1 prior to genomic stress. we show that a phosphomimetic of h2ax (h2ax - e139) is ubiquitinated in vivo, and the major site of ubiquitination is on k118 and/or k119. when brca1 levels were reduced using an antisense morpholino knockdown strategy, we observed substantially reduction in h2ax ubiquitination and increased amounts of h2ax s139 phosphorylation. these results are consistent with the hypothesis that brca1 is present in non - chromatin - associated complexes (including processive rna polymerase ii) prior to genotoxic stress ; it becomes phosphorylated, moves into a stable chromatin - associated complex containing -h2ax, and then targets -h2ax for turnover as a late phase of dna repair. h2ax, h2ax - a139, and h2ax - e139, were generated by rt - pcr from human rna using primers, described in the supplemental data (see table 1 in supplementary material available online at doi : 10.4061/2011/801594), and cloned into pcdna3.1d - v5-h6 (invitrogen) to generate epitope - tagged variants. pcr - mediated mutagenesis was performed using primers described in the supplementary data to generate additional mutations. pmt - ha - ub (hemagglutinin tagged- ubiquitin) was the generous gift of dr. damage randomly cycling hbl100 cells were treated with 4 m adriamycin or were exposed to 10 gy ionizing radiation using a cs source (mark 1 irradiator, shepherd and associates). following treatments, cells were returned to a 5% co2 incubator for the indicated amount of time. cells treated with wortmannin were first pretreated with 100 m wortmannin for 15 minutes. nuclei were prepared by extracting cells in ebc buffer (50 mm tris ph 8.0, 120 mm nacl, and 0.5% np-40) as described in. nuclei were then treated with 0.1 m hcl for 30 minutes and neutralized with 0.1 m naoh. this acid soluble chromatin fraction was centrifuged for 5 minutes at 14,000 rpm to remove insoluble material. lysates were precleared with protein a or g beads and then incubated overnight with primary antibodies to -h2ax (upstate biotechnology) or brca1 (ab-4, emd biosciences). proteins were then transferred to nitrocellulose membranes, blocked in 5% nonfat dry milk, and incubated with a primary antibody generated against brca1 (ab-4, emd biosciences), -h2ax (upstate biotechnology), h2a (h-124, santa cruz biotechnology), rna polymerase ii (n-20, santa cruz biotechnology or 8wg16 (provided by dr. michael e. dahmus (uc davis)), or -actin (sigma), followed by a goat antirabbit (or antimouse) horseradish peroxidase - conjugated secondary antibody (pierce). alexa-488-conjugated secondary antibodies (molecular probes) were used to visualize immune complexes, and photomicrographs were prepared as described in. quantitation of fluorescent -h2ax foci was accomplished using a laser scanning cytometer (lsc, compucyte). human mcf-7 or 293 t cells were treated with 5 m adriamycin for 1 hour. following adriamycin treatment, the cells were washed then treated with or without lactacystin (sigma) or a serine / threonine phosphatase inhibitor cocktail [(-)-p - bromotetramisole oxalate, cantharidin and microcystin - lr ] (emd biosciences) for 4 hours, after which the cells were fixed in 4% pfa. quantitation of fluorescent -h2ax foci was accomplished using a laser scanning cytometer (lsc, compucyte). two antisense morpholino oligos (gene tools, inc.) 20 l of each of the two 500 m morpholino antisense oligos were delivered into the cells using the epei delivery solution according to the manufacturer 's protocol. the remaining cells were treated with a serum - free media control, epei delivery solution (gene tools, inc.), or 40 l of a 500 m scrambled control oligo (5-cctcttacctcagttacaatttata-3). after a two - hour treatment, the media were replaced with serum - containing media. the cells were allowed to recover for 24 hours at which time they were lysed with ebc buffer. brca1 and -h2ax form a physical complex on chromatin following dna damage. before dna damage, small amounts of brca1 are present in the chromatin fraction, consistent with the percentage of s - phase nuclei in this population of cells [4, 7, 26 ]. after treatment with the dna - damaging agent adriamycin, there is a substantial increase in acid - stable nuclear brca1 : -h2ax complexes (figure 1(a)). stable interaction is minimal at early times following dna damage then increases to the maximum at about 60 