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the initial manifestations are neurologic in 40% of patients, hepatic in 40% and psychiatric in 15% of patients. wd is an autosomal recessive condition characterized by inability of the liver to transport and store normally absorbed dietary copper, resulting in abnormal deposition of copper in the basal ganglia, eyes, liver and other tissues. in 1912, kinnier wilson published a description of 12 patients who presented with extrapyramidal motor disease and, on autopsy, demonstrated softening of the lenticular nucleus and cirrhosis of the liver. he further noted that these patients exhibited emotionalism, and 2 of his 12 patients presented with schizophrenic - like psychoses. scheinberg and sternlieb wrote that 1025% of patients with wd initially present with psychiatric symptoms. psychiatric symptoms in wd may range from major depression, mania, and anti - social behavior to psychosis. g. d., a 32-year - old unmarried gentleman born of non - consanguineous parents, presented with personality change and behavioral disturbances since the last 6 months. the behavioral disturbances were in the form of emotional liability, aggressiveness and disinhibition and delusions. he had past history of jaundice at 18 years of age which resolved in 3 months. physical examination at the time of the admission revealed a kf ring obvious on naked eye examination and risus sardonicus. higher mental function examination revealed impaired attention span 7 days of week forward and 2 days of a week backward, and he was able to comprehend two - stage axial commands. he lost three points in orientation to time, two points in orientation to place, five points for calculation, one point in construction and one point in recall. he had impaired abstract thinking and could not explain the meaning of a given proverb. complete blood count (cbc) was normal ; total bilirubin : 0.6 mg% ; direct bilirubin : 0.15 mg / dl ; total protein : 8.2 g / dl ; albumin : 4.8 g / dl ; globulin : 3.4 g / dl ; serum glutamic oxaloacetic transaminase (sgot) : 35 iu ; serum glutamic pyruvic transaminase (sgpt) : 22 iu ; alkaline phosphatase : 84 the 24-hour urinary copper was significantly elevated, i.e. 635.52 g / day (normal 32 - 64 g / day). usg abdomen revealed liver parenchymal disease with minimal free fluid in the abdomen along with splenomegaly. for the severe agitation and restlessness, he was given injection haloperidol 5 mg i m in bid doses for 2 days haloperidol was replaced with quetiapine 25 mg tablet which was increased to bid over the next 2 weeks. after 2 weeks of inpatient treatment with antipsychotics and sedatives, his behavior was controlled. the patient was started on penicillamine 250 mg daily which was gradually increased to 250 mg five times a day supplemented with pyridoxine 40 mg daily. wd is an autosomal recessive disease characterized by inability of the liver to transport and store normally absorbed dietary copper, resulting in abnormal deposition of copper in the basal ganglia, eyes, liver and other tissues. he had emotional lability, disinhibition and was severely agitated, restless with delusional thoughts. since the psychiatric symptoms in wd are generally mild, patients may not present with these symptoms. of the 108 patients, only 9 (8.3%) manifested severe psychiatric symptoms that required admission. among these patients, only one patient developed bipolar disorder that began some months before the onset of neurologic disorder. our patient presented with severe neuropsychiatric symptoms and had tremors on examination which were present since 5 years. a high degree of suspicion and early detection of wd is critical because early initiation of chelation therapy can prevent a catastrophic outcome. | wilson 's disease (wd) is a relatively rare disease of copper metabolism. the diagnosis is often missed initially. the presentation is usually neurologic or hepatic, seen in 40% of patients. psychiatric presentation of wd is reported in only 15% of patients. we present a 32-year - old patient with severe psychiatric manifestations. on examination, he had mild rest and postural tremors and a kf ring was seen. serum ceruloplasmin was low and 24-hour urinary copper was elevated. the patient responded to penicillamine, lorazepam and quetiapine, and is being followed up. |
asthma is a chronic respiratory condition that affects over 7 million united states (us) children. acute episodes are characterized by airway constriction and excess production of mucus and may require medication to restore normal breathing. several different triggers have been found to set off episodes, including cold or dry air, dust, pollen, pollution, physical activity, stress, and cigarette smoke. children are more vulnerable to tobacco smoke than adults because their respiratory and immune systems are not completely developed. as early as 1992, the environmental protection agency reported that environmental tobacco smoke (ets) is causally associated with 1 million episodes and increased severity of symptoms among asthmatic children. in addition, ets is a risk factor for new asthma cases in children. in the us in 1997, figures for childhood illness and death attributed parental smoking with an estimated excess of 1.8 million outpatient visits for asthma and an excess of 14 asthma deaths. a worldwide study conducted in 2004 found that 603,000 deaths were attributable to ets exposure, 28% of which were in children. deseret mutual benefits administrators (dmba) is the health insurer for employees of the church of jesus christ of latter - day saints (lds) and their families, with electronic data for 62,000 individuals. due to religious proscription, those who work for the lds church are required to abstain from alcohol and tobacco use, creating a cohort of children not exposed to parental smoking. non - lds in utah have 1.6x (male) and 2.2x (female) higher rates of smoking - related cancers, compared to their lds counterparts. not everyone who is listed on the membership records of the lds church adheres to the teachings regarding abstinence from smoking ; however, those employed by the church, and therefore under dmba coverage, are required to state they follow the nonsmoking requirement. thus, we would expect to see a gradient of smoking - related disease, with the lowest rates found in the dmba population and the highest rates in the us. the rates for utah, with its large lds population, should be intermediate between the dmba and us rates. this study compares the childhood asthma hospitalization rates of a nonsmoking population (dmba enrollees) to utah and us hospitalization rates to determine the impact of smoking on these rates. it also examines the effect of smoking on utilization of asthma - related outpatient medical services, including emergency department (ed) visits. the dmba insurance claims database was used to estimate childhood hospitalization rates from 19972002 among a nonsmoking population. the dmba was established in 1970 to provide insurance to the employees of the lds church and their families. the dmba is a national insurance claims database that insures approximately 62,000 members a year with nearly equal numbers of males and females. utah residents under the age of eighteen comprise approximately 35% of the database, and analysis was limited to this population. the dmba population has little employment turnover, estimated at less than 5% per year, providing a stable population for analyses. the majority of turnover occurs because of the addition of newborns, the loss of young adults who marry, move out of the parental home, or reach age 26, and individuals who become eligible for medicare. the university of utah 's institutional review board approved this study as low risk. the national hospital discharge survey (nhds) is an annual national probability survey that collects information on characteristics of inpatients discharged from non - federal short - stay hospitals in the united states. the nhds collects data from a sample of approximately 270,000 inpatient records acquired from about 500 hospitals. reported data include number and rate of discharge and average length of stay by age, sex, and geographic region. only first listed diagnoses of international classification of disease, 9th revision, clinical modification (icd-9), are used to define disease. the national ambulatory medical care survey is an annual national survey that is designed to collect data on the use of ambulatory care services in the united states. results are based on a national sample of visits to nonfederally employed office - based physicians who are primarily engaged in direct patient care. specially trained interviewers visit the physicians prior to participation to train them in data collection procedures. data include information about physician diagnoses, medications ordered or provided, patient demographics, and services provided. the national hospital ambulatory medical care survey is a survey designed to collect data on the utilization of ambulatory services in hospital emergency and outpatient departments. findings are based on a national sample of visits to emergency and outpatient departments of noninstitutional and short - stay hospitals, exclusive of federal hospitals, military hospitals, veterans administration hospitals, psychiatric institutions, and long - term care facilities. collected data include patient demographics, physician diagnoses, services and procedures provided, and medication provided. the national health interview survey (nhis) is an annual survey conducted by the centers for disease control and prevention 's national center for health statistics and provides national estimates for a broad range of health measures for children less than 18 years of age. each year, a representative number of households across the united states are selected using a multistage cluster sample design. child health information, utilization of health care services, household composition, and sociodemographic characteristics are ascertained by asking a knowledgeable family member, aged 18 years or older, who resides in the household. two questions are asked to determine the prevalence and frequency of asthma : ever told had asthma, and had an asthma attack in the last 12 months. data for this study covers the years 19972002. the utah indicator - based information system for public health contains data on all hospitalizations from the 49 utah hospitals operating during the study period. billing, medical, and demographic information describing a patient, the services received, and inpatient charges are collected quarterly. hospital discharge diagnosis, based on icd-9 codes was used to identify inpatient hospitalizations for asthma. dmba asthma services prevalence was determined by dividing all individuals with an asthma claim within a calendar year by the population of those < 18 in that year. multiple claims could be generated at a single visit in order to provide for differing points of service. in our data, these claims were associated and counted as a single visit. however, if treatment for asthma was sought more than once during a year, each visit was counted separately. stratifications were made by sex, age (< 5, 511, 1217 years), calendar year, and season (mar may, jun aug, sept ambulatory visits were broken down into physician office visits, outpatient visits, and ed visits in order to establish utilization rates. utilization rates in dmba were compared to the state and national data sources by calculating rate ratios (rr) with 95% confidence intervals (ci). one - way analysis of variance (anova) was conducted using sas, version 9.1, to determine differences in age - specific seasonal asthma cases. the level of significance was set at alpha = 0.05 using a two - tailed test. the average number of health care utilizations for asthma in children was 2.03 per year (95% ci 1.742.34). age- and sex - specific hospital discharge rates for asthma are presented in table 2 for the dmba and comparison populations. a similar trend was seen in all populations, with asthma discharge rates for males 3050% higher than for females. the dmba had the lowest discharge rate for all ages across both sexes, with utah rates intermediate between dmba and us rates. hospitalizations among us children were 5 times higher (95% ci 3.656.84) than among dmba children. hospitalizations among utah children were 3.34 times higher (95% ci 2.444.47) than among dmba children. average length of stay was similar between the dmba and comparison populations, at about three days (data not shown). a similar pattern was seen for visits to ed and outpatient clinics (table 3). relative to children in dmba, emergency department visits were 4.00 (95% ci : 3.504.56) times higher for us children and 1.20 (95% ci : 1.051.37) times higher for utah children. relative to children in dmba, hospital outpatient department visits were 5.56 (95% ci : 4.646.65) times higher for us children. relative to children in dmba, physician 's office visits were 1.65 (95% ci : 1.601.71) times higher for us children. data were not available to make utah to dmba comparison for hospital outpatient or physician 's office visits. as seen in figure 1, utilization of health services for asthma was generally highest for those aged 04 years, and there was no significant variation in utilization by season in this age group (p = 0.19). there was seasonal variation and utilization for older age groups, with summer having the lowest rates for both ages 511 (p = 0.005) and 1217 (p = 0.05). the prevalence of asthma was estimated for dmba as the proportion of children with any claim for asthma - related services (icd-9 493.00493.99), and compared with nhis survey prevalence of asthma for the us (table 4). the prevalence for dmba children was relatively constant across the age groups 04 years, 511 years, and 1217 years, while there was a noticeable increase in prevalence for us children as age increased. the relative prevalence of asthma was 36 times higher for us children compared to dmba children. all measures of asthma service utilization were lower in the dmba pediatric population than in comparison groups in the us and utah. we hypothesized that asthma services utilization would follow the same trend seen for other smoking - associated diseases, with dmba having the lowest rate, utah having an intermediate rate, and the us having the highest rate, based on the prevalence of smoking in these populations. this trend was documented for asthma hospitalizations (3.3-fold and 5.0-fold higher in utah and the us, resp., compared to dmba) and ed visits (20% higher in utah and 4-fold higher in the us). for outpatient visits, data were only available for the dmba and us populations. visits to hospital outpatient departments were 5.6-fold higher for us relative to dmba children, and visits to physician 's offices were 1.7-fold higher for us relative to dmba children. the prevalence ratio for asthma services used, regardless of location of care, was higher for us children than for dmba children, ranging from 3.1 for children age 04 years to 6.4 for children age 1217 years. one of this study 's strengths is that it included males and females in a very stable population, ranging from 017 years of age. those covered under the dmba insurance have a low turnover rate, estimated at less than 5% per year, providing a stable population for analyses. the dmba population was limited to the insured employees and their families, who work for and are members of the church of jesus christ of latter - day saints. members of this religion abstain from alcohol and tobacco use, creating a cohort of children not exposed to parental smoking. this created a unique opportunity to use population - based data to study the impact of environmental tobacco smoke on childhood asthma hospitalization and health care service utilization. while there is no direct information available on smoking for individuals who are covered by the dmba, it is a requirement to abstain from smoking to retain employment. the low lung cancer rates in the dmba population support the premise that it is primarily composed of nonsmokers. additionally, studies have documented rates of smoking - related cancers in lds populations that are about half that seen in the us and about two - thirds that seen in utah [8, 15 ]. the possibility exists that despite the employed parent being required to abstain from tobacco use, a spouse or child could potentially be a smoker and still be covered by the dmba. this would reduce the exposure difference between the dmba and comparison groups, leading to an underestimate of the rr for children living in nonsmoking households and would not change our overall conclusion. the dmba is an insured population, so the use of services could potentially be higher than the general population where not all individuals have insurance coverage. the net effect of this bias would be that the rrs comparing other populations to dmba are an underestimate of the actual amount of services used in the comparison populations. employed populations often have improved health when compared to the general population, specifically because they are healthy enough to be employed. we believe that the health benefits of employment are unlikely to explain the lower childhood asthma rates, as the children themselves are, for the most part, not employed. higher socioeconomic status within the dmba population also does not explain their lower childhood asthma rates, since the majority (70.6%) of the employed individuals ' income is $ 60,000 or less per year. this study shares the limitations of all nonrandomized, observational studies, including the possibility of selection bias and unadjusted confounding. using an insurance claims database is more accurate than voluntary self - reporting of illness, since physicians rely on icd-9-cm codes for reimbursement. diagnostic discharge data were used in this study in order to reflect actual diagnosis rather than claims attempting to rule out asthma, which could have overestimated the use of services. while discharge data are subject to diagnostic inaccuracy, these errors are likely to be distributed randomly through all databases and should not bias our comparisons. comparisons made between the dmba, the state of utah, and the us were based on standard definitions of disease, and there should be no bias associated with the comparisons. comparisons in this study were made between age- and sex - specific groups to minimize confounding due to these factors. akinbami and schoendorf studied national asthma health care utilization in physician offices, hospital eds, and hospital outpatient departments. their reported asthma rates for hospitalization, ed and outpatient visits, and office visits were similar to the us rates we have reported. dmba rates were significantly lower than those reported by akinbami, as well as utah, and us rates (table 3). in a 2004 study conducted by sturm., after adjusting for sex, race, socioeconomic status, and a variety of environmental factors, those who were exposed daily to ets were 1.72 times more likely to have active, diagnosed asthma (defined as self - reported asthma symptoms in children with a previous diagnosis of asthma) and 1.86 times more likely to have experienced wheezing in the last 12 months. the risk of asthma diagnosis and increased asthma symptoms associated with ets illustrates a dose response pattern, with a small but significantly increased risk among those exposed less than once a month and a much larger risk among those exposed daily. fleming found that rates for hospital admissions and asthma episodes were highest during september and lowest during august for children in both age groups 04 and 514. our study found that rates in the dmba for children 511 and 1217 peaked in the fall (september to november), but that rates for the age group 04 were highest in the winter months (november to february). the difference between our study and that of khot and burn for the age group 04 might be attributed to regional differences between utah and great britain. in utah, children without fully developed immune and respiratory systems are likely to be more at risk due to the harsh winter climate. this could lead to a greater frequency of upper respiratory infections and asthma prevalence in the winter for the youngest age group. several studies have sought to address the effects of parental smoking and medical visits for childhood asthma. and jindal and gupta studied the impact of ets on children and found that smoking in the home was significantly associated with an increase of ed visits and hospitalizations [2022 ]. mackay. studied the influence of smoke - free legislation on admissions for childhood asthma. before legislation was implemented, they found admissions increasing at 5.2% per year ; however, after legislation implementation in march 2006, a decrease of admissions of 18.2% per year was found relative to prelegislation rates. wilson. conducted a randomized controlled trial highlighting a reduction in acute asthma medical visits and overall healthcare utilization for children whose parents were assigned to smoking - cessation classes. similar to our study, these studies document that reductions in ets exposure reduce outpatient visits, ed visits, and hospitalizations in children. utilization rates for hospitalization, ed, outpatient department, and physician office visits were significantly lower in the nonsmoking group formed by the dmba population when compared to the us utilization rates for hospitalization and ed visits for utah children were intermediate between dmba and us rates. the comparison of the dmba to state and national rates demonstrates the magnitude of the difference between populations exposed to ets versus those who are not exposed. | risk factors, such as parental smoking, are commonly associated with increased asthma symptoms and hospitalizations of children. deseret mutual benefits administrators (dmba) is the health insurer for employees of the church of jesus christ of latter - day saints and their families. due to religious proscription, employees abstain from alcohol and tobacco use, creating a cohort of children not exposed to parental smoking. calculation of hospitalization rates for dmba, utah, and the us were made in children to compare rates between a nonsmoking population and general populations. compared to dmba, rate ratios for asthma hospitalization and emergency department asthma visits were higher for the us and utah. the incidence of hospital outpatient department and physician office visits was significantly greater for the us population compared to the dmba. this study demonstrates a decreased need for health services used by children not exposed to second - hand smoke. |
inflammation is associated with atherosclerosis as well as with fatty liver disease (fld). both diseases are common in the western world and several studies indicate that there could be a link between both disorders. inflammation causes formation of vascular atheromas and thinning of the plaque 's fibrous cap ; unstable plaques can rupture and cause coronary thrombosis resulting in myocardial infarction. fatty liver disease is considered to be the hepatic manifestation of metabolic diseases, summarized as the metabolic syndrome (nonalcoholic fatty liver disease). severe forms are characterized by an inflammation of the hepatic tissue, induced by inflammatory cell invasion and cytokine production, leading to hepatocyte necrosis and fibrosis. nafld and cvd are inflammatory diseases and a number of common cytokines are involved in the inflammatory process of both diseases [5, 6 ]. a variety of potentially predictive biomarkers have been identified so far (see below) ; some of these might play an active role in the initiation and progression of cvd and might also be linked to nafld, such as adiponectin or interleukin- (il-) 6. alcohol intake is associated with the risk of all - cause mortality in u- or j - shaped manner, primarily through reduction of the risk for coronary heart disease (chd) [8, 9 ]. in several meta - analyses, the lowest risk has been found among those with light - to - moderate alcohol consumption (525 g / day) [10, 11 ]. some studies could demonstrate that moderate alcohol consumption is associated with lower concentrations of circulating inflammatory biomarkers compared to nondrinkers and those with high alcohol consumption [12, 13 ]. thus, it has been suggested that potential beneficial effects of moderate alcohol consumption might be mediated at least in part by lowering the inflammatory burden in different stages of atherosclerotic disease [14, 15 ]. as moderate alcohol consumption seems to have anti - inflammatory effects, one might speculate that it may also reduce the risk of developing nafld. further, nafld has been shown to be associated with an increased risk for cardiovascular events [16, 17 ]. there is an ongoing debate whether nafld is an independent risk factor for cardiovascular disease or a concomitant disease [5, 18 ]. given the link between these two inflammatory entities, further, it might interact with the effect of alcohol intake on markers of inflammation and thus also on the cardiovascular risk. lipoprotein oxidation, especially of low - density lipoprotein (oxldl), is a crucial step in the development of early atherosclerotic lesions. in cohort studies, low serum levels of adiponectin can be found in patients with type 2 diabetes or coronary artery disease. c - reactive protein (crp), an acute phase - protein, is produced by the liver. increased serum levels in noninfectious situations, as measured by high sensitivity assays, fibrinogen levels have been shown to be inversely correlated with alcohol consumption [12, 13 ]. elevated serum levels are thought to reflect early stages of atherosclerotic vascular disease [7, 22 ]. we examined the association between alcohol consumption and prevalence of nafld, their interaction with serum levels of different biomarkers predictive for cardiovascular disease, or its complications in a large cross - sectional population - based study. a subsample of 515 men and women aged 1849 was randomly selected according to the amount of self - reported alcohol consumption from a population - based cross - sectional survey including 2445 individuals in the area of leutkirch in southern germany. this survey was primarily designed to estimate the prevalence of echinococcus multilocularis and internal diseases in leutkirch (emil) study. a standardized interview was performed by trained personnel, including sociodemographic data, medical history, and lifestyle habits, including physical activity, drug history, and alcohol consumption. to estimate alcohol consumption, each subject was interviewed about the amount of beer, wine, and spirits he or she had consumed on the previous workday and over the last weekend. total alcohol intake was calculated by multiplying weekday consumption by five and adding this figure to weekend consumption. this recall method had been validated in a subsample of 899 male participants of the first monica augsburg survey in 1984/85. body height (in m), body weight (in kg), waist circumference, and hip circumference were measured in a standardized manner. body mass index (kg / m), waist - to - hip ratio, and daily alcohol intake were calculated. physical inactivity was defined as being physically active during leisure time neither in summer nor in winter. collection of data on physical activity was based on standardized and validated questions used in previous large epidemiological studies. all subjects gave informed, written consent, and all protocols and collection procedures were approved by the relevant institutional committees. all participants underwent a real - time ultrasonographic examination of the liver for detection of typical signs of fatty liver disease and other pathologies performed by a single investigator (hdi 5000, advanced technology laboratories, philips medical systems, bothell, wa, usa). the diagnosis of a fatty liver was based on the finding of a bright liver tissue signal with fine tightly packed echoes, making it significantly more echogenic compared to the adjacent right kidney [25, 26 ]. fasting blood was collected from the antecubital vein in the supine position with minimal suction and short - term occlusion. plasma was obtained by centrifugation at 3,000 g for 15 min and was stored at 20c within 90 min and then stored at 70c until analysis. liver enzymes were measured the same day photometrically from lithium - heparin plasma on a dimension xl (dade behring, marburg, germany). hdl - cholesterol was measured enzymatically on a dimension rxl (dade behring, marburg, germany). further measurements were performed using a highly sensitive elisa for il-6 (r&d systems, wiesbaden, germany ; range 010 pg / ml ; coefficient of variation (cv) 17.8%), an elisa for e - selectin (r&d systems, wiesbaden, germany ; range 09.69 ng / ml ; cv 7.92%), and also one for oxldl (mercodia, usa ; range 07.6 u / l ; cv 7.99%). a high - sensitivity assay (latex - enhanced nephelometry ; cv 4.26% for apolipoprotein control and 5.85% for rheuma control) was used for crp, also nephelometry for fibrinogen (dade behring, marburg, germany ; cv 5.73%). adiponectin concentrations were measured using an elisa (quantikine, r&d systems, wiesbaden, germany ; 0250 ng / ml range ; cv 8,93%). sociodemographic, clinical, and biochemical characteristics were analyzed in a descriptive manner. adjusted geometric mean for crp and adjusted arithmetic mean for the other inflammatory markers were calculated in categories of daily alcohol intake from multiple linear regression analyses. known or presumed potential confounders including age (four categories), sex, smoking status (current, ex, and never), body mass index (continuous), hdl - cholesterol (continuous), formal education, and physical activity (two categories) were forced into all models. all tests performed were two - sided, and a p value 60 serum levels of ggt as well as of alt showed a slight increase with more alcohol consumption (12 to 36 u / l for ggt p 80%) had severe fld across all categories of alcohol intake. in these subjects, the underlying metabolic syndrome might have been the driving force of hepatic pathophysiology., we found a consistent correlation between ggt serum concentrations and self - reported alcohol intake. serum levels for nondrinkers and subjects with low - to - moderate alcohol consumption were not different, indicating a low likelihood for liver damage. it is possible that elevated serum levels of ggt itself contribute to the increased risk for cardiovascular events in heavy drinkers. recently, ggt has been proposed as an independent cardiovascular risk marker and several mechanisms have been suggested to explain its active role in atherogenesis. as shown in table 3, there is an increase in serum levels in subjects with fld of all proinflammatory biomarkers, namely, fibrinogen, ox - ldl, il-6, crp, and e - selectin. the presence of fld thus constantly increases the inflammatory burden and thereby might be correlated with an increased cardiovascular risk. however, most likely fld does not alter the known association between alcohol consumption and cardiovascular risk. crp, for example, has been described to be associated with alcohol consumption in a j - shaped manner. yet, we were not able to identify a clear j - shaped association in individuals neither with nor without fld. at present, the crp lowering effect of alcohol is still discussed controversially. a meta - analysis of interventional studies by brien. including 5 studies with a total of only 186 participants was not able to find a significant reduction of crp in moderate drinkers. also, association curves for all other biomarkers did not reflect the known j - shaped association. therefore, in this study, the effect of alcohol on inflammatory biomarkers is not conclusive, which might be due, at least in part, to the small sample size in each category. a large number of cardiovascular biomarkers have been suggested to be lowered in subjects with moderate alcohol intake. all other biomarkers chosen in this study (il-6, e - selectin, and oxldl) reflect different stages of vascular inflammation. yet, it is not fully understood how moderate amounts of alcohol reduce cardiovascular mortality, although anti - inflammatory mechanisms might play a role. a meta - analysis of prospective studies on alcohol intake and its effects on biomarkers by brien. showed that high levels of hdl - cholesterol and adiponectin and low levels of fibrinogen correlated best with moderate alcohol intake. these markers might be the key to understand antiatherogenic mechanisms and the role of alcohol in other inflammatory conditions, such as fatty liver disease. finally, the most important limitation of our study is its cross - sectional design not allowing causal inferences. the present study demonstrates that low - to - moderate alcohol consumption is associated with lower prevalence of fld compared to nondrinkers and heavy drinkers. however, in our study, alcohol consumption showed no interaction with inflammatory biomarkers. assuming the same drinking habits, subjects with fld had higher levels of inflammatory biomarkers than those without. | background and aims. local and systemic inflammation represent a major feature of atherosclerotic cardiovascular disease (cvd) and are also linked to nonalcoholic fatty liver disease (nafld). studies indicate that nafld might be a risk factor for cvd whereas low - to - moderate alcohol consumption is associated with lower cardiovascular morbidity and mortality compared to abstainers and heavy drinkers. we hypothesize that fld interacts with the effect of alcohol intake on markers of inflammation, and thus potentially on cardiovascular risk. methods and results. we evaluated alcohol consumption, markers of inflammation and sonographic criteria of fld in 515 subjects, representing a subsample of a cross - sectional population based study (echinococcus multilocularis and internal diseases in leutkirch (emil) study). presence of fld was markedly reduced in subjects drinking 020 g alcohol / d (19%), compared to nondrinkers (35%) and heavy drinkers (3444.9%). serum concentrations of inflammatory markers were substantially higher in subjects with fld. however, presence of fld showed no effect on the association between alcohol consumption and inflammatory biomarkers. conclusions. based on data from a population - based sample, there is no evidence for a link between fld, alcohol consumption, and inflammatory cardiovascular risk markers. however, larger prospective studies are needed to confirm this. |
the prevalence of surgical trauma as a public health hazard in low - resource settings globally has been severely neglected. traditionally, surgery has been viewed as a resource - intense intervention that does not fit in with most effective public health models. however, surgery can provide solutions to diverse yet common crises such as appendicitis or obstructed labour, which constitute a large burden of global morbidity (1). specifically, the world health organisation recognises accidents and injuries as a rising cause of disability and mortality globally (2). burns, road accidents and domestic violence are all common situations where surgical intervention may prevent death or disability (3). of these, the focus on road accidents as a preventable cause of high mortality and disability has become central. estimates go as high as 3000 people dying every day in road accidents worldwide. the very young, the very old, cyclists and pedestrians are the most vulnerable (4). as societies globally become more automated, increasing congestion of motor transport has made this issue of critical concern. understanding the burden injury that could otherwise be treated through simple trauma surgery places on global health systems can not be accurately understood without effective means to monitor its prevalence. furthermore, evaluating the capacity to respond to such a burden also requires effective management of data. as global capacity to provide emergency surgical care develops, mechanisms to monitor and evaluate outcomes also need to be instituted concomitantly. furthermore, using trauma scores to analyse injury data can identify best practices in treatment of injury by identifying where outcomes are improved (1).. however, many now aim to move beyond mere epidemiological analysis towards managing responses to such injuries. developing tools that permit appropriate monitoring and evaluation of burgeoning surgical capacity provides a means to ensure that challenges are identified, and successes are replicated, as demonstrated in iran (5). this study showed that a standard trauma score, developed in north america, had some capacity to evaluate the care given to injury patients in the iranian context, demonstrating where treatment of trauma there was suboptimal to comparative injuries in north america. a larger study, also in iran, demonstrated that a registry system provides valuable information focusing attention on plausible public health intervention for injury prevention, specific to local contexts (6). similar studies have been done in costa rica (7) and a study of occupational injuries from a government database of such incidents was completed in turkey (8). in nigeria, a retrospective study of data from hospital records focused on the specific issue of peripheral artery injuries to better understand prevention (9). however, correlating the epidemiological data of a registry with the clinical practice data from measurement of trauma severity scores requires a further step. in north america, the trauma injury severity score (triss) is the standard tool used in hospitals to evaluate clinical performance in trauma wards. standardised tool, comparisons of performance in order to identify problems or best practices are thus possible (10). however, triss can only be applied reliably in the settings where the index has been validated, in the high - resource settings of north america. although there have been minor studies done to evaluate the adequacy of the scores in low - resource settings (5), more predictive efficacy could be reached by designing an index specific to low - resource settings. trauma surgeons in uganda and canada are already developing a trauma severity index specific to east african contexts (11). this index, the kampala trauma score (kts), was developed to be effective in areas where access to certain indicators may be costly or time - consuming. thus, patient status is evaluated by taking four vital parameters into account : blood pressure, respiratory rate, neurological status and number of serious injuries sustained. these relatively easy measurements give a global overview of a patient 's status, and in principle, can be correlated to the outcome of the case, given interventions appropriate for the patient 's status. analysis of data from uganda showed some degree of success in predicting outcome, although evaluation of its efficacy as a triage tool remains to be seen (12). this data from dar es salaam, tanzania is the first time data has been collected externally using this system of registry and trauma indexing. one round of data has already been evaluated from this collection in dar es salaam (13). the data presented in this round are the preliminary results from the second round of data being collected in dar es salaam, given the changes incorporated into the survey after the first round. research is required to evaluate that data collection is standardised and consistent, in order to improve the ability to monitor and plan trauma management protocols specific to their context effectively (14). regional reliability of trauma data has become an increasing concern in the field, even in the developed world, as populations become more mobile and social systems more integrated (15). previous experiences in data collection from developing country registries have showed variable consistency in the recording of necessary data, requiring efforts to systematise the data available in manageable ways (1). however, research in the developing world faces serious difficulties in capacity (16). the transfer of research capacity from national systems to global actors is an issue, as well as the instability of political agendas to support research in low- and middle - income countries. poor pay of research workers that require them to seek opportunities elsewhere also has an impact on research capacity in the developing world. old models of academic partnerships in and of themselves have contributed to the degeneration of research capacity globally, as local authority has been pre - empted by external control of research agendas (17). with this context in mind, this project thus sought to accomplish three primary goals : to understand the benefits maintaining a trauma registry can bring to the tanzanian context, to describe the efficacy of the kampala trauma score in monitoring injury in dar es salaam, and finally to evaluate the challenges apparent in the maintenance of such a registry for research purposes in this context. the data analysed in this paper comes from an initial two - week collection of the second round of data from tanzania. this round of data collection is derived from a survey instrument that has been modified to respond to collection challenges encountered in the year - long first round. some data in this round had to be dismissed as data collectors used the old tool for approximately twenty cases. the remaining data, involving 125 valid cases, was analysed for data quality concerns, screened for missing values and contradictory evidence. interviews were also conducted with three project managers in the hospital administration to highlight some of the most pressing challenges in trauma registry maintenance. a focus group with emergency department nurses who served as data collectors at the dar es salaam site was also conducted. these data collectors were also responsible for clinical nursing duties for patients entering the emergency department, thus engaging in acute care to ensure stabilisation of patients ' conditions. therefore, it is important to note that they were not primarily researchers steeped in the research questions and methodology, their main interest being to ensure appropriate care of the injured patient. while the data they recorded would be pertinent to routine care, the burden of filling out additional paperwork could come into conflict with their clinical duties. the data collectors were however remunerated a nominal amount for every score evaluation conducted. a visit to a rural clinic at mkuranga, 80 km outside of dar es salaam, where data collection has commenced as part of a national process, provided additional information on the nature of trauma registries in tanzania, through interviewing the project coordinator at that site. initial findings from the second round of data confirms the general understanding that men are preponderantly susceptible to visit the emergency room, involving 97 cases of the 125.. there were some inconsistencies in recording of age, as some collectors recorded year of birth rather than age. in three cases, simply adult was noted, and in four cases, age was not recorded at all. of anecdotal interest, on the initial day of the visiting researcher 's stay at the hospital, all four beds in the department devoted to serious injuries were occupied by young men (suffering head trauma from road accidents). certain parameters required to define the trauma score on the kampala scale did not pose problems. the vast majority of cases came in with normal systolic blood pressure, and this index was not scored in only one instance (see table i). eleven cases came in with poor respiratory rates, five of which were children, one unknown. two cases showed the poorest neurological signs of no response to any stimuli whatsoever, one of those cases being a child. although in interview, collectors seemed clear about what constituted a serious injury anything that required surgical intervention these discrepancies came to light as there was a repeated question on serious injury. one question, question 9, asked the collector identify the number of serious injuries. another question, question 19, asked the clinician to identify the site of serious injuries. table iv shows some of the findings of this internal cross - checking, with figures of note in bold. the table compares the initial reporting of serious injury of the case under question 9 by the data collector, against where they later record as the site of serious injury under question 19. for instance, of the 47 patients who were initially described as having no serious injuries, 8 were later noted to have the head / neck as a site of serious injury, and 26 at the pelvis or extremities. of the 55 who were initially noted to have one site of serious injury, 1 was later described as having no site of serious injury, 3 as multiple sites of injury and 9 had no site recorded at all. this inconsistency in data collection must naturally lead to inaccuracies in calculating the total kts. these problems were compounded by simpler errors, where, for example, clinicians provided a total score for four of the twelve cases which had not been scored on the serious injury scale. the kts was not calculated in eight cases but can be extrapolated from the component scores, all of them at a stable 8. in four cases where there was no serious injury score, the data collector nonetheless provided a kts score, all of them a stable 8. besides the problems with actual scoring, there were other points to note on the data collection (see table v). in three cases of the total, otherwise, 58 cases were caused by road accidents, 39 were caused by a fall, two were stabs or cuts, two were blunt force and 21 were from other causes. however, when prompted, 14 of the cases in this latter category were described as assaults, rather than by the specific nature of the assault described in the earlier categories. given the preponderance of road accidents, the survey asked for further detail regarding the circumstances of these injuries (see table vi). of the 58 road accidents, the highest proportion was those involving vehicles and pedestrians as counterparts 31 out of the 55 of the cases where the counterparts were noted by data collectors in the survey. important information regarding seatbelt use (or helmet use in the case of motorcycle or bicycle accidents) was quite well recorded where relevant (see table vii). gathering this data is essential to enhance monitoring and implementation of public health initiatives. the most critical lack of information comes from the two - week followup (see table viii). of the 125 cases, only 31 had information on the follow - up, of which 22 remained discharged, two had died and the remainder were still noted as remaining in hospital. of the 39 cases originally noted as having been admitted to the hospital, two - week follow - up was missing for 30 of them. interestingly, of the two who died, one had no kts score noted and the other had come in scored as a stable 9. firstly, the ability to monitor data in an emergency department provides valuable insight into the nature of injuries that are prevalent in an area. as such, registries may prove to be an effective tool for public health interventions, in their capacity to direct resources towards addressing the most serious concerns in preventing accident and injury. the data, although preliminary, provides an overview of the injuries that place the most burden on an orthopaedic emergency room in dar es salaam. clearly, mimicking global trends, road accidents are a major concern in the region. there is also particular concern for the health status of young men. while this statistic may be due to the enhanced risk - taking behaviours of men, it is also plausible that women in dar es salaam are less likely to seek formal care for injuries than men, thus presenting less often to the emergency room secondly, the leverage tool the registry provides in order to launch public health interventions underscores its importance. this data can be used both by national authorities in tanzania, as well as regionally and more globally, to address pressing issues of road safety and accident awareness. although human resources are insufficient to devote to this work, the interviews with hospital administration in dar es salaam underlined the necessity of bringing this data to government authorities to launch a campaign to limit the injury burden from road accidents. thirdly, the process of launching and maintaining the registry at a national hospital in a low - resource setting promotes the development of capacity to engage in research in these contexts, which is direly needed in order to make sure public health dollars are spent effectively. locally - led research ensures that funds are not misdirected to concerns that are externally determined. while these initiatives bring to light global health concerns that need redress, they also enhance global health equity in acknowledging the expertise of researchers active in low - resource settings. the hospital administration in dar es salaam shows remarkable commitment to the research process with little public support, fuelled by the energy of a few individuals. overall, the relative completeness of the components of the kampala trauma score collection shows that at least the three initial parameters (blood pressure, respiratory rate, and neurological status) are efficient indicators to collect data in clinical contexts in tanzania. whether they adequately describe patient outcomes remains to be seen, although the analysis done on the more extensive ugandan data suggests that as the tanzanian data is more rigorously collected and analysed, there will be predictive capacity (12). however, the results for the serious injury score in this preliminary tanzanian data, for example, show some of the most pressing concerns in data collection. the interviews provided some nuance to the questions where the most critical lapses occurred. the project managers noted that inadequate support for research capacity requires that nurses undertake the work. the nurses in the emergency room, in return, underlined that balancing the needs of the researcher and clinician in a busy emergency room was difficult, with the research needs suffering. this reality was particularly highlighted at the rural clinic, where the data entries (not analysed here) were much more nuanced and detailed, given the many fewer visits at mkuranga. while some of the difficulties could be ascribed to the fact that this data collection occurred early on in the round of data collection, it must be noted that the hospital had conducted one full year s round of data collection previously. introducing new data collectors to the process can naturally cause difficulties, but providing immediate feedback once these challenges have been identified early on is necessary to ensure usable data arises in the long - term. in the meantime, a stopgap measure of cross - checking data with hospital records themselves may be instituted. however, devoting human resources to this task is also an untenable drain on clinical work in the hospital. for future perspectives, the two most important areas where further improvement of data collection needs to take place are in scoring serious injury, and in recording outcomes in the two - week follow - up. adequate recording of this component is essential, as an accurate calculation of the kts is dependent on it. the former can be a challenge given the possibilities of rapid clinical evolution in emergency settings. however, refining the design of the survey in this regard may prove useful, requiring clinicians to list only the sites where intervention was required. the data analysers can then total up this elementary information to assign a serious injury score themselves, ensuring that there is no repetition of the same data in the collection. in this way, the analysers can also do the work of calculating the total kts, further relieving the burden from the data collectors so they are required only to gather the most elementary components of the data. furthermore, clear definitions of what constitutes a serious injury need to be refined further, taking into account that surgical intervention alone can not be the most reliable marker of gravity of trauma. it is conceivable that there is not even a consensus amongst data collectors on what constitutes a surgical intervention itself. sustaining the collection of outcome data is critical to harness the entire potential of a trauma registry, in order to monitor the effectiveness of treatment in each setting. in this consideration, the lack of capacity to undertake a two - week follow - up, even as minimally required by this survey instrument, was limited with the resources available to the hospital. even though most data collected by the survey instrument was also independently entered into the hospital records, there was little time to cross - check this data with hospital records, and certainly two - week follow - up was often impossible by the emergency nurses even if the patient remained in hospital. rather than research design, this problem is a more structural issue in low - resource settings of limited research capacity. clearly, addressing this difficulty can not be resolved within one project alone. in the long - term, the registry would ideally be integrated into routine care in the emergency department across east africa, perhaps incorporated into regular charting activities in order to monitor the predicted outcomes and the course of treatment of trauma patients. however, much needs to be done to verify the validity of the tool until then. the establishment of research partnerships between highresource settings and lower - resource contexts can be helpful, only if there is a transfer of both financial and human resources to local actors so that research capacity within the society is enhanced. control of research agendas and projects needs also to remain within the developing world. firstly, the ability to monitor data in an emergency department provides valuable insight into the nature of injuries that are prevalent in an area. as such, registries may prove to be an effective tool for public health interventions, in their capacity to direct resources towards addressing the most serious concerns