minutes. the association is blocked by the atm / atr inhibitor wortmannin (figure 1(b)). this data supports the hypothesis that interaction of brca1 and h2ax occurs well after dna damage, requires phosphorylation by atm or atr [3, 27 ], and provides evidence that the interaction requires the formation of a stable complex. in order to analyze the kinetics of brca1 interactions, we examined brca1 complexes present in undamaged cells and in cells responding to dna damage. in undamaged cells, we had previously demonstrated that brca1 interacts with the phosphorylated, transcriptionally active form of rna polymerase ii (rna pol ii). following -irradiation, we found that the brca1 : -h2ax complex was enhanced (figure 1(a)) whereas the brca1:rna pol ii interaction is disrupted (figure 1(c)). this shift accompanies the movement of brca1 from an easily extractable form in undamaged cells to a chromatin - associated form in damaged cells. the association between brca1 and -h2ax peaks about 3060 minutes after dna damage whereas the association between brca1 and rna polymerase ii is at the lowest 3060 minutes after dna damage (figure 1(d)). fcp1, a protein that is part of the elongating pol ii complex, has a similar pattern of association with brca1 as that of pol ii (figure 1(c)), illustrating the reciprocal functionality between brca1 : -h2ax complex and the brca1:rna pol ii complex. further analysis of the timing of the brca1 : -h2ax complex shows that this complex coincides with a decreased level of cellular -h2ax. when the brca1 : -h2ax complex is at its highest level (about 60 minutes after dna damage), the level of -h2ax has begun to decrease, as demonstrated by immunoblot of the total chromatin extract (figure 1(e)). because brca1 is an e3 ubiquitin ligase, we hypothesized that the reduction of -h2ax seen following association with brca1 could be the result of ubiquitin - mediated proteasomal degradation. if this were true, then proteasome inhibitors should result in stabilized levels of -h2ax. brca1 has been shown to ubiquitinate h2ax in vitro ; therefore, we wanted to determine if -h2ax turnover was proteasome mediated. to test whether -h2ax was degraded by the 26s proteasome, mcf-7 mammary epithelial cells were treated with the dna - damaging agent adriamycin then treated with or without the proteasome inhibitor lactacystin for an additional hour before fixation. cells were fixed and immunostained to identify -h2ax high and -h2ax low cells. as shown in figure 2(a), lactacystin increased the percentage of -h2ax high cells in the population of cells recovering from adriamycin treatment. by this -h2ax immunofluorescence assay, 20% of untreated cells were found to be positive for high levels of -h2ax (figure 2(b)). when cells were treated with adriamycin for 1 hour followed by 1 hour recovery, -h2ax immunofluorescence showed a 2-fold increase over control cells, consistent with the view that h2ax was dynamically phosphorylated in response to dna damage. the proteasome inhibitor lactacystin had a similar effect as adriamycin, when added alone, consistent with the hypothesis that -h2ax formation in normal cycling cells could be stabilized by proteasome inhibitors. when adriamycin - induced damage was followed by lactacystin treatment, there was a fourfold increase in the amount of -h2ax over control, resulting in over 80% of cells containing high levels of -h2ax by two hours. addition of a phosphatase inhibitor cocktail also stabilized -h2ax level, suggesting that there may be additional ways to attenuate h2ax phosphorylation in cells. however, the phosphatase inhibitors alone did not cause stabilization or activation of -h2ax, indicating that proteasome - mediated turnover was a general mechanism to be addressed in further detail. these results provide evidence that -h2ax is degraded by the proteasome and potentially by other mechanisms requiring dephosphorylation of one or more components after dna damage. previous reports have shown that histone h2a is polyubiquitinated on k119, and h2b had shown evidence of ubiquitination on k120. to examine the role of ubiquitin modifications in -h2ax turnover we created a series of modifications intended to model phosphorylation at s139 and the roles of various lysine (k) residues in the c terminus (figure 3(a) and supplementary figure 1). h2ax - v5-h6 was first mutagenized to replace s139 with alanine (a) or glutamic acid (e), to mimic the negative charge of phosphorylation at s139. as predicted, h2ax - s139-v5-h6 localized to chromatin but was moderately less stable than the wild - type h2ax - v5-h6 or a139 mutation products (not shown). cotransfection of h2ax - s139-v5-h6 with a cytosolic form of brca1 lacking a nuclear localization sequence (brca1-11-gfp) resulted in nuclear localization of both proteins and rapid turnover (not shown). for these reasons, we hypothesized that h2ax - s139 was mimicking s139 phosphorylation and recruiting binding of brca1. we then replaced each lysine (k) residue found in the conserved c - terminus of h2ax - e139 with arginine (r) and tested for ubiquitination in vivo (figure 3). k118 and k119 (which align with k119 and k120 of h2a) were mutated individually (figure 3(c), lanes 6 and 7) and in combination (lane 8). k133 and k134 were also mutated individually (figure 3(c), lanes 9 and 10) and in combination (lane 11) to arginine. mutation of k128 to r128 was also prepared as part of this series but had no effect (data not shown). 293 t cells were transfected with wild - type or mutagenized pcdna3-h2ax - v5-h6 in addition to ha - tagged ubiquitin. h6-tagged complexes were purified using nickel - chelated beads, and bound proteins were analyzed by immunoblotting for the presence of ha - tagged h2ax (ub - h2ax). as shown, v5 immunoblotting identified modified and ubiquitinated h2ax only when extracts from cotransfected cells were purified and blotted (figure 3(b)). reprobing these blots with anti - ha antisera revealed that the upper band contained ubiquitin (figure 3(b)). while the major band migrated in a fashion consistent with that of monoubiquitinated h2ax, there were additional bands of immunoreactivity at higher molecular weights that suggested polyubiquitination as well (figure 3(b) arrowheads). h2ax mutated at both k118 and k119 showed no ubiquitination (lane 8), indicating that h2ax is ubiquitinated at either k118 or k119, whereas other point mutations showed levels of ubiquitination similar to wild type. these results show that h2ax - e139 is ubiquitinated at either k118 or k119. to functionally examine the interaction between brca1 and -h2ax, we used antisense morpholino oligonucleotides to knockdown brca1 expression. treatment of cells with brca1 antisense oligonucleotides reduced the amount of brca1 to under 3% of normal levels (figure 4(a), and supplemental data). reduction of brca1 protein resulted in increased levels of -h2ax expression by both immunofluorescence (figure 4(a)) and immunoblotting (figure 4(b)). cells treated with antisense oligos directed at brca1 mrna were transfected with h2ax - v5-h6 and ha - tagged ubiquitin. the epitope - tagged h2ax product was then isolated using nickel - chelated beads and analyzed by immunoblotting for the presence of ha - tagged ubiquitin. cells treated with brca1 antisense oligos had a reduction in the global amount of h2ax ubiquitination detected (figure 4(c)). efforts to examine the role of double - strand break repair on this process are underway but are complicated by the extreme sensitivity of brca1 deficient cells to genotoxic agents. it has been shown previously that brca1 deficiency is associated with g2/m checkpoint and other defects [2932 ]. to examine the relationship between brca1 and genotoxic stress more carefully, we examined cells treated with brca1 antisense oligos for evidence that -h2ax stabilization correlated with replicative defects. we found that early in the response to brca1 knockdown, -h2ax expression was confined to cells in g2/m or late s - phase of the cell cycle (figure 4(d)). most cells with 2n dna, as measured by dapi fluorescence, had normal -h2ax staining patterns. these results suggest that brca1 deficiency is associated with defective clearance of -h2ax from cells following replication and other types of genotoxic stress (figure 5). we propose that brca1 interacts with processive rna pol ii in undamaged cells as part of a role in genomic surveillance (figure 5(a)) [25, 33 ]. following genotoxic stress, phosphorylation of brca1 by atm / atr and potentially by chk1 results in its dissociation from stalled rna pol ii complexes. we propose that an early repair complex forms on dna as a consequence of atm / atr phosphorylation. early targets for atm / atr