in preventing accident and injury. the data, although preliminary, provides an overview of the injuries that place the most burden on an orthopaedic emergency room in dar es salaam. clearly, mimicking global trends, road accidents are a major concern in the region. there is also particular concern for the health status of young men. while this statistic may be due to the enhanced risk - taking behaviours of men, it is also plausible that women in dar es salaam are less likely to seek formal care for injuries than men, thus presenting less often to the emergency room secondly, the leverage tool the registry provides in order to launch public health interventions underscores its importance. this data can be used both by national authorities in tanzania, as well as regionally and more globally, to address pressing issues of road safety and accident awareness. although human resources are insufficient to devote to this work, the interviews with hospital administration in dar es salaam underlined the necessity of bringing this data to government authorities to launch a campaign to limit the injury burden from road accidents. thirdly, the process of launching and maintaining the registry at a national hospital in a low - resource setting promotes the development of capacity to engage in research in these contexts, which is direly needed in order to make sure public health dollars are spent effectively. locally - led research ensures that funds are not misdirected to concerns that are externally determined. while these initiatives bring to light global health concerns that need redress, they also enhance global health equity in acknowledging the expertise of researchers active in low - resource settings. the hospital administration in dar es salaam shows remarkable commitment to the research process with little public support, fuelled by the energy of a few individuals. overall, the relative completeness of the components of the kampala trauma score collection shows that at least the three initial parameters (blood pressure, respiratory rate, and neurological status) are efficient indicators to collect data in clinical contexts in tanzania. whether they adequately describe patient outcomes remains to be seen, although the analysis done on the more extensive ugandan data suggests that as the tanzanian data is more rigorously collected and analysed, there will be predictive capacity (12). however, the results for the serious injury score in this preliminary tanzanian data, for example, show some of the most pressing concerns in data collection. the interviews provided some nuance to the questions where the most critical lapses occurred. the project managers noted that inadequate support for research capacity requires that nurses undertake the work. the nurses in the emergency room, in return, underlined that balancing the needs of the researcher and clinician in a busy emergency room was difficult, with the research needs suffering. this reality was particularly highlighted at the rural clinic, where the data entries (not analysed here) were much more nuanced and detailed, given the many fewer visits at mkuranga. while some of the difficulties could be ascribed to the fact that this data collection occurred early on in the round of data collection, it must be noted that the hospital had conducted one full year s round of data collection previously. introducing new data collectors to the process can naturally cause difficulties, but providing immediate feedback once these challenges have been identified early on is necessary to ensure usable data arises in the long - term. in the meantime, a stopgap measure of cross - checking data with hospital records themselves may be instituted. however, devoting human resources to this task is also an untenable drain on clinical work in the hospital. for future perspectives, the two most important areas where further improvement of data collection needs to take place are in scoring serious injury, and in recording outcomes in the two - week follow - up. adequate recording of this component is essential, as an accurate calculation of the kts is dependent on it. the former can be a challenge given the possibilities of rapid clinical evolution in emergency settings. however, refining the design of the survey in this regard may prove useful, requiring clinicians to list only the sites where intervention was required. the data analysers can then total up this elementary information to assign a serious injury score themselves, ensuring that there is no repetition of the same data in the collection. in this way, the analysers can also do the work of calculating the total kts, further relieving the burden from the data collectors so they are required only to gather the most elementary components of the data. furthermore, clear definitions of what constitutes a serious injury need to be refined further, taking into account that surgical intervention alone can not be the most reliable marker of gravity of trauma. it is conceivable that there is not even a consensus amongst data collectors on what constitutes a surgical intervention itself. sustaining the collection of outcome data is critical to harness the entire potential of a trauma registry, in order to monitor the effectiveness of treatment in each setting. in this consideration, the lack of capacity to undertake a two - week follow - up, even as minimally required by this survey instrument, was limited with the resources available to the hospital. even though most data collected by the survey instrument was also independently entered into the hospital records, there was little time to cross - check this data with hospital records, and certainly two - week follow - up was often impossible by the emergency nurses even if the patient remained in hospital. rather than research design, this problem is a more structural issue in low - resource settings of limited research capacity. clearly, addressing this difficulty can not be resolved within one project alone. in the long - term, the registry would ideally be integrated into routine care in the emergency department across east africa, perhaps incorporated into regular charting activities in order to monitor the predicted outcomes and the course of treatment of trauma patients. however, much needs to be done to verify the validity of the tool until then. the establishment of research partnerships between highresource settings and lower - resource contexts can be helpful, only if there is a transfer of both financial and human resources to local actors so that research capacity within the society is enhanced. control of research agendas and projects needs also to remain within the developing world. the current process of data collection in tanzania provides very interesting descriptive data on the nature of injury in a particular setting, which provides evidence upon which to build public health interventions. however, to improve clinical outcomes, further refinements in the data collection process need to be instituted through modifications to the survey tool, and particularly, further resource allocation to bolster clinical research capacity in emergency rooms in the country. once this capacity to monitor outcomes is developed, further research into the correlation of patient status and outcome, depending on interventions used, needs to be undertaken in order to develop an analysis of the clinical capacity to respond to trauma in this context. the ultimate goal is, after all, to refine practice to improve patient outcome. especially given the increasingly high burden injuries are placing on global health systems given the advent of motorised traffic worldwide, this work is becoming imperative to protect those most vulnerable to these accidents. however, the process requires a solid base of evidence upon which to design both treatment and prevention interventions. these challenges can not be overcome unless there is serious investment in clinical research in low- and middle - income countries. | the prevalence of surgical trauma as a global public health hazard has been severely neglected. trauma surgeons in uganda and canada have developed the kampala trauma score (kts), a trauma severity index specific to east african contexts. hospitals in tanzania have begun to use this tool to measure their own trauma management protocols in order to measure the validity of this index regionally. this study sought to enhance analysis of data collected through the kts, by highlighting the efficacy and the lacunae of this registry through evaluation of the data quality of one ongoing round of data collection at an orthopaedic emergency room in dar es salaam, tanzania. the data was screened for missing values that would have impact on prediction of clinical evolution and also analysed for contradictory evidence. interviews were conducted with data collectors on the main challenges involved in data gathering and analysis for this project. analysis of the initial round of data collection confirms road accidents cause the most trauma in dar es salaam, with pedestrians being particularly vulnerable. however, critical sources of information such as serious injury scores and two - week followup were inconsistently recorded, thereby limiting outcome measurement. the lack of research resources, both financial and human, had a major impact on the ability to sustain the data collection. while the results of this study demonstrate the public health value of having a mechanism to record trauma, research capacity must be supported in low - resource settings in order to enhance clinical care to accident and injury patients. |
when the combined oral contraceptive pill (coc) was introduced over six decades ago,1,2 it was designed to be taken in a regimen that mimicked the normal menstrual cycle. this requires active pills to be taken for 21 days followed by 7 days without hormones, resulting in a withdrawal bleed. it has been common practice for many years to prescribe modified pill - taking regimens if unwanted symptoms such as withdrawal headaches3 or painful menstruation4,5 occur during pill - free days. these modified regimens entail taking active pills for more than 21 days and/or shortening the hormone - free interval. such regimens are commonly termed tricyling, extended, or tailored use of the pill. more recently, interest has focused on tailored or extended coc regimens for reasons of individual preference.6 evidence suggests that many western european women would prefer either amenorrhea or a longer interval between periods.612 more than 90% of health care providers also support extended pill use.13,14 in a previous study where pills were prescribed for 84 days followed by a 6-day, pill - free interval,12 82% of the participating women welcomed having fewer periods and many found the regimen easier to follow. the clinical effectiveness unit of the faculty of sexual and reproductive health, which publishes guidelines for recommended standards in contraception provision, endorses the prescription of extended coc use in its most recent coc prescribing guideline,15 although cautions this as an off - license use of the product.16 whilst extended use is viewed as acceptable, there is no clear consensus over which of the many different extended regimes in use is preferable. it is also unclear what knowledge practitioners hold about extended pill regimes, to what extent these are being used in routine practice, and which various regimens are being used most frequently. in the uk, the latter are also responsible for training doctors and nurses in contraceptive care, so play a key role in promoting innovation, service improvement, and delivering gold standard17 contraceptive care. we wanted to ascertain whether extended use was common or routine practice in these specialist settings, and whether there was significant variation in prescribing characteristics across different clinics. to explore these questions, we selected three specialist contraceptive services out of 15 faculty of sexual and reproductive health registered services providing training in london which are expected to be early adopters of innovation in practice, namely, counseling women about extended use of oral contraception. we used purposive sampling (based on known service characteristics) with the intention of revealing a range of clinical practice across specialist services, rather than aiming for a representative sample. we selected three contrasting specialist contraception services in london which had varying policies on extended coc use : the margaret pyke centre, chosen immediately following the clinic s involvement in a study of extended coc use ; the enfield clinic, where a guideline on extended pill use had been introduced in association with staff training (2010) ; and king s college hospital, which is an example of a newly developed fully integrated and forward - looking service, but where extended use of the coc has not been formally introduced. in april 2012, we invited all doctors and nurses in the three services who prescribe, counsel, or provide the coc to complete an adapted18 online survey (https://docs.google.com/spreadsheet/viewform?formkey=ddhtrnuwcmtvs0xxytf5q2ezowzhtle6mq). the link was distributed through a designated staff member at each of the three locations. the questionnaire was pilot - tested in the study of gerschultz.18 the average intraclass correlation was 0.69 (range 0.301.00), showing good reliability. we asked about frequency of coc prescription, the reasons and conditions for which extended use would be considered, their preferred regimens, and any concerns expressed by patients or staff regarding extended use. we excluded 24/4 or 26/2 regimens (24 or 26 consecutive pill days followed by 4 or 2 pill - free days) as none were available at any of the three clinics at the time of the survey. we present descriptive analyses of knowledge and provision of extended pill use, and the relationship between participant characteristics and prescribing patterns. odds ratios were calculated to estimate differences between clinics in providing information to patients about the extended use. all analyses were performed using stata / se 12 (statacorp, college station, tx, usa). a total of 67/105 (64%) doctors and nurses completed the survey. out of these, nine of the 38 staff who did not respond were locum doctors, and six respondents could not comment on extended use in the survey because, having come from a stand - alone genito - urinary medicine service, they had not been trained to prescribe oral contraceptives. of those who provided coc, 84% had ever provided information to women about extended use. however, only 29% offered this information to more than half of their patients and this proportion varied ten - fold across the three clinics (p < 0.001). there was no difference between behavior of doctors and nurses, but frequent counseling about extended coc use was more likely to be provided by younger clinic staff and in the clinic that had developed specific local guidance. no other factors, such as age, ethnicity, or gender of health care professional were significantly different between those who prescribed extended use compared with those who did not (see table 1). these included 62% of clinicians who would prescribe if patients want to miss a bleeding because of a holiday, examination, or for cultural reasons (for patient preference, figure 1). there was likely to be an overlap between giving patient preference as a reason for prescribing and all the other reasons given in figure 1. patients seem to like the option of more than one way to take pills especially when going on holiday, travelling to a hot country, or taking exams and removing the period pain and pmt symptoms. there were no significant differences in the reasons given between the different clinics, and so data from the three clinics were combined. a wide range of different regimens was prescribed, of which the most common was taking three pill packets together followed by a pill - free interval, or tricycling (figure 2). however, there was variation in recommended length of the pill - free interval following the 63 consecutive days of pill - taking. twenty survey participants advised a 3-day, pill - free interval (63/3 cycle) most commonly, 14 recommended a longer pill - free break of 7 days, and three recommended a 4-day or 5-day break, while 12 survey participants did not specify a recommended length of break. the 63/3 regimen was regarded as simple to explain to patients, with the added benefit of greater contraceptive efficacy by shortening the hormone - free interval. four of five respondents stated that the lack of a monthly bleed was the most common concern raised by their patients, followed by concerns about fertility (41%) and breakthrough bleeding (38%). overall, 34% of respondents felt more comfortable prescribing the standard 21/7 regimen for the coc, mostly because of lack of familiarity with other regimens. there was a desire for more evidence on prescribing recommendations, including the long - term effects of extended coc use, and for better information leaflets for users. a total of 67/105 (64%) doctors and nurses completed the survey. out of these, nine of the 38 staff who did not respond were locum doctors, and six respondents could not comment on extended use in the survey because, having come from a stand - alone genito - urinary medicine service, they had not been trained to prescribe oral contraceptives. of those who provided coc, 84% had ever provided information to women about extended use. however, only 29% offered this information to more than half of their patients and this proportion varied ten - fold across the three clinics (p < 0.001). there was no difference between behavior of doctors and nurses, but frequent counseling about extended coc use was more likely to be provided by younger clinic staff and in the clinic that had developed specific local guidance. no other factors, such as age, ethnicity, or gender of health care professional were significantly different between those who prescribed extended use compared with those who did not (see table 1). these included 62% of clinicians who would prescribe if patients want to miss a bleeding because of a holiday, examination, or for cultural reasons (for patient preference, figure 1). there was likely to be an overlap between giving patient preference as a reason for prescribing and all the other reasons given in figure 1. patients seem to like the option of more than one way to take pills especially when going on holiday, travelling to a hot country, or taking exams and removing the period pain and pmt symptoms. there were no significant differences in the reasons given between the different clinics, and so data from the three clinics were combined. a wide range of different regimens was prescribed, of which the most common was taking three pill packets together followed by a pill - free interval, or tricycling (figure 2). however, there was variation in recommended length of the pill - free interval following the 63 consecutive days of pill - taking. twenty survey participants advised a 3-day, pill - free interval (63/3 cycle) most commonly, 14 recommended a longer pill - free break of 7 days, and three recommended a 4-day or 5-day break, while 12 survey participants did not specify a recommended length of break. the 63/3 regimen was regarded as simple to explain to patients, with the added benefit of greater contraceptive efficacy by shortening the hormone - free interval. four of five respondents stated that the lack of a monthly bleed was the most common concern raised by their patients, followed by concerns about fertility (41%) and breakthrough bleeding (38%). overall, 34% of respondents felt more comfortable prescribing the standard 21/7 regimen for the coc, mostly because of lack of familiarity with other regimens. there was a desire for more evidence on prescribing recommendations, including the long - term effects of extended coc use, and for better information leaflets for users. our findings show that routine use of extended coc regimens is generally low, but highly variable between settings even in the context of specialist contraceptive services. however, reasons for prescribing extended use were more uniform across the three sites and respondents expressed a desire for more knowledge and guidelines about extended use. development of local clinic guidance does seem to have had a positive influence on extended coc prescribing in this context, although it is well known that guidelines alone often have little impact on practice.19 this was a small study of london - based clinicians that limits the generalizability of our findings. the response rate was lower than expected at 64% (which partly reflects non - response from locum doctors) and varied across settings, which limits the potential for cross - site comparisons especially in view of the small sample size. furthermore, some clinical staff working in fully integrated services but previously working for a genito - urinary medicine service had not had the training to provide contraceptives. despite evidence from other studies that an increasing number of women would prefer to menstruate less frequently,6 most clinicians believe that the commonest concern among patients using extended cycles is that it is unhealthy not to have a monthly period. in a recently completed randomized trial of extended versus standard use of an coc containing 0.03 mg ethinylestradiol and 0.15 mg levonorgestrel,20 we showed that tailored pill use suited some women very well and was an acceptable alternative to the standard pill. qualitative interviews conducted during the trial21 showed that reduced bleeding on extended use clearly suited some women very well, while others disliked the unpredictability of bleeding. to our knowledge, the only study that has analyzed clinicians attitude to withdrawal bleeding and manipulating bleeding patterns in order to reduce pain or inconvenience associated with menses in the uk was conducted in 1977.12 overall, the respondents in our survey appeared to be more enthusiastic about extended regimens than in 1977 when half of the 24 doctors and nine nurses taking part in a trial of extended pill use preferred to prescribe the standard 21/7 pill regimen, but change has clearly not been rapid. there are now several published trials and a cochrane review providing evidence in support of extended or tailored pill use, and this has recently been backed in the uk by national guidance from the faculty of sexual and reproductive healthcare. the cochrane review of eight randomized trials showed similar outcomes with respect to participant satisfaction, contraceptive efficacy, and discontinuation rates for women taking a variety of extended regimens compared with traditional 21/7 regimes of coc use.22 studies also support the safety of extended use.15,22,23 regimens supported by the uk faculty of sexual and reproductive healthcare include tricycling (63/7), shortened pill - free interval (21/4, or 63/4) extended use (21 +) with a shortened (4 days) or regular (7 days) pill - free interval, of which just the 21/7 regimen is licensed in the uk. anecdotally, primary care providers are more cautious about prescribing non - licensed contraceptive regimens, but specialist contraceptive services are well placed to lead innovation in clinical practice. to build on evidence from randomized controlled trials, we need health services or implementation research to show how guidance on extended coc use can be incorporated successfully into routine practice in busy clinics. building on the findings of this targeted survey, we are now evaluating methods for routine implementation of extended pill guidance, including tricycling, and tailored and standard use, for all women requesting coc. in conclusion, despite growing evidence backed by national guidance, routine provision of information about extended coc use is very variable even within specialist innovative contraceptive services. targeted training, use of service guidelines, and implementation research will be needed to extend patient choice of different coc regimens and increase the pace of change in clinical practice. | backgroundthe purpose of this study was to determine attitudes to, and provision of, extended regimens for taking the combined oral contraceptive pill (coc) by specialist contraception practitioners from three contrasting specialist contraception services in london.methodsan online cross - sectional survey was administered to all doctors and nurses, who counsel, provide, or prescribe the oral contraceptive pill at each clinic.resultsa total of 105 clinicians received the questionnaire and 67 (64%) responded. only one of three clinics initiated and maintained guidelines for extended coc use. in that service, 60% of staff prescribing coc advised more than 50% of patients regarding alternative coc regimens. in the other two services, this was discussed with 20% and 6% of patients, respectively (p < 0.001). the reasons for prescribing extended use included cyclic headaches, menorrhagia, patient request, menstrual - related cramps, and endometriosis, and did not differ between the three different settings. the most common extended regimens were 63 pills or continuous use until bleeding occurs, followed by a hormone - free interval. concerns highlighted by providers and patients were unhealthy not to have a monthly bleed, future fertility, and breakthrough bleeding. such comments highlight the need for further information for providers and patients.conclusionthere is growing evidence, backed by national guidance, about extended coc use, but routine provision of this information is patchy and varies ten - fold, even within specialist family planning services. targeted training, use of service guidelines, and implementation research will be needed to extend patient choice of different coc regimens and change clinical practice. |
the implantable collamer lens (icl) is a flexible, posterior chamber phakic intraocular lens. it is implanted in the ciliary sulcus and is vaulted to avoid contact with the crystalline lens. icl implantation is an effective option for the treatment of refractive errors, offering an optical quality superior to that of corneal refractive surgeries. according to data from the icl in treatment of myopia study group, icl implantation is a safe and effective option for patients with moderate to high myopia. the icl 's designs and materials were refined in several clinical studies through a series of prototypes. recent advances in ophthalmic anterior segment imaging such as scheimpflug photography, very high - frequency ultrasonography, and anterior segment optical coherence tomography (as - oct) have been used to evaluate the position and vault of posterior chamber phakic intraocular lens and their dynamics with intraocular changes [46 ]. ultrasound biomicroscopy (ubm) is a high - frequency ultrasound technology that can provide in vivo high resolution (near microscopic resolution) cross - sectional images of anterior segment and objective quantitative measurements of anterior segment parameters in living patients [7, 8 ]. it provides a unique tool to noninvasively evaluate the relations of these implants within the posterior chamber and helps to analyze the mechanisms of crystalline lens and iris complications. however, it is a contact technique that requires topical anesthesia and an immersion bath with a coupling medium with a risk of infection or corneal injury. noncontact imaging devices including as - oct and scheimpflug photography are fast and easy for the examiner and patient and are especially better for examining patients in early postoperative period. however, these devices have an important limitation that they can not measure any object behind opaque structure. on the other hand, ubm can obtain clear images through opaque structures including the iris and allows visualization of the ciliary sulcus. the purpose is to evaluate the anterior segment, the anatomical position of icl, and its relationship to adjacent ocular structures using ubm. 142 eyes of 93 patients implanted with spherical visian icl (model v4 staar surgical ag, nidau, switzerland) for correction of myopia were enrolled in this study. preoperative evaluation of the patients was done using pentacam (oculus) to measure white to white diameter (w - w), keratometric (k) readings, and internal anterior chamber depth (acd) from the corneal endothelium to the crystalline lens. although ubm is the most accurate method for preoperative determination of w - w, this requires a 40 mhz machine that allows full sulcus to sulcus measurement in one image. unfortunately, the machine used in this study is a 50 mhz one that provides better resolution but smaller angular field. pentacam was used to measure w - w as it is more objective than caliper measurement and the icl diameter was ordered accordingly. topical ocular hypotensive was used in the early postoperative period if the intraocular pressure was elevated. postoperative clinical examination included slit - lamp biomicroscopic examination of the anterior segment to evaluate the icl position, patency of iridotomies, iris configuration, and clarity of the crystalline lens. other examinations included measurement of the intraocular pressure (iop), postoperative refraction, and uncorrected visual acuity (ucva). ubm study was done for all patients in the ophthalmology department of minia university hospital using immersion technique with paradigm ubm six months after icl implantation between march 2010 and january 2015. ubm examination technique includedaxial scanning through the center of the cornea, anterior chamber, center of the pupil, icl, and anterior lens capsule (figure 1),radial scanning in the four quadrants to image the angle of the anterior chamber, iris, posterior chamber, icl haptics, and ciliary body (figure 2). axial scanning through the center of the cornea, anterior chamber, center of the pupil, icl, and anterior lens capsule (figure 1), radial scanning in the four quadrants to image the angle of the anterior chamber, iris, posterior chamber, icl haptics, and ciliary body (figure 2). ubm examination was done under the same ordinary room lighting condition and while the patient was fixating on the ceiling to alleviate the effect of illumination and accommodation on the anatomical relations and measurements. the ubm machine calibers were used to measure the minimum central distance between the icl and anterior lens capsule (vault) and the vertical central distance between the corneal endothelium and the icl (e - icl). the relation between icl and the posterior surface of the iris and the position of icl haptics together with the overall iris configuration were evaluated. scans were taken in upper nasal, upper temporal, lower nasal, and lower temporal quadrants where the haptics of icl were suspected to rest. this study was approved by the local ethics committee and adhered to the tenets of the declaration of helsinki. all patients agreed to be enrolled in the study following a thorough explanation of the purpose of the study and the methodology used. data were analyzed using spss version 20.0 for windows (statistical package for the social sciences). quantitative data were presented by mean and standard deviation, while qualitative data were presented by frequency distribution. kolmogorov - smirnov test was applied for all variables and resulted in nonsignificant outcomes indicating the normality of data distribution. the probability of less than 0.05 was used as a cut - off point for all significant tests. the mean preoperative spherical error of refraction was 14.1 4.4 diopters (d) and the mean cylindrical error was 2.3 1.2 d. the mean white to white diameter was 11.6 0.6 mm (range 11 to 13 mm). keratometric readings ranged from 39 to 48.25 d with a mean of 43.2 1.9 d for k1 and from 40 to 51.6 d with a mean of 44.6 2.1 d for k2. the mean central corneal thickness was 519.043 m and the mean internal acd was 3200 0.27 m. the mean power of implanted icl was 15.5 3.7 d and the mean implant diameter was 12.6 0.5 mm (table 2). the mean icl vault was 376 105 m (range 192 to 402 m). the mean e - icl was 2826 331 m (range 2159 to 3079 m). the lens haptics could be imaged resting in the ciliary sulcus (figure 2) in 112 eyes (78.87%) and at least one haptic rested on the lens periphery and zonules in 30 eyes (21.12%). the overall iris configuration (figure 3) was flat in 89 eyes (62.76%) with central anterior convexity in at least one quadrant in 41 eyes (28.87%) and peripheral iris bombe in at least one quadrant in 12 eyes (8.45%). contact between icl and the posterior surface of the iris was present in all eyes (figure 4). this contact was limited to the central third of the iris in 78 eyes and extended more peripherally in 64 eyes. one eye developed persistent increase in the iop that necessitated the use of topical antiglaucoma medications. on ubm imaging there was marked forward displacement of the optic and optic haptic junction (figure 5) with very narrow angle. the vault was less than 200 m while in the third one the vault was 264 m. there was a high statistically significant strong correlation between internal acd and the summation of e - icl and vault (p < 0.001, r = 0.78). there was a high statistically significant fair correlation between vault and the preoperative internal acd (p = 0.001, r = 0.28) and a statistically significant fair correlation between vault and preoperative spherical error (p = 0.01, r = 0.25). nonsignificant week correlation was found between vault and w - w (p = 0.8, r = 0.02). in this study the 50 mhz paradigm ubm was used for in vivo evaluation of visian 4 icl. the biologic nature of the icl 's soft collamer material allows good quality imaging unlike the pmma iols that cause many reflections. the high - frequency ultrasound can penetrate the iris and visualize the sulcus and the lens within the posterior chamber ubm is done in supine position and requires a plastic or silicone eyecup to hold a coupling medium (methyl cellulose or saline solution) and this can induce pressure on the eyeball and may influence the results. however, it has long been known that the immersion technique is more accurate than direct contact technique which may exert more pressure. study the mean vault was 376 105 m and the mean e - icl was 2826 331 m. several previous studies used anterior segment imaging devices to measure vault and e - icl with relatively comparable results. using ubm, pitault., found that the mean central vault was 402 194 m and the mean distance between icl and the central endothelium was 2398 203 m. zhang. used both ubm and anterior segment oct in evaluation of cornea to icl and central vault measurement in myopic eyes implanted with visian icl. they found that the mean central vault was 440 190 m and e - icl distance was 2490 250 m when measured by ubm. vault measured with oct was significantly higher than that obtained with the ubm while the cornea to icl distance measured with two devices was not statistically significantly different. in the current study ubm examinations were done in the ordinary room lighting condition while, in the study of zhang., the examinations were performed in a dark room with ambient illumination below 5 lx. du. used ubm to study the changes in acd and the vaulting between the visian icl and the crystalline lens during pharmacologic accommodation (with topical pilocarpine). they found that there was a significant decrease in vault accompanied by a significant increase in e - icl distance after instillation of pilocarpine (p < 0.01). they concluded that, during pharmacologic accommodation, the icl and the crystalline lens came closer as the icl was pushed backward by the iris as a result of pupillary constriction. simultaneously, the anterior surface of the crystalline lens became more convex and moved forward. they found contact between the icl and the crystalline lens in 15.6% of the eyes and anterior subcapsular opacification developed in 13.0% of eyes. compared to the previous study, our results showed significantly less percentage of eyes that developed cataract. only three eyes (2.11%) had cataract. the vault was less than 200 m in two eyes while in the third eye the vault was 264 m. although decreased vault is an important risk factor for cataract formation, other factors including operative trauma, postoperative inflammation, and myopia itself may have a role. most of the previous similar studies focused on the relation between icl and the anterior surface of the crystalline lens. the classic concept is that undersizing of icl leads to small vault and hence the increased incidence of cataract formation and oversizing of icl ; on the other hand, increased vault will push the iris forward increasing the risk of decreased angle width and glaucoma. however, previous studies did not focus on the relation between icl and the posterior surface of the iris. in the current study we found contact between icl and the posterior epithelium of the iris in all examined eyes. this contact was unrelated to increased vault. such contact theoretically increases the risk of pigment dispersion from the posterior surface of the iris especially after physical exercise or accommodation like what happens in pigment dispersion syndrome. long term follow - up of the iop, corneal endothelium, and angle pigmentation is recommended to prove or to contradict this possibility. ubm can provide valuable anatomical information that allows detailed postoperative in vivo assessment of icl. | purpose. to evaluate the anterior segment, the anatomical position of the implantable collamer lenses (icl), and its relationship to adjacent ocular structures using ultrasound biomicroscopy (ubm). methods. in a prospective study, 142 myopic eyes of 93 patients implanted with visian icl were subjected to ubm examination between march 2010 and january 2015. the relative position of icl to the adjacent structure and the overall iris configuration were evaluated. the machine calibers were used to measure the minimum central distance between the icl and anterior lens capsule (vault) and the vertical central distance between the corneal endothelium and the icl (e - icl). results. the mean icl vault was 376 105 m. the mean e - icl was 2826 331 m. contact between icl and the posterior epithelium of the iris was present in all eyes. the overall iris configuration was flat in 89 eyes. central anterior convexity was present in 41 eyes and mild peripheral iris bombe in 12 eyes. the haptics could be imaged in the ciliary sulcus in 112 eyes and at least one haptic resting on the lens periphery and zonules in 30 eyes. conclusion. ubm can provide valuable anatomical information that allows detailed postoperative in vivo assessment of icl. |
image denoising is a common preprocessing step in many magnetic resonance (mr) image processing and analysis tasks, such as segmentation, registration or parametric image synthesis. many filtering methods use the signal averaging principle which is based on the natural spatial pattern redundancy in the images. in this sense, gaussian filters have been largely used in some applications such as functional mr imaging (fmri). however, they have the disadvantage of blurring edges due to the averaging of nonsimilar patterns. in order to avoid this problem, probably the most well - known filter is the anisotropic diffusion filter (adf) [5, 6 ]. however, such filtering usually erases small features and transforms image statistics due to its edge enhancement effect resulting in unnatural images. modern wavelet - based filters have also been applied to mr image denoising [7, 8 ]. such filters, although effective, are prone to introduce characteristic artifacts (small spots) that can hamper the image analysis process. many existing filters used in mri work using a single image component or volume without taking into consideration the multicomponent intrinsic nature of mr studies. a typical mr study is comprised by many different types of images of the same patient (e.g., t1,t2, flair, etc.), where after a registration process each voxel can be seen as vector with as many components as image types in the study. there are few methods that use this multicomponent information as a basis for the denoising process. one of the first attempts to use this information was the multicomponent adf proposed by gerig.. in this method, the authors proposed using the gradient information of different image sequences of the same subject to constrain the diffusion process. in the context of wavelet thresholding, a new denoising technique for multicomponent images, exploiting interscale and intercomponent correlations was recently proposed by scheunders and backer. this technique was demonstrated to outperform similar single and multicomponent wavelet thresholding techniques. on the other hand, a partial volume modelling - based approach has been also recently proposed by thacker and pokric where the filtering was performed using multidimensional data and a partial volume data density model. this approach abandons altogether local smoothness constraints and achieves noise reduction by enforcing agreement between measured data using underlying tissue proportions computed from a physics - based image formation model. in the present work, a novel method for multicomponent mr this method is inspired on a new filter recently proposed by buades. the main hypothesis in this work is that when multiple mr images of different type or different acquisition times are available, the filtering process can be improved by using the additional correlated information in such images. the proposed method will work on both spatial and intercomponent domain. in the next sections, the proposed filter is fully described and its application in real and simulated multicomponent mr data is evaluated and compared with related previous state - of - the art methods. the nlm filter is a neighbourhood filter which achieves denoising by averaging similar image pixels according to their intensity similarity. the main difference between the nlm and previous related filters is that the similarity between pixels has been made more robust to the noise level by using region comparison rather than pixel comparison ; furthermore, pattern redundancy has been not restricted to be local (nonlocal). that is, pixels far from the pixel being filtered are not penalized due to its distance to the current pixel, as for example, happens in the bilateral filter. given an image y, the filtered value at pixel p using the nlm method is computed as a weighted average of all the pixels in search area within the image : (1)nlm(y(p))=qw(np, nq)y(q),0w(p, q)1 qw(np, nq)=1, where y(p) is the pixel being filtered and y(q) represents each one of the pixels in the image. the weights w(np, nq) are based on the similarity between the neighbourhoods np and nq of pixels y(p) and y(q). the neighbourhood ni is defined as a square window centered on pixel i with a user - defined radius rsim. the region is defined as a squared region surrounding the pixel being processed of radius rsearch. although the original method claims that this region can be the entire image due to computational reasons this region has to be restricted to be of smaller size. the similarity w(np, nq) is calculated as (2)w(np, nq)=1z(p)ed(np, nq)/h2,z(p)=qyed(np, nq)/h2, where z(p) is the normalizing constant, h is a exponential decay control parameter and d is a gaussian weighted squared euclidian distance (with standard deviation 1) of all the pixels of each neighbourhood as defined in : (3)d(np, nq)=(y(np)y(nq))rsim2. this measure penalizes pixels far from the center of the neighbourhood window giving more weight to pixels near the center of the window in the distance computation. in (2) there is a special case when p = q. as the self distance is zero, it can produce an over - weighting effect. to solve this problem, buades proposed to calculate d(np, np) as the minimum distance of the rest of the pixels in the image : (4)d(np, np)= min (d(np, nq), qp). in figure 1, an example of the nlm estimated weights for a small squared region is displayed. as can be noticed, the nlm method finds successfully as similar to the current pixel (small red square) other edge pixels within the search window. as in medical mr imaging the acquisition of multiple images with different acquisition parameters is a common practice, the above method can be extended to be used in a multicomponent framework. effectively, the similarity measure can be better estimated by combining information not only from the surrounding pixels but also using information of the different components in a similar manner as performed on colour image denoising using the rgb space. therefore, the multicomponent similarity function is computed as follows : (5)w(np, nq)=1z(p)e(i=1c(d(npi, nqi)/hi2)/c), where (6)z(p)=qe(i=1c(d(npi, nqi)/hi2)/c), where c is the number of components and h parameter is related with the noise standard deviation of each image. the mnlm algorithm, as in the single component case, has three free parameters, and the filtering results highly depend on their correct setting. the first parameter, rsearch, is the radius of the search window. although the original method claimed to use all the pixels in the image by taking the weighted average of every pixel, this is inefficient if the only similar locations are relatively nearby. besides therefore, the search window has to be reduced to a local window of smaller size. the second parameter, rsim, is the radius of the neighbourhood window used to compute the similarity between two pixels. if the value of rsim is increased the similarity measure will be more robust but fewer similar neighbourhoods will be found. the third parameter, h, is related to the decay of the exponential curve and controls the degree of smoothing. if h is too small, little noise will be removed while if h is set too high, the image will become blurry. in our experiments, rsearch was set to 10 (this is a 21 21 search window), which has been found experimentally as a good compromise between noise reduction and computational burden. for 2d processing an rsim of 2 has been found to be a good choice for typical noise levels in mr imaging. however, for multicomponent imaging the additional information from other components allows the use of a smaller similarity region (in our experiments an rsim = 1 was used, i.e., a 3 3 similarity region) dealing in a more point specific similarity measure and therefore increasing the number of similar patches. finally, the parameter hi was set to i2, being i the noise standard deviation in each image component (this value was found to be experimentally the best choice). although the mnlm filter obtains remarkable results, a number of optimizations can be done to increase its accuracy. in (3) the distance between two equal noisy patches will have an average distance equal to 2. therefore, its associated weight will not be equal to 1 as expected (assuming h = 2) but 1/e (see (2) and (5)). this can be easily solved by simply subtracting 1 from exponent in the weight computation ; so similar pixels will have a weight close to 1 (this has the same effect than subtracting 2 to distance as calculated in (3)). to avoid negative values due to the subtraction operation, we calculate the normalized distance as the maximum of the distance after the subtraction and 0. this optimization is specially effective on low - noise conditions : (7)w(np, nq)=1z(p)e(max ((i=1cd(npi, nqi)/hi2/c)1,0)). in this case, the multicomponent similarity function is computed using the same definition as in (5) but subtracting 1 to averaged distance to obtain a weight close to 1 when computing the distance of two equal patches. another useful improvement is to perform a pixel preselection in order to save useless computations and to improve filtering results by excluding nonsimilar pixels in the averaging process. several methods of preselection have been already proposed using gradient information or local image moments. in the present work, we propose selection of those pixels with a difference of their first local moment (mean value of a 3 3 image patch) smaller than the k i / n (being i the noise standard deviation in the image i and n the number of pixels used to compute the mean) in each image : (8)w(np, nq)={1z(p)ed(p, q),if (|piqi|<ki / n),i[1,c],0,otherwise, where (9)d(p, q)=max (i=1cd(npi, nqi)/hi2c1,0). in single image denoising the value of k can be set to 3 which correspond to the third quantile of a standard normal distribution. patches with mean value differences higher than this threshold have a very small probability to be similar to the current patch. in multicomponent data this threshold was found experimentally to be too restrictive. in our experiments, we have set k = 4 which was found to be a better option. experiments using the local variance were performed to improve the preselection step but no significant improvements were found. we will refer to the optimized mnlm method including optimizations 1 and 2 as omnlm. in the presented method, noise reduction is achieved by averaging similar pixels in the spatial domain. however, a higher noise reduction can be obtained by using information in the intercomponent domain as well. principal component analysis and related approaches have been previously used to reduce noise in the images [18, 19 ]. in this context, noise removal is done by decomposing the signal into the local principal components, attenuating less relevant components and reconstructing again. the simplest approach for multicomponent image denoising is to perform pca decomposition of the image series and to remove all the components that have a variance similar to the image noise. however, although effective, this approach requires the number of images to be higher than the number of significant components of the image. besides, image inhomogeneities typically present in mri make it more difficult to obtain noise - related components due to a higher number of significant components required to represent the data. this limits the applicability of the technique to studies that acquire large image series such us fmri. this problem can be overcome by performing pca decomposition over small local windows instead of the whole image. the basic idea is that every pixel of the image can be denoised by decomposing the local surrounding square window of a given radius for the different images in the corresponding components and attenuating the less significant ones. in the proposed approach for each pixel, the pca decomposition of a local matrix of n k data is calculated, n being the number of pixels of the local window (in our experiments, n = 9 was used, i.e., a 3 3 local window) and k the number of components. the obtained components are processed (using hard or soft thresholding) before recomposing the original matrix. this means that, for example, a local window containing a single tissue can be well approximated by its mean value and therefore all components can be suppressed dealing in a much stronger noise reduction than in the global case. finally, after reconstruction the filtered intensity value for a particular pixel can be obtained by averaging estimates of multiple overlapping windows. such averaging allows removal of more noise, in a similar way to the translation invariant denoising proposed by coifman and donoho. the attenuation can be done using hard or soft thresholding similar to wavelet - based denoising. in our case, we use soft thresholding where each component is multiplied by a factor f defined as. this process is applied as a postprocessing step after the application of the omnlm filter to reduce noise in the images by using information of the spatial domain as well as in the intercomponent domain. we will refer to the combination of omnlm method and pca postprocessing as omnlm - pca. spatial denoising, apply the omnlm to the noisy data to reduce noise in the spatial domain. pca postprocessing, perform a final local pca denoising over the spatially filtered images using an estimate of the local remaining noise. to evaluate the proposed method over synthetic cases, a slice of simulated t1, t2, and two pd (low and high flip angles) weighted images (1 mm voxel resolution and 8 bit quantization) from the brainweb phantom was used (see figure 2). noise in mri can be gaussian or rician distributed depending on the image type (real or magnitude data). in our experiments, all images were corrupted with gaussian - distributed random noise with different levels since at brain tissues noise distribution can be generally well approximated by a gaussian distribution. as our main interest is to remove noise from the region of interest (typically the brain), in our experiments all the measures have been obtained from a region corresponding to the brain parenchyma since noise removal at background can bias the measures. the accuracy of the proposed filter (omnlm and omnlm - pca versions) over usual noise levels (1 to 9% of the maximum t1-weighted image intensity) was evaluated and compared with the mnlm method. also, experiments were performed to evaluate the effect of adding multiple images in the image denoising process. approaches the root mean squared error (rmse) and multicomponent root mean squared error (mrmse) were used : (11)rmse=1mi=1m(x(i)f(i))2,mrmse=1cmj=1ci=1m(x(i, j)f(i, j))2, where x(j) is the jth noise free image component and f(j) is its denoised image version, both containing m pixels. this measures was calculated only from data within a mask comprising the brain tissues. in table 1, a comparison of the basic mnlm filter and the proposed omnlm and omnlm - pca methods is presented. it can be noticed that the proposed optimizations improved significantly the accuracy of the basic filter for all the noise levels. the omnlm - pca version achieved the better results for all noise levels. to evaluate the effect of adding new images to the filtering process only the rmse of the t1-weighted image results comparing the basic mnlm filter and the proposed omnlm - pca filter can be observed in table 2. curiously, in the basic mnlm filter, the best results were obtained when using only two images (t1 and t2), probably because these images present good contrast between different tissues. in this case, the inclusion of the two pd images in the similarity measurements, did not improve the results but made them slightly worse behaving as a confounding