include phosphorylation of h2ax to form -h2ax [34, 35 ]. -h2ax then serves as a template to aid in the recruitment of early and late components of the repair machinery, including 53bp1 (figure 5(b)). we propose that brca1 is recruited at later times, potentially after break repair has been affected. one target for brca1 recruitment is -h2ax, which is directly or indirectly ubiquitinated on k118 or k119 and degraded through the actions of the 26s proteasome (figure 5(c)). we have shown that brca1 is in a biochemical complex with -h2ax after dna damage. this interaction is dependent on the dna - pk family of kinases (atm and/or atr), as the interaction is disrupted by the inhibitor wortmannin. this data agrees with previous data that brca1 colocalizes with repair proteins and suggests a function for brca1 in the chromatin fraction of nuclei. brca1 becomes part of the brca1-associated surveillance complex (basc), which includes many proteins involved in dna repair, including msh2, msh6, mlh1, atm, blm, and the rad50-mre11-nbs1 protein complex. brca1 and the basc complex are localized at the site of dna damage (nuclear foci). the repair factors are thus at the site of damage where they can perform their particular enzymatic activities and repair the dna. using a mutant from of h2ax (h2ax - e139) designed to mimic phosphorylation at s139 in -h2ax, we performed mutagenesis of several conserved lysine residues in the c - terminal end of h2ax. results from these experiments suggest that ubiquitination is suppressed following mutation of both k118 and k119, but not by mutation of other lysines either alone or in combination. previously reports also showed that brca1 was capable of supporting ubiquitination of h2ax, but those studies were carried out in cell - free reactions in vitro. ubiquitination of k118 and k119 agrees with tryptic peptide data that showed ubiquitination between residues 118 and 127. we propose that a major function of brca1 is to decrease the levels of -h2ax in cells as a mechanism for signaling an end to early events in dna repair. defective production of brca1 in tumor cells would be expected to result in less ordered diminution of the repair signal and lead to problems in progression though g2/m phases of the cell cycle. | following genotoxic stress, the histone h2ax becomes phosphorylated at serine 139 by the atm / atr family of kinases. the tumor suppressor brca1, also phosphorylated by atm / atr kinases, is one of several proteins that colocalize with phospho - h2ax (-h2ax) at sites of active dna repair. both the precise mechanism and the purpose of brca1 recruitment to sites of dna damage are unknown. here we show that brca1 and -h2ax form an acid - stable biochemical complex on chromatin after dna damage. maximal association of brca1 with -h2ax correlates with reduced global -h2ax levels on chromatin late in the repair process. since brca1 is known to have e3 ubiquitin ligase activity in vitro, we examined h2ax for evidence of ubiquitination. we found that h2ax is ubiquitinated at lysines 119 and 119 in vivo and that blockage of 26s proteasome function stabilizes -h2ax levels within cells. when brca1 levels were reduced, ubiquitination of h2ax was also reduced, and the cells retained higher levels of phosphorylated h2ax. these results indicate that brca1 is recruited into stable complexes with -h2ax and that the complex is involved in attenuation of the -h2ax repair signal after dna damage. |
perhaps the most fundamental question to arise from these and other studies is whether tlr activation is critical for both the initiation and perpetuation of autoantibody production. mammalian dna or rna must enter discrete endocytic compartments within the cell in order to interact with intracellular tlrs (such as tlr3, 7, 8, and 9). the only way that this has been achieved experimentally without the use of transfection is through engagement of igg nucleoprotein complexes with the antigen receptor on b cells or fc receptors on dcs (8). furthermore, the most robust responses were observed when the target cells were first primed by exposure to ifn- or cd40 ligand (cd40l) (1, 2), a phenomenon explained in part by the ifn- or cd40l - induced up - regulation of tlr7. from these in vitro studies, one can infer that during immune activation and after the production of antinucleoprotein autoantibodies in