factor. conversely, the proposed method presented a consistent improvement of the results as the number of images was increased. this was found to be mainly due to the preselection optimization which avoids averaging the current pixel with nonsimilar pixels. the described method was compared with the state - of - the art multicomponent denoising methods. specifically, the compared methods were the multidimensional partial volume modelling method (mpvm) proposed by thacker. and the multicomponent method based on wavelet thresholding using gaussian mixture modelling (mgmm) recently proposed by scheunders and backer. these methods were applied with their default parameters (sym4 wavelet type and depth equal 3 for the mgmm method and a 6 tissue model for the mpvm method). the mgmm method showed good results for low - noise levels while for medium and high noise levels this method tends to blur the images (figure 3). the execution times of the compared methods were 10 seconds for the mvpm method, 6 seconds for mgmm method and 33 seconds for the omnlm - pca method. to evaluate effectiveness of the different multicomponent approaches on real image conditions, four real clinical images were used (irtse, pd, t2, and flair) (figure 4). all images were acquired with a 1.5-t system (acs - nt, with powertrack 6000 gradient subsystem ; phillips medical systems, hamburg, germany) with a birdcage head coil receiver. data was in contiguous 3 mm thick sections throughout the brain, with an in - plane resolution of 0.89 mm (matrix, 256 204, field of view, 230 184 mm). all images were registered with in - house software developed in our lab. the noise level in each image was estimated using the lne technique which is based on the area of the probability distribution p of the second - order derivatives of the image (see (12)) : (12)noise=(di=ddp(i))0.63 where d = arg min (i=ddp(i)0.63). to evaluate the efficiency of the different filters, two measures were used. one based on the image residuals (i.e., difference between the noisy and denoised images) and the other based on an estimation of the noise level after filtering (in this case a noise free image is not available for direct comparison). these measures were estimated from all the pixels belonging to the intracranial cavity as in this area the random noise can be approximately considered as gaussian - distributed. the first measure was the remaining noise fraction (rnf) : (13)rnf=denoisedoriginal, where denoised is the standard deviation of the noise after filtering and original is the standard deviation of the noise in the original noisy image. both standard deviations were estimated using the lne technique this measure was the number of outliers in the residuals, defined as the number of pixels with values lying 3 times beyond the standard deviation of the image residuals (for gaussian - distributed noise this is expected to be around the 0.05% of the data). the first measure relates with the amount of noise removed while the second is devised to measure the quality of the filtering process. as can be observed, mpvm method tends to overcorrect the data, as the number of outliers is much higher than the expected for an optimal filter (this can be noticed in figure 5 where image edges in the residuals are clearly visible). this is probably related to the modelling of each pure tissue as one grey - level value across the entire image. it has to be considered that the proportion of outliers measured for mpvm method represents an upper bound due to the removal of spatially correlated residuals introduced by field inhomogeneity artifacts. on the other hand, mgmm method performed well showing a good agreement with results obtained for the same level of noise (2%) on synthetic images. it removed the highest quantity of noise (77% in average) while showing a number of outliers very close from the theoretically expected for an optimum filter (60 in this case). figures 4 and 5 show the filtered images with the compared methods and the corresponding residuals for visual comparison. in this paper, a new method for multicomponent mr image denoising has been proposed and evaluated using both synthetic and real clinical data. the proposed method has been compared with related state - of - the art methods. it has been demonstrated that using multicomponent images to denoising image series presents important benefits over single image techniques due to the increased data redundancy. our proposed filter removes noise in the image domain by averaging similar patches around the image by using a robust multicomponent similarity measure which has been shown to improve the results of the mnlm method. besides, the proposed filter also removes noise in the intercomponent domain by using a local pca - based decomposition. the proposed method has shown a consistent improvement in the results when the number of images increases in contrast with the erratic behaviour of the basic version. this can be attributed to a more cautious image formation model, which was designed to avoid the use of spatial smoothness constraints for some clinical applications. the mgmm method showed numerically a good performance but a significant blurring effect was present in the filtered images. there are a number of factors that can influence the denoising results of the proposed method such as image inhomogeneities, incorrect image registration or spatially dependent noise. as the denoising is performed using a local region surrounding the processed pixel the image inhomogeneities are expected to have a low impact in the final results. however, if an inhomogeneity correction is performed prior the denoising process, this effect can be minimized at the expense of spatially modulating the noise amplitude. however, it has been observed that as the similarity measure is computed from several images mainly the incorrectly registered image(s) will be affected by this factor due to the robustness of the similarity measure. besides, if registration errors are present, this will not cause an incorrect denoising but a suboptimal one since the preselection step only will reduce the number of similar patches as it is based on the intersection of distances of the local means. finally, it has to be noticed that modern mri is increasingly acquired using parallel imaging which leads to spatially - dependent and variable noise pattern. in such case the proposed method only requires an estimation of the local image noise and therefore an automated local noise estimator is required. in this sense, there are several works dealing with this issue [24, 25 ]. other possibility is to obtain local noise estimations from the less significant local principal components. the implementation of the proposed method in the 3d case can potentially improve the results by increasing the number of similar pixels in the local surrounding volume and by using a more specific local similarity volume. in this work, we have chosen a 2d implementation to ease the comparison with other multicomponent state - of - the art methods. besides, it has to be noticed that in 3d the execution time may be an issue for some implementations. however, as the proposed method is highly parallelizable the processing time associated with this technique can be dramatically reduced using distributed computing and related techniques. there are a number of possible applications of the proposed method to improve the quality of the images acquired on dynamic series such as fmri, perfusion and diffusion weighted imaging or mr relaxometry. the application of the proposed methodology in these fields has to be addressed with further research. | magnetic resonance images are normally corrupted by random noise from the measurement process complicating the automatic feature extraction and analysis of clinical data. it is because of this reason that denoising methods have been traditionally applied to improve mr image quality. many of these methods use the information of a single image without taking into consideration the intrinsic multicomponent nature of mr images. in this paper we propose a new filter to reduce random noise in multicomponent mr images by spatially averaging similar pixels using information from all available image components to perform the denoising process. the proposed algorithm also uses a local principal component analysis decomposition as a postprocessing step to remove more noise by using information not only in the spatial domain but also in the intercomponent domain dealing in a higher noise reduction without significantly affecting the original image resolution. the proposed method has been compared with similar state - of - art methods over synthetic and real clinical multicomponent mr images showing an improved performance in all cases analyzed. |
antidepressants are widely used in the treatment of depression, which is the most common psychiatric disorder with an enormous burden globally. for a long time, tricyclic antidepressants (tcas) were the mainstay of drug treatment of depression. thromboembolic phenomena are also very common and need treatment with anticoagulants. of the oral anticoagulants, studies of drug interaction between warfarin and antidepressant drugs show that bleeding is the most common and also the most feared manifestation of the interaction. we report a patient who was started on warfarin for treatment of deep vein thrombosis (dvt), who was receiving bupropion for depression. while the international normalized ratio (inr) had been stable at the desired level, bupropion was stopped and this resulted in an alarmingly high inr. higher values of inr than the desired levels for adequate anticoagulation indicate a high risk of bleeding. to our knowledge, there is no reported case so far, of drug interaction between bupropion and warfarin. a 55-year - old male was on treatment for dysthymic disorder (dsm iv tr) since 12 years. he was mostly on bupropion, in doses ranging from 150 mg/ day to 300 mg / day. he responded well to treatment, had few depressive symptoms intermittently, and his smoking had decreased to about 6 - 8 cigarettes a day. a year back, he developed dvt, at which time, the dose of bupropion was 300 mg / day. he was put on warfarin and the desired level of inr, that is, 2 - 3, was achieved. at this time, the surgeon did not know that the patient was taking bupropion 300 mg / day. after a couple of weeks, when the patient informed him of this, bupropion was abruptly stopped. the patient was under observation, and the surgeon put him back on bupropion, 300 mg / day. it is among the 15 most prescribed drugs in the united states, with more than 1 million prescriptions / year and 75.7% of those on warfarin are elderly. considering that antidepressant drugs are so widely prescribed across all age groups need to study the drug interactions between warfarin and antidepressants is obvious. warfarin has a narrow therapeutic range, and for most patients on this drug the target inr usually is 2.0 - 3.0. a higher target inr of 3.0 - 4.0 is recommended for patients with the mechanical prosthetic heart valves. above a level of 4.0, the risk of bleeding is very high, especially intracranial hemorrhage. in our patient, the average dose of warfarin to achieve the desired level of inr ranges from 2 mg / day to 10 mg / day. the dose of warfarin can vary according to the individual sensitivity to it. in a sensitive individual if there is warfarin resistance, a dose in excess of 20 mg may be required. the list of drugs and other factors like diet and food supplements containing vitamin k, that affect the action of warfarin, is prodigious and expanding ; for example, acetaminophen, barbiturates, phenytoin, rifampin, phenylbutazone, sulfinpyrazone, metronidazole, disulfiram, allopurinol, cimetidine, and ingestion of large amounts of alcohol. there are four hypothetical mechanisms by which antidepressants or any other drugs can alter warfarin levels : by decreasing absorption ; by inhibiting cytochrome p2c9 (cyp2c9) isoenzyme - causing an increase of inr ; by enzyme induction of cyp2c9 causing decrease in inr ; by increased protein binding of the drug, resulting in dislodging warfarin, which is highly bound to plasma albumin. while more than 40 hepatic enzymes have been identified, only six isoenzymes (1a2, 3a4, 2c9, 2c19, 2d6, and 2e1) significantly affect the metabolic clearance of more than 90% of all drugs. for warfarin, the isoenzyme 2c9 is the most important, followed by 1a2. in recent studies, specific serotonin reuptake inhibitors (ssris) have been studied the most and tcas the least in terms of interaction with warfarin. as mentioned earlier, about three - fourth of all patients on warfarin are older people. in this population, reported drug interactions between antidepressants and warfarin, of which bleeding is the commonest, are seen most with fluoxetine and fluoxamine. tcas, serotonin norepinephrine reuptake inhibitors venlafexine, duloxetine and desvenlafexine, and mirtazapine, are relatively, safe and have no clinically significant interaction with warfarin. this perhaps is due to their minimal or no inhibitory action on relevant cyp450 isoenzymes, 2c9 and 1a2. there are no reports or empirical data suggesting that there is any risk in using the combination of bupropion and warfarin. it needs to be mentioned though that bupropion has been much less studied compared to ssris. this makes the bupropion - warfarin interaction, in this case, difficult to explain in the light of available evidence. it is possible that there is a hitherto unknown mechanism of action that causes an adverse interaction with warfarin. | depressive illness and thromboembolic disorders are both highly prevalent. warfarin is frequently combined with an antidepressant drug, the choice of which depends mainly on the risk of a hemorrhagic complication. patients requiring the warfarin are often in the older age group, where the newer antidepressants with a better safety profile are preferred over tricyclic antidepressants. we report herein, a patient who was on bupropion for depression, when he developed deep vein thrombosis high - risk. warfarin was started. while on this combination bupropion was abruptly stopped. this caused a more than two - fold elevation of international normalized ratio (inr) above the level, which is considered a high - risk for a hemorrhagic complication. inr reverted back to the desired level on reintroduction of bupropion. this indicates that a bupropion - warfarin combination should be used with the caution, though there has been no reported interaction so far. |
basaloid squamous cell carcinoma (bscc) as defined by the world health organization is an aggressive, high - grade, variant of squamous cell carcinoma (scc) composed of both basaloid and squamous components. the tumor arises most frequently in the head and neck region, the most common sites being epiglottis, piriform sinus and base of the tongue. other less common sites of origin include the floor of the mouth, oral mucosa, palate, tonsils, sinonasal tract, nasopharynx and trachea. head and neck bscc tends to have an aggressive clinical course than stage - matched conventional scc. we report two cases of bscc, one in the floor of the mouth and the other arising on the lateral border of the tongue. a 54-year - old male patient reported to the department with a complaint of burning sensation and diffuse pain under the tongue since last 8 months [figure 1 ]. on examination, an ulceroproliferative growth of size 3 cm 2 cm, with indurated margins, on the anterior floor of the mouth in relation to 31, 32, 41 and 42 regions was noticed. the lesion was tender on palpation. left submandibular lymph nodes were palpable, nontender and mobile. incisional biopsy was performed and histopathology revealed a moderately collagenous connective tissue stroma infiltrated with nests and islands of tumor epithelial cells. the tumor cells exhibited a basaloid appearance with hyperchromatic nuclei and scanty cytoplasm and were arranged in a lobular configuration. numerous mitotic figures were also noticed amidst the tumor cells [figures 2 and 3 ]. photograph showing the ulceroproliferative lesion with indurated margins in the floor of mouth photomicrograph showing nests and islands of infiltrating basaloid cells. occasional squamous differentiation is also seen (h&e stain, 40) photomicrograph showing basaloid cells with nuclear atypia. large number of mitotic figures are also noted (h&e stain, 400) a 57-year - old male patient reported with the complaint of ulcer of 1-month duration on the lateral aspect of the tongue. he reported a habit of tobacco smoking for more than 15 years. on examination, an ulceroproliferative growth of size 2 cm 2 cm on the left lateral border of the tongue, covered by necrotic slough was noticed [figure 4 ]. incisional biopsy was performed and histopathological examination revealed a dysplastic stratified squamous epithelium infiltrating into underlying moderately collagenous connective tissue [figure 5 ]. occasional areas also showed a peripheral palisading of cells and comedo necrosis [figure 6 ]. photomicrograph showing an ulceroproliferative lesion on lateral border of tongue with necrotic slough and indurated margins photomicrograph showing dysplastic stratified squamous epithelium infiltrating into the connective tissue. basaloid appearance of the tumor cells and comedo necrosis can also be appreciated (h&e stain, 40) photomicrograph showing tumor cells with high nuclear - cytoplasmic ratio and peripheral palisading (h&e stain, 100) a 54-year - old male patient reported to the department with a complaint of burning sensation and diffuse pain under the tongue since last 8 months [figure 1 ]. on examination, an ulceroproliferative growth of size 3 cm 2 cm, with indurated margins, on the anterior floor of the mouth in relation to 31, 32, 41 and 42 regions was noticed. the lesion was tender on palpation. left submandibular lymph nodes were palpable, nontender and mobile. incisional biopsy was performed and histopathology revealed a moderately collagenous connective tissue stroma infiltrated with nests and islands of tumor epithelial cells. the tumor cells exhibited a basaloid appearance with hyperchromatic nuclei and scanty cytoplasm and were arranged in a lobular configuration. numerous mitotic figures were also noticed amidst the tumor cells [figures 2 and 3 ]. photograph showing the ulceroproliferative lesion with indurated margins in the floor of mouth photomicrograph showing nests and islands of infiltrating basaloid cells. occasional squamous differentiation is also seen (h&e stain, 40) photomicrograph showing basaloid cells with nuclear atypia. a 57-year - old male patient reported with the complaint of ulcer of 1-month duration on the lateral aspect of the tongue. he reported a habit of tobacco smoking for more than 15 years. on examination, an ulceroproliferative growth of size 2 cm 2 cm on the left lateral border of the tongue, covered by necrotic slough was noticed [figure 4 ]. incisional biopsy was performed and histopathological examination revealed a dysplastic stratified squamous epithelium infiltrating into underlying moderately collagenous connective tissue [figure 5 ]. occasional areas also showed a peripheral palisading of cells and comedo necrosis [figure 6 ]. photomicrograph showing an ulceroproliferative lesion on lateral border of tongue with necrotic slough and indurated margins photomicrograph showing dysplastic stratified squamous epithelium infiltrating into the connective tissue. basaloid appearance of the tumor cells and comedo necrosis can also be appreciated (h&e stain, 40) photomicrograph showing tumor cells with high nuclear - cytoplasmic ratio and peripheral palisading (h&e stain, 100) first described by wain. in 1986 in the upper aerodigestive tract (uadt), bscc is a rare, aggressive variant of scc with varying proportions of basaloid and squamous components. it represents 2% of head and neck cancers with an increased tendency to be multifocal, deeply invasive and metastatic even at the initial presentation. tobacco, alcohol abuse and a previous history of radiation to the head and neck region are considered as strong risk factors. although both of our patients failed to give a history of previous irradiation or alcohol consumption, they were chronic tobacco chewers (betel leaf, areca nut, slaked lime and tobacco) with occasional smoking. the role of viruses in the etiology of bscc is still controversial, but many authors have reported a higher frequency of human papillomavirus and herpes simplex virus in basaloid tumors than in conventional scc. the origin for bscc has been suggested to be from a totipotent cell capable of divergent differentiation located in the basal zone of the surface epithelium or in the minor salivary glands of the submucosa. some authors also believe that basaloid pattern occurring anywhere in the body represents an attempt at glandular differentiation. bscc usually presents as a centrally ulcerated mass with extensive submucosal induration, often being confused with a minor salivary or soft tissue tumor. histologically, it is considered to be a bimorphic variant of scc, with basaloid cells and an scc component which can be either in situ carcinoma or invasive keratinizing scc. the closely packed basaloid cells growing in a solid pattern with a lobular configuration often exhibit prominent peripheral palisading and comedo - type necrosis. this was not discernible in our cases, most probably due to small size of the specimen received. distinctive features of bscc, not found in scc, are small cystic spaces containing periodic acid - schiff and alcian blue positive material and stromal hyalinization. reported that wain 's criteria (peripheral palisading, association with scc, high nuclear - cytoplasmic ratio, high mitotic rate and solid growth), anti-34be12 and ck 5/6 staining and absence of neuroendocrine markers are mandatory for the diagnosis of bscc. the immunoprofile of bscc shows consistent positive staining to high molecular weight cytokeratin antibody 34e12, kl1, p63 and mnf116, and focal staining for vimentin, ema, cam5.2, ck7, cea, s100 and gfap and negative immunostaining for ck20, chromogranin, synaptophysin, bcl2 and ber - ep4. the histopathological differential diagnosis for bscc includes solid variant of adenoid cystic carcinoma (acc), small cell neuroendocrine carcinoma (scnc), adenosquamous carcinoma, basal cell adenocarcinoma, salivary duct carcinoma and basal cell ameloblastoma. bscc can be differentiated from acc by the absence of myoepithelial cells, the presence of basement membrane such as material positive for laminin and type iv collagen in the microcystic spaces, dot - like vimentin expression and negative ck7 staining. moreover, acc shows no dysplasia or continuity with the surface epithelium and often shows less pleomorphism, mitosis and necrosis than bscc. emanuel. reported that p63 immunostaining constitutes a specific and accurate means of distinguishing bscc from acc with diffuse p63 positivity and staining of nearly 100% of tumor cells in the former and a consistently compartmentalized pattern within tumor nests in the latter. to differentiate bscc from scnc, it is important to note that scnc shows characteristic nuclear molding, crushing artifact, lack of stromal mucinous - myxoid changes and hyalinosis and is rarely connected to the surface mucosa. furthermore, bscc is negative for chromogranin, synaptophysin and glial - fibrillary acid protein. the recognition of mucin positivity and true ductul - acinar differentiation in adenosquamous carcinoma differentiates it from bscc. focal necrosis and squamous differentiation usually seen in bscc are not seen in basal cell adenocarcinoma. eosinophilic cytoplasm and irregular - shaped cystic spaces lined by papillary projections seen in salivary duct carcinomas help to distinguish them from bscc. since the tumor shows a propensity for early metastases to regional lymph nodes and visceral locations, a multimodality treatment approach including radical surgical excision, neck dissection, radiotherapy and often chemotherapy is preferred. distant metastasis involving the lung, bone, skin and brain develops in up to 44% of cases and is six times higher in cases of bscc compared to scc. suggests that since lungs is the most frequent site of bscc metastasis, all patients diagnosed with bscc of the uadt systematically should undergo a chest computed tomography scan to rule out the lung metastasis and a 2-deoxy-2-fluoro - d - glucose positron emission tomography to detect the presence of extrapulmonary asymptomatic metastatic lesions. despite more aggressive therapy, bscc the parts of dysplastic squamous epithelium infiltrating into connective tissue were evident and the tumor cells showed a basaloid appearance with peripheral palisading, comedo necrosis and occasional squamous differentiation, which prompted us to give a final diagnosis of bscc. although the clinical diagnosis is simply that of an oral scc (oscc), it should be a routine to diagnose the case histologically as any of its variants. the diagnosis of these cases as bscc is essential as the lesion has a different clinical course, prognosis and treatment aspects when compared to the conventional oscc. since the tumor is almost always associated with an aggressive course and poor prognosis, once diagnosed with bscc, the patient should be extensively worked up to rule out the occurrence of subclinical metastatic lesions. the possibility of finding a second primary tumor in any site should also be kept in mind. | basaloid squamous cell carcinoma (bscc) is an aggressive, high - grade, variant of squamous cell carcinoma (scc), which is uncommon in the oral cavity but slightly more common in the oropharynx. we present two cases of bscc, one arising in the floor of the mouth and the other arising on the lateral border of the tongue. the diagnosis of this subtype of scc is important owing to its particular behavior, with an aggressive course, a high incidence of local recurrence, regional lymph node metastases and mortality rate. |
pain is one of the most commonly experienced symptoms in dentistry and is a major concern to the dentist. pain, in many instances, is considered as a caution signal. in practice of dentistry, pain is no more a warning signal, instead it is an evil to be conquered. one of the most important aspects of the practice of dentistry is the control or elimination of pain. the most essential skill of all dental practitioners is the ability to provide safe and effective local anesthesia. a revolutionary advancement of the late 1800s was the discovery of local anesthetics that facilitated pain prevention without the loss of consciousness. lidocaine hydrochloride is the most commonly used anesthetic agent since its clinical availability in 1948. it is labeled as gold standard due to its efficacy, low allergenicity, and minimal toxicity. it has a more profound level of anesthesia and greater ability to diffuse through tissues. anesthesia of maxillary anteriors and premolars can be achieved by infraorbital nerve block (ionb) or anterior superior alveolar (asa) which involves deposition of the anesthetic solution at the infraorbital foramen. anatomic variations in anterior maxilla are very common, which reduces the success of anesthesia. multiple needle penetrations will be necessary to ensure an adequate volume of anesthetic solution which is deposited at the target site. the ideal maxillary injection should produce a rapid onset of profound pulpal anesthesia for multiple teeth from a single needle penetration. the literature does not explain a single injection site that would produce pulpal anesthesia to the majority of the maxillary teeth without collateral anesthesia of the face, lip, and muscles of expression. alternate nerve blocks are anterior middle superior alveolar nerve block (amsanb) and palatal approach to the asa. friedman and hochman suggested that administration of anesthetic solution midpalatally using slow, constant pressure would access to the asa and middle superior alveolar nerve and their plexuses. this technique induces anesthesia from the incisor to the premolar and possibly to the mesiobuccal root of the first molar. thus, the aim of this study is to comparatively evaluate anesthetic efficacy of articaine 4% and lidocaine 2% with amsanb and ionb for anesthesia of maxillary anteriors and premolars. the study was conducted in the department of conservative dentistry and endodontics, and institutional ethical clearance was obtained. patients were administered 0.1 ml of the anesthetic agent (intradermal allergy test) on the dorsal part of their arms and were observed for hypersensitivity reactions. the patients (participants) were not disclosed about the anesthetic agent as well as the technique. patients undergoing root canal treatment of maxillary anteriors and premolars were included. before administration of nerve blocks, the pulpal response was tested with the electric pulp tester (digitest, parkell, inc., patients aged between 18 and 65 years in good health (asa i or ii)patients reporting with spontaneous pain ranging from 7 to 9 on numerical reading scalepatients who granted informed consent. patients aged between 18 and 65 years in good health (asa i or ii) patients reporting with spontaneous pain ranging from 7 to 9 on numerical reading scale patients who granted informed consent. known case of allergy to local anesthetic agentshistory of significant medical problem (asa iii or greater)patients who had taken central nervous system depressants including alcohol or any analgesic medication within the last 48 hpregnancyinability to give informed consent known case of allergy to local anesthetic agents history of significant medical problem (asa iii or greater) patients who had taken central nervous system depressants including alcohol or any analgesic medication within the last 48 h inability to give informed consent the patients were randomly divided into four groups of ten each : group i : patients receiving amsanb with articaine hcl 4% with 1:100,000 adrenaline (septodont, france)group ii : patients receiving ionb with articaine hcl 4% with 1:100,000 adrenalinegroup iii : patients receiving amsanb with lidocaine hcl 2% with 1:80,000 adrenaline (indoco remedies, india)group iv : patients receiving ionb with lidocaine hcl 2% with 1:80,000 adrenaline. group i : patients receiving amsanb with articaine hcl 4% with 1:100,000 adrenaline (septodont, france) group ii : patients receiving ionb with articaine hcl 4% with 1:100,000 adrenaline group iii : patients receiving amsanb with lidocaine hcl 2% with 1:80,000 adrenaline (indoco remedies, india) group iv : patients receiving ionb with lidocaine hcl 2% with 1:80,000 adrenaline. patients received 0.91.2 ml of anesthetic agent for classic ionb using conventional luer lock 2 ml syringe with 26-gauge, 1 inch needle using aseptic measures, and sterile techniques. for amsanb, 0.61.4 ml of the anesthetic agent was deposited using slow, constant pressure in the palatal mucosa. the nerve blocks given with articaine 4% were administered with standard cartridges (1.7 ml) and an aspirating syringe equipped with 27-gauge, 1 inch needle. the following data were collected for interpretation of results : onset of anesthesia : it was assessed from the time lapse between the end of the nerve block and onset of symptoms of subjective anesthesia (feeling of heaviness at the site of injection). it was calculated in secondsextent of pulpal anesthesia : the response to electrical pulp tester was obtained at three intervals - before the administration of nerve block, after the onset of anesthesia, and 30 min after anesthesia. the numerical readings were recordedpain assessment : the pain on injection was rated by vas. onset of anesthesia : it was assessed from the time lapse between the end of the nerve block and onset of symptoms of subjective anesthesia (feeling of heaviness at the site of injection). a standard digital stop clock was used. it was calculated in seconds extent of pulpal anesthesia : the response to electrical pulp tester was obtained at three intervals - before the administration of nerve block, after the onset of anesthesia, and 30 min after anesthesia. the numerical readings were recorded pain assessment : the pain on injection was rated by vas. the readings were statistically analyzed using spss software version 20.0 (ibm corp, armonk, ny, usa) by two - way analysis of variance (anova) test, tukey 's multiple post hoc procedures, paired t test, and mann whitney u - tests. patients aged between 18 and 65 years in good health (asa i or ii)patients reporting with spontaneous pain ranging from 7 to 9 on numerical reading scalepatients who granted informed consent. patients aged between 18 and 65 years in good health (asa i or ii) patients reporting with spontaneous pain ranging from 7 to 9 on numerical reading scale patients who granted informed consent. known case of allergy to local anesthetic agentshistory of significant medical problem (asa iii or greater)patients who had taken central nervous system depressants including alcohol or any analgesic medication within the last 48 hpregnancyinability to give informed consent known case of allergy to local anesthetic agents history of significant medical problem (asa iii or greater) patients who had taken central nervous system depressants including alcohol or any analgesic medication within the last 48 h inability to give informed consent the patients were randomly divided into four groups of ten each : group i : patients receiving amsanb with articaine hcl 4% with 1:100,000 adrenaline (septodont, france)group ii : patients receiving ionb with articaine hcl 4% with 1:100,000 adrenalinegroup iii : patients receiving amsanb with lidocaine hcl 2% with 1:80,000 adrenaline (indoco remedies, india)group iv : patients receiving ionb with lidocaine hcl 2% with 1:80,000 adrenaline. group i : patients receiving amsanb with articaine hcl 4% with 1:100,000 adrenaline (septodont, france) group ii : patients receiving ionb with articaine hcl 4% with 1:100,000 adrenaline group iii : patients receiving amsanb with lidocaine hcl 2% with 1:80,000 adrenaline (indoco remedies, india) group iv : patients receiving ionb with lidocaine hcl 2% with 1:80,000 adrenaline. patients received 0.91.2 ml of anesthetic agent for classic ionb using conventional luer lock 2 ml syringe with 26-gauge, 1 inch needle using aseptic measures, and sterile techniques. for amsanb, 0.61.4 ml of the anesthetic agent was deposited using slow, constant pressure in the palatal mucosa. the nerve blocks given with articaine 4% were administered with standard cartridges (1.7 ml) and an aspirating syringe equipped with 27-gauge, 1 inch needle. the following data were collected for interpretation of results : onset of anesthesia : it was assessed from the time lapse between the end of the nerve block and onset of symptoms of subjective anesthesia (feeling of heaviness at the site of injection). it was calculated in secondsextent of pulpal anesthesia : the response to electrical pulp tester was obtained at three intervals - before the administration of nerve block, after the onset of anesthesia, and 30 min after anesthesia. the numerical readings were recordedpain assessment : the pain on injection was rated by vas. onset of anesthesia : it was assessed from the time lapse between the end of the nerve block and onset of symptoms of subjective anesthesia (feeling of heaviness at the site of injection). a standard digital stop clock was used. it was calculated in seconds extent of pulpal anesthesia : the response to electrical pulp tester was obtained at three intervals - before the administration of nerve block, after the onset of anesthesia, and 30 min after anesthesia. the numerical readings were recorded pain assessment : the pain on injection was rated by vas. the readings were statistically analyzed using spss software version 20.0 (ibm corp, armonk, ny, usa) by two - way analysis of variance (anova) test, tukey 's multiple post hoc procedures, paired t test, and mann whitney u - tests. a significant difference was observed in the onset of anesthetic action between articaine 4% and lidocaine 2% by two - way anova test (p < 0.05) [table 1 and graph 1 ]. the onset of action was the fastest for articaine 4% with amsanb (group i) and the slowest for lidocaine 2% with ionb (group iv) by tukey 's multiple post hoc procedures (p < 0.05) [table 2 ]. comparison of two main (lidocaine 2% with adrenaline and articaine 4% with adrenaline) and two subgroups (infraorbital nerve block and alveolar nerve block) with onset of anesthesia (s) by two - way anova comparison of two main (lidocaine 2% with adrenaline and articaine 4% with adrenaline) and two subgroups (infraorbital nerve block and anterior middle superior alveolar nerve block) with onset of anesthesia (in seconds) pair - wise comparison of two main (lidocaine 2% with adrenaline and articaine 4% with adrenaline) and two subgroups (infraorbital nerve block and alveolar nerve block) with onset of anesthesia (mm) by tukey 's multiple post hoc procedures the mean electric pulp testing (ept) readings before the nerve blocks were 13 for articaine with amsanb, 12.3 for articaine 4% with ionb, 13.25 for lidocaine 2% with amsanb, and 16 for lidocaine 2% with ionb, respectively. no response was observed on maximum stimulation (reading = 64) at the time of onset and after 30 min among all the groups. a significant change was observed in the ept readings before the injection and at the onset of anesthesia by paired t - test (p < 0.05) in all the groups [graph 2 ]. comparison of electric pulp testing scores preoperative, at onset and after 30 min among four groups the mean pain scores for group i, group ii, group iii, and group iv were 0.60, 0.22, 0.75, and 0.82, respectively. pain perception was least (0.22) for ionb with articaine 4%. in this study, the pain experienced was mild as expressed by patients on vas which corresponded with the scores of numerical rating scale (nrs) [graph 3 ]. comparison of two main (lidocaine 2% with adrenaline and articaine 4% with adrenaline) and two subgroups (infraorbital nerve block and anterior middle superior alveolar nerve block) with vas scores local anesthetics are the effective drugs available for the prevention and the management of pain. during endodontic treatment, lidocaine, also known as lignocaine, is an amide anesthetic that has been described as the most commonly used local anesthetic for dental use. lidocaine provides pulpal anesthesia for approximately 1 h and soft tissue anesthesia for 35 h. articaine was originally synthesized in 1969. articaine, the second most commonly used dental anesthetic, was first introduced to the european market in 1976 and entered the us market in 2000. pulpal anesthesia duration is approximately 1 h. the soft tissue anesthesia is approximately 2.25 h for maxillary infiltrations. according to malamed, articaine was well tolerated in clinical trials and had the toxicity profile comparable to that of lidocaine. dentistry 's clinical experience with articaine with epinephrine formulations through the years supports the assertion that the risk of systemic toxicity with articaine is low. articaine 4% with epinephrine 1:100,000 is a safe local anesthetic for use in clinical dentistry and can be effectively used in both adults and children. lidocaine 2% is commonly used by most dentists with the conventional needle technique, and the study intended to check the efficacy in that technique only and not by using cartridges unlike with articaine 4%. the administration of local anesthetic with the cartridge is technique - sensitive and needs a learning curve. the present study showed that articaine 4% had a faster onset as compared to lidocaine 2%. articaine is unique among amide local anesthetics, in that it contains a thiopentone group instead of the benzene ring found in lidocaine and other amide local anesthetics. the thiopentone ring contains a methyl ester side linkage that contributes to articaine 's rapid conversion to articainic acid, its primary metabolite. alam. reported more profound level of anesthesia and a greater ability to diffuse through tissues. borchard and drouin found that a lower concentration of articaine was sufficient to block an action potential when compared with other amide anesthetics (benzene derivatives). found out in a study of rat sensory nerve conduction that 4% articaine was superior to 2% lidocaine in blocking nerve conduction. brandt. reported that articaine was 3.81 times more likely to achieve anesthetic success when infiltration mode of administration is used. in the present study, amsanb with 4% articaine had a faster onset as compared to ionb with 2% lidocaine. it improves patient comfort through the elimination of repetitive transmucosal punctures which reduces the total amount of delivered vasoconstrictor and proves useful for cardiovascular compromised patients requiring maxillary anesthesia. it gives an outstanding hemostatic control, avoidance of undesirable collateral anesthesia, and a reduced number of cumulative injections. the ept readings taken at three intervals proved to be a clinically efficient tool for assessing the technique sensitivity of the nerve blocks. the ept readings of the present study confirmed that the electric pulp tester served as a valuable aid in determining clinical analgesia. the pain rating on the vas as interpreted by nrs was found to be of mild range. the ept readings and the pain scores were clinically significant to confirm that the nerve blocks provided definite pulpal anesthesia with least amount of soft tissue anesthesia. in the present study, traditional syringes were used and patients experienced mild pain only during the palatal injections. the amsa injection using the wand resulted in similar pain ratings for needle insertion but statistically lower pain ratings on anesthetic solution deposition. according to fukayama. and goodell gg. concluded that a conventional atraumatic syringe injection technique was superior to a controlled injection pressure system in pain perception and pain tolerance and in reducing postinjection dental anxiety. another important factor is cost effectiveness of using traditional syringes and wand, wherein cost factor was definitely decreased with traditional syringe making it an affordable technique. this study is unique in that the two important and clinically useful anesthetic agents were compared and found to be clinically significant. ionb and amsanb were efficient to provide pulpal anesthesia for maxillary anterior teeth and premolars. amsanb offers numerous advantages and is an effective alternate to the classic ionb. within the limitations of the study, articaine 4% with amsanb | background : the ideal maxillary injection should produce a rapid onset of profound pulpal anesthesia for multiple teeth from a single needle penetration. the main objective is to compare the efficacy of articaine 4% and lidocaine 2% and to compare anterior middle superior alveolar nerve block (amsanb) and infraorbital nerve block (ionb) for anesthesia of maxillary teeth.materials and methods : forty patients undergoing root canal treatment of maxillary anteriors and premolars were included and randomly divided into four groups of ten each. group i : patients receiving amsanb with articaine, group ii : patients receiving ionb with articaine, group iii : patients receiving amsanb with lidocaine, group iv : patients receiving ionb with lidocaine. the scores of onset of anesthesia and pain perception were statistically analyzed.results:onset of action was fastest for articaine with amsanb and slowest for lidocaine with ionb by tukey 's test. a significant change was observed in the electrical pulp test readings at onset and at 30 min by paired t - test. all patients experienced mild pain during the procedure recorded by visual analog scale.conclusion:articaine 4% proved to be more efficacious than lidocaine 2%, and amsanb was more advantageous than ionb in securing anesthesia of maxillary anteriors and premolars. |
surgery is the main treatment strategy for gastric cancer ; only r0 resection anticipates a potential cure. the prognosis of initially unresectable gastric cancer is poor ; approximately 50% of patients have recurrent disease after a curative resection. this suggests that micrometastases are already present, in many cases, at the time of surgery1). neoadjuvant chemotherapy has been attempted for the treatment of gastric cancers in patients with advanced t and n stage disease for the purpose of downstaging, improving resectability and survival2). these regimens have demonstrated a response rate (rr) of 3 4~69% and a resectability of 36~60%, with a median survival of 16~28 months for initially unresectable or stage iii - iv cases. however, in 19~33% of patients, the associated adverse effects of up to grade 3 - 4 have impeded the use of these agents3 - 6). therefore, the selection of patients with a good performance status is the first consideration for effective neoadjuvant chemotherapy. s-1 is a fourth - generation oral fluoropyrimidine based on the combination of tegafur with two biochemical modulators, 5-chloro-2,4-dihydroxypyridine (cdhp) and potassium oxonate (oxo). s-1 mimics the protracted continuous infusion of 5-fluorouracil (5-fu) with enhanced efficacy and safety7). one earlier and two more current, phase ii trials with monotherapy for advanced gastric cancer, in japan, achieved a remarkable rr of 45% and 54% respectively. these results are consistent with the commonly used combination regimens containing cisplatin or anthracyclines8, 9). s-1 is also known to have less toxicity than other active agents in the treatment of gastric cancer. nevertheless, there are only a few reports evaluating its efficacy for neoadjuvant treatment. in retrospective analyses, s-1 in combination with cisplatin achieved a rr of 44% and 79% and a downstaging rate of 47% and 85%, respectively10, 11). furthermore, s-1 monotherapy as neoadjuvant treatment has recently been reported to induce a pathologically complete response (cr)12, 13) and curative resection. based on these data, we report herein s-1 monotherapy as neoadjuvant treatment in a patient with locally advanced gastric cancer and poor performance status who achieved a curative resection. a 68-year - old man with epigastric pain, anorexia and general weakness for 6 months was admitted to our hospital. there was neither history of pre - existing chronic disease nor a family history of gastric cancer. his performance status was 2 according to the criteria of the eastern cooperative oncology group (ecog). endoscopic examination demonstrated a diffuse - infiltrative lesion along the great curvature of the upper body of the stomach ; the biopsy confirmed a moderately differentiated adenocarcinoma (figure 1a, 1b). abdominal - pelvic computed tomography (ct) showed a gastric cancer with massive lymph node enlargement in the perigastric area, upper retroperitoneum close to the celiac axis and the splenic hilum with no evidence of distant or peritoneal metastases (figure 1c, 1d, 1e). the clinical stage of the patient was ct3n2h0p0m0 according to the staging system of the japanese gastric cancer association (jgca).. however, significant weight loss, advanced age and a relatively poor general condition prevented full - dose administration of a multi - drug systemic chemotherapy regimen. alternately, we planned s-1 monotherapy at a dose of 35 mg / m twice a day for 4 consecutive weeks followed by a 2-week resting period. follow - up endoscopy after 4 cycles showed a decrease in the extent of infiltrative lesions of the stomach (figure 2a). the ct scan demonstrated the disappearance of lymph node enlargement around the perigastric area, celiac axis and also a decrease in the size of the splenic hilar lymph nodes along with a reduction in the stomach mass (figure 2b, 2c, 2d). according to the recist criteria, a pr with a 72% decrease of the target lesions (sum of target lesion, 90 mm to 26 mm) was achieved. during the 4 cycles of treatment the patient had grade 1 fatigue and nausea and grade 2 skin rashes without any grade 3 - 4 toxicity. the performance status improved to ecog 1 after the administration of s-1 and the patient gained 7 kg of body weight. the diagnostic laparoscopy showed a serosa - involving lesion on the upper body of the stomach extending from the anterior to the posterior wall with severe adhesions and surrounding tissues. macroscopically, the excised specimen showed a diffuse infiltrative lesion with serosal surface exposure (se) measuring 180130 mm. an adequate resection margin was obtained on both ends and there was no microscopic involvement at the margins identified. according to becker 's criteria, more than 50% of the tumor bed was composed of tumor cells ; therefore, the pathological response was defined as minor14). the final pathological stage was pt3n2m0 (h0p0), stage iiib, according to both the jgca and the american joint committee on cancer (ajcc) systems. after an additional 3 cycles of s-1 monotherapy were administered as adjuvant chemotherapy, the patient is without any evidence of recurrence after 6 months of follow - up.. early initiation of systemic therapy may result in disease downstaging, eliminate micrometastases and enable a curative resection. furthermore, neoadjuvant chemotherapy provides prognostic information due to the in vivo chemosensitivity testing. however, there are also potential risks involved. by delaying definitive local therapy, in addition, considerable tumor shrinkage is required in a short period for the chemotherapeutic regimen to obtain effective downstaging with the neoadjuvant treatment. therefore, neoadjuvant therapy may not be feasible in some circumstances such as in patients with an unfavorable performance status or advance age. in the clinical trials using cisplatin or anthracycline - based regimens, although downstaging was attained, only 20~54% of the patients completed the planned treatment. as many as one - third of the patients experienced grade 3 - 4 adverse events3 - 6). therefore, novel chemotherapeutic agents such as taxanes, irinotecan and oral fluoropyrimidines might be candidates for future neoadjuvant trials with effects on tumor shrinkage comparable to and more favorable toxicity profiles than traditional agents. among the above cited agents, s-1 might be a useful treatment option because of the results of several phase ii trials on advanced gastric cancer. two japanese retrospective analyses of s-1, in combination with cisplatin as neoadjuvant treatment in stage iii - iv disease, achieved a rr of 44% and 79% and a downstaging rate of 47% and 85%, respectively. however, these high responses are mainly due to a high pr not a pathological cr possibly implicating a survival benefit10, 11). recently, preoperative administration of s-1 alone has been shown to induce favorable responses in patients in poor clinical condition. the total disappearance of peritoneal dissemination was observed with s-1 and cisplatin, and there was a pathological cr in a 78-year old patient who was treated with s-1 alone12). our patient was not a candidate for full - dose multi - drug systemic chemotherapy due to his poor health status. although downstaging was not observed in our case, probably due to a very large initial tumor burden, there was marked resolution of the massive lymphadenopathy around the celiac axis. this is consistent with other phase ii studies on s-1 monotherapy that reported a higher efficacy for the abdominal lymph nodes (75%) compared to the primary gastric mass (28%) or visceral organs (30%)9). another aspect of neoadjuvant chemotherapy to consider is safety, which in turn is associated with treatment compliance. in this respect neutropenia and anemia were the main adverse events reported in asian patients9, 15) and the neoadjuvant combination with cisplatin showed only 7% of cases with grade 3 - 4 adverse events10, 11). moreover, the patient reported a sense of well being, had an improved appetite and gained weight. therefore, s-1 monotherapy was safe and effective treatment for this patient and might be considered in the future as neoadjuvant treatment for gastric cancer. in conclusion, the results of this case study suggest that s-1 might be a safe and effective treatment option for neoadjuvant chemotherapy in locally advanced gastric cancer, especially when accompanied byperigastric lymphadenopathy and a poor general health status in patients of advanced age. the role of s-1 in the neoadjuvant setting for the treatment of gastric cancer remains to be established by clinical trials. | s-1, a novel oral fluoropyrimidine, is an effective therapeutic agent for gastric cancer. herein, we report a case with locally advanced gastric cancer that achieved a curative resection after s-1 monotherapy as neoadjuvant treatment. a 68-year - old man was diagnosed with gastric cancer and massive lymphadenopathy involving the perigastric, celiac axis and splenic hilum. his clinical stage was ct3n2h0p0m0. considering his relatively poor performance (ecog 2, severe weight loss) and advanced age, we started the patient on s-1 monotherapy at a dose of 35 mg / m2 bid for 4 consecutive weeks followed by a 2-week rest. follow - up study after 4 treatment cycles revealed disappearance of the lymphadenopathy of the perigastric and celiac axis with diminished extension of the stomach mass. the patient had a partial response (pr) with a 72% tumor reduction, according to the response evaluation criteria in solid tumors (recist). his performance status was improved to an ecog 1 and he gained 7 kg. a curative (r0) resection was achieved with a radical total gastrectomy and d2 dissection. the pathological stage was pt3n2m0, stage iiib. in conclusion, s-1 neoadjuvant chemotherapy aided in the treatment of gastric cancer in this patient. |