other words, when tolerance has already been broken the pathways described in these articles are likely to perpetuate immune activation and autoimmunity in vivo. viglianti. (9) showed that chromatin ingestion can occur through direct binding of chromatin to the bcr on b cells derived from transgenic mice expressing a dna - specific bcr, and that this chromatin ingestion triggers b cell proliferation. if b cell proliferation equates with maturation and autoantibody production (as yet unproven assumptions, as discussed below), then the uptake of nucleoproteins directly through the bcr may initiate autoantibody production. however, as low - affinity antibodies specific for self - dna or rna are thought to be abundant in the peripheral circulation (10), the avidity of interaction between the bcr and nucleoprotein, as well as the downstream signaling responses triggered by this interaction, and/or other environmental influences may be critical in determining whether pathogenic autoantibodies are produced. perhaps exposure to ifn- or other inflammatory cytokines, produced in response to inflammatory proteins released by dying cells (11), are necessary coconspirators that are required to prime the immune system toward exaggerated responses. it is also possible that this process is sustained by defective clearance of apoptotic cells (12) (fig. failure to establish self - tolerance during early b cell development allows the escape of autoreactive b cells, as recently shown in a small number of untreated sle patients (13). if these b cells have sufficient avidity for self - nucleoproteins with repetitive epitopes that cross - link the bcr, the cells could become activated, endocytose the nucleoprotein, and be stimulated through their tlrs (fig (a) a virus infection is sensed by tlrs in plasmacytoid dendritic cells (pdcs) resulting in the production of large concentrations of type i interferon (ifn-/). ifns prime the adaptive immune system to respond to other signals that may include nucleoprotein antigens released from dead and dying cells. virus persistence and/or defective clearance of apoptotic cells might drive chronic, self - perpetuating autoimmunity through uptake of nucleoprotein antigen antibody complexes (fig. (b) defective b cell tolerance leads to capture of nucleoprotein antigens by autoreactive b cells, thereby triggering b cell activation, tlr stimulation, and antigen presentation to t cells. apoptotic cells are abundant in germinal centers, and nucleoprotein antigens may be released at other sites due to abnormal cell death or defective cell clearance. (1) the u1 snrnp binds to the bcr on a b cell or fcr on a dc. (2) (3) the receptor is endocytosed, and the endosome matures into a late endosome / lysosome containing tlr7 and 8. (4) the u - rich rna, initially protected by the smrnp proteins and/or the antibody, now engages the tlr triggering activation of irf and nf-b signal transduction pathways. (5) the protein component dissociates and is degraded by lysosomal enzymes with membrane fusion to vesicles containing mhc class ii. (6) activated transcription factors induce expression of proinflammatory cytokines such as type i ifns, il-12, il-6, and tnf which (7) up - regulate mhc class ii and costimulatory molecule expression. cell activation may also lead to cell proliferation. throughout the decades after the discovery of autoantibodies in patients with lupus and related diseases, interest in these antibodies has waxed and waned. although often dismissed as epiphenomena, the remarkable disease specificity of these autoantibodies, as well as the association between certain autoantibodies and disease manifestations (10), suggest that an understanding of how these antibodies are generated might provide insight into the mechanisms of disease. as autoantibodies in sle patients recognize components of chromatin (such as dsdna, histones, and nucleosomes) and u - rich ribonucleoproteins (such as rnp and sm)all particles that are now known to activate tlrs (10)it can be proposed that selection of these particles for immune attack is based on their intrinsic ability to activate the tlrs the toll hypothesis. this hypothesis would explain why most autoantigens in sle and related diseases are nucleoproteins the nucleic acid component could serve as the adjuvant that stimulates cytokine production and the up - regulation of costimulatory molecules, thus facilitating the presentation of the associated peptides to t cells (fig. sm / rnp is a particularly appropriate model antigen, as the highly conserved domains of sm that make up the hexameric or heptameric rings of the protein (fig. 2) bind to the oligo - u consensus sequence rau5gr (where r is any purine) on u snrnas (for review see reference 14). if the toll hypothesis is correct, other common rnp antigens that are targeted in sle, such as ro (ssa) and la (ssb), should also be tlr agonists. la associates with the 3 termini of a variety of newly synthesized small rnas (including the rna polymerase iii transcripts, ro y rnas, u6, pre - trnas, pre-5s, and 7sl rna, as well as small viral rnas) and, at least in yeast, with polymerase ii transcripts (15). a unifying explanation for the binding of rnps to these nascent transcripts is recognition of a short 3 oligo u tract, uuu - oh. it remains to be experimentally tested whether ro, la, and other ribonucleoprotein antigens that are targeted in sle are preferential activators of tlr7/8. but the large number of possible u - rich regions in rnas of the snrnp family (which includes at least 12 members), of small nucleolar rnas (which number in the hundreds), and of mrnas that contain 3 u - rich regions, suggest that the property of being u, ug, or g rich is itself unlikely to be a sufficient explanation for autoantibody selection through tlr activation. could there be stimulatory sequences within these rnas similar to those described for dna and, more recently, for small interfering rnas (16) ? could one or more of the more than 100 known biochemical modifications of rna, such as methylation or pseudouridylation, explain differences in activation potential ? recently, it was shown that several of the modifications that occur primarily in mammalian, but not in bacterial, rnas protected transfected cells from intracellular tlr stimulation (17). finally, the high abundance of u1 rna (10 copies per nucleus) and the tight binding between the nucleic acid and the core proteins may confer a relative insensitivity to nucleases and the persistence of the particles both outside and inside the cell. if immunostimulatory dna and rna sequences or structures help to account for autoantibody specificity in sle, and very likely in sjogren 's syndrome (18), a prediction is that the dominant nucleoprotein autoantigens in other systemic diseases, such as systemic sclerosis and polymyositis, will also stimulate tlrs. if self nucleic acids are capable of activating tlrs, how does the immune system avoid autoimmune activation of the sort described by lau. (1) and vollmer. the answer likely lies in the intracellular and intraendosomal location of tlr3, 7, 8, and 9, and the presence of highly abundant extra- and intracellular nucleases. serum contains high levels of dnase and rnase activity, which presumably help dissolve nucleic acids that leak out of dead and dying cells, particularly at sites of inflammation. a plethora of nucleases also exist within the cell some that assist in the processing of nucleic acid intermediates and others that appear to degrade foreign or ectopic nucleic acids. type ii dnases function optimally in an acidic environment (such as in endosomes / lysosomes), the dnase1-like (l) family have been variously proposed to act in the nucleus, cytosol, and extracellular space, and several other dnases (such as caspase - activated dnase and endonuclease g) are required for cell - autonomous dna degradation (1921). the inability to degrade dna in dnase ii mice results in high levels of ifn- production, although interestingly the inefficient removal of engulfed endogenous dna stimulates innate immunity through a tlr9-independent pathway (22). extracellular or secretory rnases of the rnase a superfamily have a wide range of activities as demonstrated by differential catalytic activities on substrates such as single or double stranded rna and poly - c or poly - u (for review see reference 23). smrnp itself, as part of the spliceosome, excises intronic rna from pre - mrna by transesterification reactions. many rnases function in processing events at the 5 or 3 region of the rna, whereas others are selectively catalytic for rna / dna hybrids (such as the rnase h family) or double stranded rna (such as the rnase iii superfamily), which