changes in hepatic contour that mimic cirrhosis radiographically, but lack the classic pathological attributes of cirrhosis, are referred to as pseudocirrhosis. features of portal hypertension, such as portosystemic venous collaterals and ascites, as well as hepatic surface nodularity can be seen on computed tomography (ct). pseudocirrhosis has been described previously in patients with cancer with metastases to the liver, both in those who underwent prior systemic chemotherapy and those who did not. in particular, it has been reported almost exclusively in patients with breast cancer with liver metastases [2 - 4 ]. we report on a case of a patient with breast cancer with liver metastases who developed cirrhotic changes during disease progression. a 47-year - old woman visited our hospital in january 2003 because of a painful right breast mass with skin dimpling. on initial evaluation, chest ct revealed the presence of a heterogeneous enhancing breast mass measuring 9 cm with skin invasion and multiple conglomerated lymph nodes in the right axillary area. in addition, multiple metastatic pleural masses with malignant pleural effusion and mediastinal lymph node enlargement were also observed. accordingly, she was diagnosed with stage iv right breast cancer with multiple pleural and bone metastases. follow - up ct scan performed after four cycles of chemotherapy showed partial regression of the breast mass and multiple metastatic masses in the pleura and axillary area. she then underwent palliative total mastectomy of her right breast because of an ulcerated skin lesion. pathological examination showed invasive ductal carcinoma with nuclear grade 2 and lymphatic and perineural invasion. immunohistochemistry studies showed positive staining for the estrogen receptor (er) protein, progesterone receptor protein, and human epidermal growth factor receptor 2 (her2) (score 3). a follow - up ct scan showed stable disease and she then started taking tamoxifen (20 mg daily) in july 2003. two years later, in november 2005, a surveillance breast ultrasound showed an irregular circumscribed mass measuring 1 cm on theright mastectomy site. although previous metastatic lesions showed stable disease, a new chest wall lesion had developed ; therefore, her treatment was switched from tamoxifen to the non - steroidal aromatase inhibitor anastrozole. at that time, she was postmenopausal, based on her serum follicle - stimulating hormone levels. in january 2007, a bone scan showed new increased uptake in the right second rib, the third anterior rib, and the right acetabulum. she had progressive disease of the bone ; therefore, capecitabine (an oral prodrug of 5-fluorouracil ; 2,500 mg / m / day) was started (two weeks on, one week off). after nine cycles of chemotherapy, she had stable disease and was off chemotherapy for approximately four years with no evidence of progression. however, follow - up ct scan performed in june 2011 showed multiple newly developed peripheral enhancing nodules in the liver (fig. she received six cycles of chemotherapy until november 2011, and ct scan showed that her metastatic hepatic lesions were stable. since then, her chemotherapy has been discontinued because of grade 4 neutropenia and osteomyelitis of the mandible. four months later, follow - up ct scan showed ill - defined heterogeneous enhancing lesions in the entire liver with surface nodularity. the liver had a nodular contour consistent with cirrhosis, as well as moderate ascites (fig. f18 fluorodeoxyglucose (fdg) positron emission tomography / computed tomography (pet - ct) also showed disseminated and innumerable lesions throughout almost the entire liver with increased fdg uptake (maximum standardized uptake value [suvmax ], 7.3), suggesting diffuse liver metastasis (fig. she denied a history of liver disease, alcohol use, and risk factors for viral hepatitis. serologies for viral or autoimmune etiologies of cirrhosis were negative and the serum level of alpha - fetoprotein was normal. an ultrasound - guided liver biopsy was performed in order to confirm the cause of the cirrhotic changes. pathological examination showed that the hepatic parenchyma was diffusely infiltrated by poorly differentiated carcinoma cells. hepatocytes were almost replaced by carcinoma cells and extensive fibrosis between clusters of cancer cells was observed (fig. 3a and b). immunohistochemical staining was positive for gross cystic disease fluid protein-15, moc 31, er, and her2 (score 3), but negative for hepatocytes (fig. 3c and d). follow - up pet - ct showed progression of liver metastases with increased fdg uptake (suvmax, 9.0) since the scan performed in june 2011. therefore, we concluded that the hepatic metastases from breast cancer had progressed and led to cirrhotic changes in the liver. subsequently, her chemotherapy regimen was changed to trastuzumab and docetaxel. however, bleeding of esophageal varices occurred on the sixth day after initiation of chemotherapy, and hepatic failure progressed gradually. cirrhosis is the end result of liver injury characterized by distortion of the hepatic architecture by extensive fibrosis and formation of regenerative nodules. cirrhosis is defined by its typical pathological features : 1) presence of regenerating nodules of hepatocytes and 2) presence of bridging fibrosis between these nodules. ct findings of cirrhosis usually include diffuse hepatic surface nodularity and/or signs of portal hypertension such as splenomegaly, ascites, or portosystemic varices. the term ' pseudocirrhosis ' is used in the following cases : radiographic findings that resemble macronodular cirrhosis, but in which histopathological specimens fail to show the typical findings of cirrhosis. the most frequently reported cause of pseudocirrhosis is breast cancer with liver metastases treated with chemotherapy ; however, cirrhotic changes have also been reported in hepatic metastasis of a variety of cancers, including pancreatic cancer, esophageal cancer, small - cell lung cancer, and thyroid cancer [7 - 10 ]. reported follow - up ct results of 91 patients undergoing chemotherapy for breast cancer metastatic to the liver. they found that 16 (17%) patients showed diffuse nodularity of hepatic contour and eight (9%) patients showed signs of portal hypertension on serial ct. another study reported that various degrees of hepatic capsular retractions (ranging from 1 to 10 mm in depth) were observed in 29 (50%) of 58 patients with hepatic metastases from breast cancer. based on previous studies, the causes of pseudocirrhosis in the setting of cancer have been classified according to two categories : 1) hepatic response to chemotherapeutic agents and 2) fibrosis combined with metastatic infiltrating tumors. the first category is assumed to be a result of the hepatotoxic effect of systemic chemotherapy or a response to chemotherapy by tumor tissues. reported that five patients with breast carcinoma that was metastatic to the liver who had undergone chemotherapy and hormonal therapy developed pseudocirrhosis, despite a decrease in the size of their liver lesions. hepatic histology in this setting is consistent with nodular regenerative hyperplasia with subsequent compression and atrophy of intervening parenchyma without fibrosis. systemic chemotherapy can result in hepatic capsular retraction by tumor shrinkage and subsequent scar formation around the treated metastases, thus resembling macronodular cirrhosis. alternatively, nodular regenerative hyperplasia in response to chemotherapy - induced hepatic injury has been proposed as the mechanism of pseudocirrhosis. young. reported on the pathological findings of 22 patients who had findings of pseudocirrhosis on ct. six of the seven patients for whom tumor tissue could be obtained had nodular regenerative hyperplasia without fibrosis. another form of pseudocirrhosis occurs in cases in which hepatic histology shows evidence of extensive fibrosis representing a profound desmoplastic response to the infiltrating tumor. nascimento. reported that two patients with pseudocirrhosis exhibited severe desmoplastic fibrosis and extensive tumor infiltration. in our case, we also confirmed diffuse infiltration of tumor cells and extensive fibrosis that developed during progression of liver metastases. first, cirrhotic changes may hamper interpretation of the evaluation of disease response during ct imaging. currently, histological evaluation of liver biopsy specimens is critical for determining whether chemotherapeutic response or liver metastasis is the cause of pseudocirrhosis ; however, this is an invasive procedure. in our case, a pet - ct scan showed progression of liver metastases with increased fdg uptake consistent with pathological findings. reported different results regarding the role of pet - ct scan in the liver of a patient with autopsy evidence of diffuse infiltration by tumor cells. in their case of diffuse desmoplastic metastatic breast cancer simulating cirrhosis, pet - ct scans showed inhomogeneous uptake consistent with cirrhosis, but no focal areas of increased uptake suggestive of fdg - avid malignancy. therefore, further studies of the role of pet - ct scan in evaluation of the response to chemotherapy in patients with pseudocirrhosis are needed. second, there is a need for awareness of the risk of hepatic decompensation and the complication of portal hypertension. the clinical manifestations of portal hypertension, such as hepatic encephalopathy and variceal bleeding, which can bringon a life threatening condition, can be similar to those seen in classic cirrhosis. third, some unresolved challenges remain, such as the relationship between any individual agent and development of pseudocirrhosis (which is more common in breast cancer than in other malignancies) and the relationship between the clinicopathological characteristics of breast cancer and pseudocirrhosis. fennessy. reported that hepatic capsular retraction in breast cancer with liver metastasis was associated with larger metastases and both an increase and a decrease in the size of subjacent lesions. however, it is unrelated to lesion number, histopathology, receptor status, or chemotherapeutic regimen. therefore, further studies are required in order to address these problems and to determine the impact of changes in liver on patient survival and for development of appropriate strategies for treatment of patients with pseudocirrhosis. in conclusion, pseudocirrhosis can occur in breast cancer patients with liver metastasis treated by chemotherapy. in this histopathological examination and pet - ct may be helpful in determining the direction of treatment. appropriate management of portal hypertension, as well as treatment of the cancer, are also important. | pseudocirrhosis refers to a condition that shows changes in hepatic contour that mimic cirrhosis radiographically in the absence of the typical histopathological findings of cirrhosis. this condition has been observed in patients with cancer metastatic to the liver, both in those who have undergone prior systemic chemotherapy and those who have not. pseudocirrhosis may cause difficulty in interpretation of the response to chemotherapy and hepatic decompression and complication of portal hypertension have a negative effect on the prognosis. we report on a case of breast cancer with liver metastases that showed cirrhotic changes during disease progression. progression of liver metastases was confirmed by f18 fluorodeoxyglucose positron emission tomography / computed tomography (pet - ct). we also performed ultrasound - guided liver biopsy and confirmed tumor infiltration with severe desmoplastic fibrosis. this case suggests the pathogenesis of pseudocirrhosis through histopathological findings and the role of pet - ct in evaluation of the response to chemotherapy in patients with pseudocirrhosis. |
bhopal gas leak disaster is the biggest industrial disaster in human history. on the night of 2/3 december 1984, about 40 tons of methyl isocyanate (mic) from tank 610 of union carbide india limited (ucil) factory at bhopal, in central india, leaked into the surrounding environment. this leak of an extremely hazardous chemical which occurred over a short span of few hours killing > 2000 people, covered the city of bhopal in a cloud of poisonous gas. the union carbide factory at bhopal was part of india 's response to the severe food shortages in 1960 s. in 1969, bhopal is the capital city of the state of madhya pradesh. in 1984, the population of bhopal was about 700,000. the city was chosen, for setting up of the pesticide plant, on the basis of its central location in the country, railway services connecting the city to rest of india and the availability of a large natural lake to provide adequate water supply. the chemical plant was located only about 2 km from the railway station and not far from the residential quarters. until 1979 mic is one of the many intermediates used in the production of the powerful pesticide sevin. it is also twice as heavy as air and as a result, in a free environment, it remains close to the ground. ironically, only in 1983, the indian government had (1-year prior to the disaster) extended the plant 's license for 7 years after a promise that the plant would secure from its parent company the technology to handle emergency situations like toxic gas release, sometimes accompanied with fire, endangering the safety of the community. there are reports that 4 months before the tragedy, the us multinational had decided to dismantle its bhopal installations to relocate in brazil and indonesia. on the night of december 2/3 1984, the gas spread and covered about 7 km radius of the plant and directly affected about 200,000 population. more than 2000 (about 1% of directly exposed) died on the night of the disaster. the cause is thought to be due to the entry of water into tank with mic or the spontaneous polymerization (in the absence of inhibitors) of the liquid of mic, which had been in storage for over a month, a longer period than normal. in addition to this, (i) the gauges measuring the temperature and pressures were not functioning properly ; (ii) the refrigeration unit for keeping the tank of mic cool had been shut off for sometime ; (iii) the gas scrubber had been shut off for maintenance ; and (iv) the flare tower, which could have burned off the escaping mic, was not functional. thus, the disaster was the result of a combination of a number of factors of negligence and poor operational procedures. though the estimated number of persons who died immediately was around 2000, in the following years it is estimated to have killed > 25,000 persons. in addition, at least 200,000 population who were exposed to the gas leak and survived are experiencing a wide variety of health problems and disabilities. the major milestones in the legal responsibility were the passing of the bhopal gas relief act in 1985 and the settlement of government of india and the company for the 1-time compensation of $ 470 million. however, the legal battles for the rights and relief to the survivors continued to occupy public space. in addition, the issues of the health damage to the population, legal liability of the company and the continuing need of the affected population continued to be active issues in india and internationally. august 2012, supreme court judgment relating to health needs of the population is a milestone. this judgment both recognized the rights of the survivors and directed for implementation of specific measures to provide health care to the survivors. the 30 anniversary, in december 2014, focused on all aspects of the disaster as reflected in the 13 part series of articles in the statesman newspaper covering deficiencies in response with regard to law, health, factory regulation, rehabilitation, compensation, and human rights. the indian council of medical research (icmr) new delhi, responded immediately to the disaster by giving importance to understand the health effects on a priority basis. the council brought together a large group of health researchers from different parts of india to study a wide variety of health effects - ranging from the immediate effects on the eye, the lungs, the gastrointestinal system, the gynecological problems and mental health effects. these research efforts were part of a larger national initiative to understand the various aspects of the bhopal disaster on the people, the environment, and the legal aspects of the chemical industry. bhopal disaster is the first disaster in india to be studied systematically for the mental health effects. earlier reports on the mental health impact of disasters were descriptive and related to the cyclones in andhra pradesh and circus tragedy in bengaluru. rao and zubair reported that the majority (77.5%) of the studied patients affected from cyclone in andhra pradesh were suffering from neurotic disorder. the mental health research of the bhopal disaster can be considered under four periods of time. the direct involvement of the psychiatrists / neurologists at the field level did not occur till about 8 weeks after the disaster. this delay was in spite of the recognition of the importance of mental health effects of the disaster within the first fortnight of the disaster. by coincidence, the fourth advisory committee on mental health of icmr was meeting on december 1214, 1984. the experts in the meeting recognized the need of the affected population as follows : the recent developments at bhopal involving the exposure of normal human beings to substances toxic to all the exposed and fatal to many, raises a number of mental health needs. the service needs and research the acute needs are the understanding and provision of care for confusional states, reactive psychoses, anxiety - depression reactions and grief reactions. long - term needs arise from the following areas, namely : (i) psychological reactions to the acute and chronic disabilities, (ii) psychological problems of the exposed subjects, currently not affected, to the uncertainties of the future, (iii) effects of broken social units on children and adults, and (iv) psychological problems related to rehabilitation. however, in spite of this early recognition of the need for mental health interventions there was a delay of 8 weeks before mental health professionals were involved. an important reason for this was the absence of mental health professionals in the state of madhya pradesh and the city of bhopal in 1984. at that point of time, none of the five medical colleges had a psychiatrist on the staff. during this period of 10 years, there were a large number of studies, both as part of the general health surveys and specific mental health studies. as part of the general health studies, andersson. reported the first community survey within 2 weeks of the disaster, in eight exposed areas and two nonexposed clusters of households with a 2 months follow - up. though the focus of the survey was eye and lung problems, the study authors noted that the pupillary reflex was normal and they conclude the fact that this reflex was normal in all groups can not be taken as evidence that neurotoxicity did not occur. report on 33 adult patients treated during the acute phase at the medical college hospital. they found that symptoms of severe cough and dyspnea were followed by fainting in 55% of the patients. one patient, who had suffered from prolonged unconsciousness, had myoclonic jerks localized to the right upper extremity and generalized hyperreflexia, suggestive of encephalopathy. three patients who had prolonged unconsciousness and brisk deep tendon jerks and extensor plantar response. mild to moderate headache (55%), giddiness (46%), burning sensation in hands and feet (9%) and hypoanesthesia (3%) were also reported. at the 3 months follow - up of this group of patients, gupta. studied systematically 687 affected persons of various age groups and from different affected areas, 2 months after the disaster and an another 592 persons after the 4 months period. the behavioral studies were carried out in 350 adults. the gas exposed groups, especially the females had poor scores in the auditory memory tests. the exposed male group showed significant low visual memory as compared to controls and females. cullinan. carried out an epidemiological study of a representative gas - exposed population, 9 years after the disaster, in january 1994. of this sample, 76 were subjected to detailed neurological testing which included vestibular and peripheral sensory function and tests for short - term memory. in this study, a high proportion of subjects reported a wide variety of neuropsychiatric symptoms such as abnormal smell, abnormal taste, faintness, headache, difficulty to stay awake and abnormal balance. when compared to 50% in the control population. neurological examination showed that a high proportion was judged to have clinical evidence of central, peripheral or vestibular neurological disease. the mean short - term memory scores were lowest among those heavily exposed (1.0 vs. 3.0). in this group, the psychological symptoms reported were fatigue (88%), anxiety (65%), difficulty in concentration (64%). difficulty in decision - making was reported in 80% as compared to 35% in the control population. there was a consistent gradient across the separate exposure groups for all symptoms except depression. approximately, 25% reported symptoms of depression. specific mental health studies started following the initial 1-week exploratory visit of, in the 1 week of february 1985, by dr. srinivasa murthy, of the national institute of mental health and neurosciences (nimhans), bengaluru, and professor sethi, of k.g. medical college (kgmc), lucknow. the team visited bhopal and examined the general population and patients attending the general health facilities. they also interacted with the medical personnel to understand the magnitude and nature of the mental health problems in the affected population. their observations, following a week 's work, were based on clinical and unstructured interviews. these initial observations led to an estimate of the magnitude of mental health needs of the population at 50% of those in the community and of about 20 - 30% of those attending medical facilities. immediately following these observations, during february - april 1985, a psychiatric team from kgmc, lucknow carried out systematic studies. as a first step, 10 general medical clinics in the disaster - affected area were chosen. a team consisting of a psychiatrist, a clinical psychologist, and a social worker visited one clinic a day, by rotation in a randomized fashion, on three occasions and screened all the newly registered adult patients with the help of a psychiatric screening questionnaire namely, self - reporting questionnaire (srq). subjects identified as probable psychiatric patients were then evaluated in detail by the psychiatrist with the help of a standardized psychiatric interview, the present state examination (pse). clinical diagnosis was based on the international classification of diseases (9 revision) (icd-9) (who, 1975). during a period of 3 months (february may 1985), of the 855 patients screened at the 10 clinics, on the basis of their srq scores, of these potentially mentally ill people, 44 could not be evaluated, and 215 were assessed using the pse. the confirmed number of psychiatric patients was 193, yielding a prevalence rate of 22.6%. most of the patients were females (8.11%) under 45 years of age (74%). the main diagnostic categories were anxiety neurosis (25%), depressive neurosis (37%), adjustment reaction with prolonged depression (20%), and adjustment reaction with predominant disturbance of emotions (16%). cases of psychosis were rare, and they were not related to the disaster. during the same period, in the 3 month of the postdisaster period, this was a survey of the gas - affected patients admitted to the various hospitals in the bhopal city. evidence of involvement of the central nervous system was present in three patients in the form of stroke, encephalopathy and cerebellar ataxia. many patients reported transitory symptoms like loss of consciousness (50%), muscle weakness, tremors, vertigo, ataxia and easy fatigability. of the 47 gas affected children, loss of consciousness at some time or other occurred in half of the patients. mental regression was observed in one child who had commenced speaking in sentences but stopped talking after the disaster. an important observation by the doctors who had examined the children during the early phase of illness was generalized hypotonia and weakness. floppy with weakness of limb movements and had difficulty in getting up from the ground. of the 3 patients who had central nervous system involvement, the patient with stroke died. his autopsy showed intense congestion and petechial hemorrhages of the gray and white matter with frank hemorrhage in the circle of willis area, perhaps indicating the sustained microvascular damage by the circulating mic. longitudinal epidemiological study of mental health effects was initiated by the icmr, new delhi. this was part of the total medical research involving the bhopal gas affected population, the bhopal gas disaster research centre. two mental health studies and one training intervention were taken up, during 19851994 period. the objectives of the epidemiological study was to (i) study the prevalence of psychiatric disorders in mic exposed and nonexposed areas ; (ii) study the factors associated with psychiatric disorders ; (iii) study the course and outcome of disease in identified cases (at first survey) and (iv) carry out annual (2, 3, 4 and 5 year) prevalence surveys on independently drawn samples. screening tool was used for initial screening, followed by psychiatrists / psychologists administering pse to arrive at the diagnosis. a random sample of 700 families from each area, that is, severely exposed area, mildly exposed area and control area, was administered to the head of the family as well as information on the same schedule, regarding other adult member of the family (aged 16 +) was collected. if any member of the family was rated positive on three or more items, that individual was further was examined in detail. a semi - structured proforma regarding psychiatric history, personal history, premorbid personality etc. subjects diagnosed as having psychiatric problems were assessed using the pse and they were referred to psychiatrist of hamidia hospital, bhopal for further medical care. the prevalence rate of psychiatric morbidity was about 4 times higher in the exposed area in comparison to nonexposed area. the result showed that exposure to mic gas was an important factor for the emergence of psychiatric disorders. prevalence rate of psychiatric disorders was higher among those persons who were present in their houses in the night of gas leakage. the prevalence rate in the severely exposed area was 139.2/1000 and in the mildly exposed area 80.8/1000, whereas, in nonexposed area it was 26.8/1000. similarly, the people, who were sleeping outside their houses, had higher prevalence rate of psychiatric morbidity (145.8/1000) in comparison to those people who were inside the house (108.5/1000). among the demographic variables income, was an important factor. it was observed that in the initial survey people belonging to lower income group (per capita income less than rs. 50/-/month) had highest prevalence rate of psychiatric disorders (269.2/1000), whereas, prevalence rate in the middle - income group was comparatively lower (122.9/1000). the psychiatric morbidity in relation to religion it was found that the prevalence rate was higher among the muslim community in comparison to hindus. the prevalence rate of psychiatric disorders during almost every rotational survey has been higher among muslims than hindus. the similar condition has been observed in exposed and nonexposed areas and also during the rotational surveys. all the persons diagnosed with psychiatric disorders were yearly followed - up to ascertain, any change in the mental status of patients (whether the patient had a remission of symptoms for a period of at least 30 days since the initial evaluation ? 