include the recently recognized dicer the enzyme required for the generation of small interfering rnas. two enzymes, rnase l and isg20, help to degrade viral rnas and are of particular interest, as they are induced by type i ifns. as mentioned previously, u - rich rnas in smrnp may be relatively resistant to nuclease attack. endosomal location can not be the only explanation for this resistance, as snrnps are assembled in the cytoplasm before their reimport into the nucleus. whether resistance is conferred by high - affinity binding of rnas to proteins or by specific patterns of protein shielding, nucleoprotein stability may be an important requirement for tlr stimulation. (1) reported that immune complexes containing rna stimulated b cell proliferation, but they did not show that stimulation led to b cell maturation and autoantibody production. although lymphocyte proliferation is often followed by maturation, for example, cd8 t cell proliferation in response to self - antigen is followed by death studies on the fate of b cells activated by nucleoprotein complexes are eagerly awaited. in the study by vollmer. (2), the major consequence of tlr7/8 activation by u - rich rnas was the production of ifn-, a cytokine long known to be elevated in patients with sle. although the low ifn response in tlr7 mice suggests a dominant role for this receptor in response to u1 rna, it is relevant to note that both viral and mammalian nucleic acids may stimulate ifns through tlr - independent routes, as recently shown (2527). of particular interest, rna helicases containing caspase - recruiting domains (cards), such as rig-1 and mda5, stimulate ifn production in response to intracellular, double stranded viral rna in a tlr - independent manner (28, 29). regardless of whether tlr engagement is required for the initiation and/or progression of autoantibody production, modulation of certain tlr pathways impacts autoantibody production, at least in murine models of sle. although studies performed to date have used mice with a mixed genetic background, they have consistently shown that both myd88 and tlr9 deficiencies reduced the level of serum autoantibodies (1, 30), suggesting that these pathways are required for high titer autoantibody production. although tlr9 deficiency reduced some anti - dna antibodies, it did not protect mice from glomerulonephritis (30). this could occur either because the lack of tlr9 altered the isotypes or affinity of the anti - dna antibodies (not tested in this study), or because nephritis was caused by a different subset of autoantibodies. since oligonucleotide - based inhibitors of both tlr9 and 7 have been developed (31), their therapeutic application for treatment of sle is an exciting prospect (32). there is currently no unifying hypothesis to explain autoantigen selection in systemic autoimmune disorders. here, we propose that a necessary property for the selection of nucleoprotein autoantigens is their ability to activate intracellular tlrs. according to the toll hypothesis, the nucleic acid component of rnps serves as an adjuvant, and the protein component is processed by the activated apc and presented to t cells (fig. other critical abnormalities that sensitize immune cells and provide a constant source of antigen, or that facilitate uptake of nucleoproteins (fig. nevertheless, at least part of the answer to the 50-yr - old puzzle of autoantibody specificity now seems to be solved. | like the immune response itself, our efforts to understand the rules for self nonself discrimination are constantly evolving. the discovery of pattern recognition receptors the toll - like receptor (tlr) family in particular shifted the emphasis of self nonself recognition from lymphocytes functioning in the adaptive immune system to antigen - presenting cells (apcs) functioning in the innate immune system. two new articles, one in a recent issue (1) and one in this issue (see vollmer. [2 ] on p. 1575), demonstrate that antigen antibody complexes containing rnas activate b lymphocytes and dendritic cells (dcs) through interaction with tlr7 and/or tlr8. from these and other papers, one begins to see how specific types of autoantigens by virtue of their capacity to act as tlr ligands favor autoantibody production. this is known as the toll hypothesis. |
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