279 cases in the severely exposed area, 148 in the mildly exposed area and 47 in the nonexposed area. in the first follow - up, there were 230 patients actually followed - up, remaining 47 patients had either migrated of died. 3.9% patients were also in the episode of symptom, but they were not in a continuous state. seven patients out of 230 were rated symptom - free. during first, second, third and fourth follow - up the percentage of patients in continuous illness were 89.6, 66.8, 56.8 and 47.4 respectively. remission of symptoms were not present in the majority of patients during the first follow - up, and it gradually comes down in the fourth follow - up. it is interesting to mention here that the majority of patients (58.3%) took treatment for psychiatric disorders from the general physician during the first follow - up. whereas 37.4% patients did not take treatment from any source at the time of follow - up. during second, third and fourth follow - up the rate of recovery also increased gradually in the mildly exposed area and in nonexposed area. in the mildly exposed area, it increases from 7.6% to 44.4% and in nonexposed area it is from 14.3% to 36.6%. significantly point to note is the nearly half of the patients, continuing to be ill at the end of 5 years of follow - up. a pilot psychiatric study of children (015 years) affected by mic in bhopal, with aim to compare the frequency and types of psychiatric disorders and intellectual levels of children (016 years) of 100 families (having at least one child between 0 and 16 years) randomly selected from one area severely affected by mic in bhopal and 100 families (having at least one child between 0 and 16 years) from the control area was carried out. the rate of psychiatric disorders in exposed area was 12.66% when compared to 2.4% in the control area. another activity of this period was the training of medical officers in mental health care in the bhopal city to address the need for urgent mental health care in the city. one of the challenges faced by the team of psychiatrists was the provision of psychiatric services to the affected population. for a total population of 700,000 and the affected population of about 200,000 the basic aim of the training was to enhance the sensitivity of the medical officers to the emotional needs of individuals and to provide the skills to recognize, diagnose, treat and refer (when required) the mental health problems. the training was practical utilizing case studies and group discussions, along with audio - visual, audio taped material of the affected population with maximum learner involvement. a manual was prepared for this training on the basis of experience of training on the basis of experience of training primary care physicians medical officers at nimhans, bengaluru. a revised manual incorporating the experience of the training and the needs of the medical officers was prepared subsequently and distributed to all the doctors working with the gas affected population. the comments of the participants in the posttraining evaluation supported the usefulness of the training. most of them felt that with the training, they were more capable of treating psychiatric illness and other patients having medical problems as well. some doctors expressed that earlier, they used to give the patients only symptomatic treatment, but after the training they were able to think and diagnosed the condition in terms of a psychological approach. some doctors mentioned that earlier to the training, they were not aware of any psychiatric problems and were of the opinion that the patients were malingering and giving vague symptoms to evoke a sympathetic response and get more medicines. the important development of this period was the setting up of the department of psychiatry, as part of the bhopal memorial hospital and research centre (bmhrc) in 2000, providing mental health care to survivors. one qualitative study of the mental health needs of the population was also carried our during this period. this study explored qualitatively the state of mental health of survivors in 2003 and the adequacy of mental health services provided to them. twenty - six people suffering from various mental health needs were the subject of detailed interviews. the salient findings of this study were the following : based on the interviews with victims (survivors) themselves, as well as with professionals, it highlights the fact that despite the continuing suffering of the victims, no systematic effort has been made to tackle the mental health problems that were generated as an impact of the gas leak. following the court judgment of june 2010, and the high dissatisfaction expressed by the survivors, there was a revival of the health care and research. there was a higher level of compensation to survivor family members of the dead persons, to those with chronic kidney diseases and cancer diagnoses. the national institute for research in environmental health (nireh) was set up in bhopal. the mental health studies of nireh, focused on understanding of the continuing mental health needs of the population and developing a training program and manual for training in mental health for the general medical officers of bhopal. the first effort was a follow - up assessment for the health status of the psychiatric patients of the 19851994 was completed that showed the chronicity of the mental disorders in the majority of the patients and the limited mental health care patients had received. in an another study of treatment utilization of the psychiatric patients, cared for during 2010 at bmhrc. this analysis of the routine clinical records pointed to the limited utilization of the available mental health care, and the problems patients had in utilizing the services. the third effort, was with 500 families in one area, to understand the prevalence of mental disorders in 2012. this study was a two - stage screening for mental disorders and showed about 20% had potential mental disorders and the rates were much higher in those living in socioeconomically difficult life situations and with medical conditions. the fourth activity was to develop a mental health manual for the medical officers involving the psychiatrists of bhopal city. the final activity, during this period, was to carry our five training programs of 3 days each (for groups of 615 medical officers) of the gas rahat health department and the doctors working in the mini units of bmhrc. a revised mental health manual to address the continuing mental health needs has been developed. a striking positive development in the mental health care situation in the city, during the last three decades, have been the increase in the mental health professionals both in the government and private sectors along with in - patient care facilities at gandhi medical college, all india institute of medical sciences, bhopal, all the three private medical colleges in the city, bmhrc, bhopal, beml and in the private sector. continuing mental health needs of the population, in 2014, are : (i) people with postdisaster anxiety - depression, posttraumatic stress disorder, adjustment disorder conditions, directly related to the disaster of 1984 ; (ii) people with psychiatric disorders, attributable to the various life changes, family and occupational status, resulting directly (e.g., unemployment due to poor health condition) and indirectly (e.g., loss of head of the family in disaster) from the disaster experiences ; (iii) people with chronic physical conditions like chronic obstructive pulmonary disease, diabetes, hypertension, cancer, with associated psychiatric disorders such as depression, adjustment disorders ; and (iv) people with psychiatric disorders, not directly related to the disaster. the direct involvement of the psychiatrists / neurologists at the field level did not occur till about 8 weeks after the disaster. this delay was in spite of the recognition of the importance of mental health effects of the disaster within the first fortnight of the disaster. by coincidence, the fourth advisory committee on mental health of icmr was meeting on december 1214, 1984. the experts in the meeting recognized the need of the affected population as follows : the recent developments at bhopal involving the exposure of normal human beings to substances toxic to all the exposed and fatal to many, raises a number of mental health needs. the service needs and research can be viewed both in the short - term and long - term perspectives. the acute needs are the understanding and provision of care for confusional states, reactive psychoses, anxiety - depression reactions and grief reactions. long - term needs arise from the following areas, namely : (i) psychological reactions to the acute and chronic disabilities, (ii) psychological problems of the exposed subjects, currently not affected, to the uncertainties of the future, (iii) effects of broken social units on children and adults, and (iv) psychological problems related to rehabilitation. however, in spite of this early recognition of the need for mental health interventions there was a delay of 8 weeks before mental health professionals were involved. an important reason for this was the absence of mental health professionals in the state of madhya pradesh and the city of bhopal in 1984. at that point of time, none of the five medical colleges had a psychiatrist on the staff. during this period of 10 years, there were a large number of studies, both as part of the general health surveys and specific mental health studies. as part of the general health studies, andersson. reported the first community survey within 2 weeks of the disaster, in eight exposed areas and two nonexposed clusters of households with a 2 months follow - up. though the focus of the survey was eye and lung problems, the study authors noted that the pupillary reflex was normal and they conclude the fact that this reflex was normal in all groups can not be taken as evidence that neurotoxicity did not occur. report on 33 adult patients treated during the acute phase at the medical college hospital. they found that symptoms of severe cough and dyspnea were followed by fainting in 55% of the patients. one patient, who had suffered from prolonged unconsciousness, had myoclonic jerks localized to the right upper extremity and generalized hyperreflexia, suggestive of encephalopathy. three patients who had prolonged unconsciousness and brisk deep tendon jerks and extensor plantar response. mild to moderate headache (55%), giddiness (46%), burning sensation in hands and feet (9%) and hypoanesthesia (3%) were also reported. at the 3 months follow - up of this group of patients, depression and irritability were the commonly reported symptoms. gupta. studied systematically 687 affected persons of various age groups and from different affected areas, 2 months after the disaster and an another 592 persons after the 4 months period. the behavioral studies were carried out in 350 adults. the gas exposed groups, especially the females had poor scores in the auditory memory tests. the exposed male group showed significant low visual memory as compared to controls and females. cullinan. carried out an epidemiological study of a representative gas - exposed population, 9 years after the disaster, in january 1994. of this sample, 76 were subjected to detailed neurological testing which included vestibular and peripheral sensory function and tests for short - term memory. in this study, a high proportion of subjects reported a wide variety of neuropsychiatric symptoms such as abnormal smell, abnormal taste, faintness, headache, difficulty to stay awake and abnormal balance. when compared to 50% in the control population. neurological examination showed that a high proportion was judged to have clinical evidence of central, peripheral or vestibular neurological disease. the mean short - term memory scores were lowest among those heavily exposed (1.0 vs. 3.0). the psychological symptoms reported were fatigue (88%), anxiety (65%), difficulty in concentration (64%). difficulty in decision - making was reported in 80% as compared to 35% in the control population. there was a consistent gradient across the separate exposure groups for all symptoms except depression. approximately, 25% reported symptoms of depression. specific mental health studies started following the initial 1-week exploratory visit of, in the 1 week of february 1985, by dr. srinivasa murthy, of the national institute of mental health and neurosciences (nimhans), bengaluru, and professor sethi, of k.g. medical college (kgmc), lucknow. the team visited bhopal and examined the general population and patients attending the general health facilities. they also interacted with the medical personnel to understand the magnitude and nature of the mental health problems in the affected population. their observations, following a week 's work, were based on clinical and unstructured interviews. these initial observations led to an estimate of the magnitude of mental health needs of the population at 50% of those in the community and of about 20 - 30% of those attending medical facilities. immediately following these observations, during february - april 1985, a psychiatric team from kgmc, as a first step, 10 general medical clinics in the disaster - affected area were chosen. a team consisting of a psychiatrist, a clinical psychologist, and a social worker visited one clinic a day, by rotation in a randomized fashion, on three occasions and screened all the newly registered adult patients with the help of a psychiatric screening questionnaire namely, self - reporting questionnaire (srq). subjects identified as probable psychiatric patients were then evaluated in detail by the psychiatrist with the help of a standardized psychiatric interview, the present state examination (pse). clinical diagnosis was based on the international classification of diseases (9 revision) (icd-9) (who, 1975). during a period of 3 months (february may 1985), of the 855 patients screened at the 10 clinics, on the basis of their srq scores, 259 were identified as having a potential mental disorder. of these potentially mentally ill people, 44 could not be evaluated, and 215 were assessed using the pse. the confirmed number of psychiatric patients was 193, yielding a prevalence rate of 22.6%. most of the patients were females (8.11%) under 45 years of age (74%). the main diagnostic categories were anxiety neurosis (25%), depressive neurosis (37%), adjustment reaction with prolonged depression (20%), and adjustment reaction with predominant disturbance of emotions (16%). cases of psychosis were rare, and they were not related to the disaster. during the same period, in the 3 month of the postdisaster period, neurological studies were carried out. this was a survey of the gas - affected patients admitted to the various hospitals in the bhopal city. evidence of involvement of the central nervous system was present in three patients in the form of stroke, encephalopathy and cerebellar ataxia. many patients reported transitory symptoms like loss of consciousness (50%), muscle weakness, tremors, vertigo, ataxia and easy fatigability. of the 47 gas affected children, loss of consciousness at some time or other occurred in half of the patients. mental regression was observed in one child who had commenced speaking in sentences but stopped talking after the disaster. an important observation by the doctors who had examined the children during the early phase of illness was generalized hypotonia and weakness. floppy with weakness of limb movements and had difficulty in getting up from the ground. of the 3 patients who had central nervous system involvement, the patient with stroke died. his autopsy showed intense congestion and petechial hemorrhages of the gray and white matter with frank hemorrhage in the circle of willis area, perhaps indicating the sustained microvascular damage by the circulating mic. longitudinal epidemiological study of mental health effects was initiated by the icmr, new delhi. this was part of the total medical research involving the bhopal gas affected population, the bhopal gas disaster research centre. two mental health studies and one training intervention were taken up, during 19851994 period. the objectives of the epidemiological study was to (i) study the prevalence of psychiatric disorders in mic exposed and nonexposed areas ; (ii) study the factors associated with psychiatric disorders ; (iii) study the course and outcome of disease in identified cases (at first survey) and (iv) carry out annual (2, 3, 4 and 5 year) prevalence surveys on independently drawn samples. screening tool was used for initial screening, followed by psychiatrists / psychologists administering pse to arrive at the diagnosis. a random sample of 700 families from each area, that is, severely exposed area, mildly exposed area and control area, were surveyed, for each rotational survey independently. the mental health item sheet of verghese. was administered to the head of the family as well as information on the same schedule, regarding other adult member of the family (aged 16 +) was collected. if any member of the family was rated positive on three or more items, that individual was further was examined in detail. a semi - structured proforma regarding psychiatric history, personal history, premorbid personality etc. subjects diagnosed as having psychiatric problems were assessed using the pse and they were referred to psychiatrist of hamidia hospital, bhopal for further medical care. the prevalence rate of psychiatric morbidity was about 4 times higher in the exposed area in comparison to nonexposed area. the result showed that exposure to mic gas was an important factor for the emergence of psychiatric disorders. prevalence rate of psychiatric disorders was higher among those persons who were present in their houses in the night of gas leakage. the prevalence rate in the severely exposed area was 139.2/1000 and in the mildly exposed area 80.8/1000, whereas, in nonexposed area it was 26.8/1000. similarly, the people, who were sleeping outside their houses, had higher prevalence rate of psychiatric morbidity (145.8/1000) in comparison to those people who were inside the house (108.5/1000). among the demographic variables income, it was observed that in the initial survey people belonging to lower income group (per capita income less than rs. 50/-/month) had highest prevalence rate of psychiatric disorders (269.2/1000), whereas, prevalence rate in the middle - income group was comparatively lower (122.9/1000). the psychiatric morbidity in relation to religion it was found that the prevalence rate was higher among the muslim community in comparison to hindus. the prevalence rate of psychiatric disorders during almost every rotational survey has been higher among muslims than hindus. the similar condition has been observed in exposed and nonexposed areas and also during the rotational surveys. all the persons diagnosed with psychiatric disorders were yearly followed - up to ascertain, any change in the mental status of patients (whether the patient had a remission of symptoms for a period of at least 30 days since the initial evaluation ? 279 cases in the severely exposed area, 148 in the mildly exposed area and 47 in the nonexposed area. in the first follow - up, there were 230 patients actually followed - up, remaining 47 patients had either migrated of died 3.9% patients were also in the episode of symptom, but they were not in a continuous state. seven patients out of 230 were rated symptom - free. during first, second, third and fourth follow - up the percentage of patients in continuous illness were 89.6, 66.8, 56.8 and 47.4 respectively. remission of symptoms were not present in the majority of patients during the first follow - up, and it gradually comes down in the fourth follow - up. it is interesting to mention here that the majority of patients (58.3%) took treatment for psychiatric disorders from the general physician during the first follow - up. whereas 37.4% patients did not take treatment from any source at the time of follow - up. during second, third and fourth the rate of recovery also increased gradually in the mildly exposed area and in nonexposed area. in the mildly exposed area, it increases from 7.6% to 44.4% and in nonexposed area it is from 14.3% to 36.6%. significantly point to note is the nearly half of the patients, continuing to be ill at the end of 5 years of follow - up. a pilot psychiatric study of children (015 years) affected by mic in bhopal, with aim to compare the frequency and types of psychiatric disorders and intellectual levels of children (016 years) of 100 families (having at least one child between 0 and 16 years) randomly selected from one area severely affected by mic in bhopal and 100 families (having at least one child between 0 and 16 years) from the control area was carried out. the rate of psychiatric disorders in exposed area was 12.66% when compared to 2.4% in the control area. another activity of this period was the training of medical officers in mental health care in the bhopal city to address the need for urgent mental health care in the city. one of the challenges faced by the team of psychiatrists was the provision of psychiatric services to the affected population. for a total population of 700,000 and the affected population of about 200,000 the basic aim of the training was to enhance the sensitivity of the medical officers to the emotional needs of individuals and to provide the skills to recognize, diagnose, treat and refer (when required) the mental health problems. the training was practical utilizing case studies and group discussions, along with audio - visual, audio taped material of the affected population with maximum learner involvement. a manual was prepared for this training on the basis of experience of training on the basis of experience of training primary care physicians medical officers at nimhans, bengaluru. a revised manual incorporating the experience of the training and the needs of the medical officers was prepared subsequently and distributed to all the doctors working with the gas affected population. the comments of the participants in the posttraining evaluation supported the usefulness of the training. most of them felt that with the training, they were more capable of treating psychiatric illness and other patients having medical problems as well. some doctors expressed that earlier, they used to give the patients only symptomatic treatment, but after the training they were able to think and diagnosed the condition in terms of a psychological approach. some doctors mentioned that earlier to the training, they were not aware of any psychiatric problems and were of the opinion that the patients were malingering and giving vague symptoms to evoke a sympathetic response and get more medicines. the important development of this period was the setting up of the department of psychiatry, as part of the bhopal memorial hospital and research centre (bmhrc) in 2000, providing mental health care to survivors. one qualitative study of the mental health needs of the population was also carried our during this period. this study explored qualitatively the state of mental health of survivors in 2003 and the adequacy of mental health services provided to them. twenty - six people suffering from various mental health needs were the subject of detailed interviews. the salient findings of this study were the following : based on the interviews with victims (survivors) themselves, as well as with professionals, it highlights the fact that despite the continuing suffering of the victims, no systematic effort has been made to tackle the mental health problems that were generated as an impact of the gas leak. following the court judgment of june 2010, and the high dissatisfaction expressed by the survivors, there was a revival of the health care and research. there was a higher level of compensation to survivor family members of the dead persons, to those with chronic kidney diseases and cancer diagnoses. the national institute for research in environmental health (nireh) was set up in bhopal. the mental health studies of nireh, focused on understanding of the continuing mental health needs of the population and developing a training program and manual for training in mental health for the general medical officers of bhopal. the first effort was a follow - up assessment for the health status of the psychiatric patients of the 19851994 was completed that showed the chronicity of the mental disorders in the majority of the patients and the limited mental health care patients had received. in an another study of treatment utilization of the psychiatric patients, cared for during 2010 at bmhrc. this analysis of the routine clinical records pointed to the limited utilization of the available mental health care, and the problems patients had in utilizing the services. the third effort, was with 500 families in one area, to understand the prevalence of mental disorders in 2012. this study was a two - stage screening for mental disorders and showed about 20% had potential mental disorders and the rates were much higher in those living in socioeconomically difficult life situations and with medical conditions. the fourth activity was to develop a mental health manual for the medical officers involving the psychiatrists of bhopal city. the final activity, during this period, was to carry our five training programs of 3 days each (for groups of 615 medical officers) of the gas rahat health department and the doctors working in the mini units of bmhrc. a revised mental health manual to address the continuing mental health needs has been developed. a striking positive development in the mental health care situation in the city, during the last three decades, have been the increase in the mental health professionals both in the government and private sectors along with in - patient care facilities at gandhi medical college, all india institute of medical sciences, bhopal, all the three private medical colleges in the city, bmhrc, bhopal, beml and in the private sector. continuing mental health needs of the population, in 2014, are : (i) people with postdisaster anxiety - depression, posttraumatic stress disorder, adjustment disorder conditions, directly related to the disaster of 1984 ; (ii) people with psychiatric disorders, attributable to the various life changes, family and occupational status, resulting directly (e.g., unemployment due to poor health condition) and indirectly (e.g., loss of head of the family in disaster) from the disaster experiences ; (iii) people with chronic physical conditions like chronic obstructive pulmonary disease, diabetes, hypertension, cancer, with associated psychiatric disorders such as depression, adjustment disorders ; and (iv) people with psychiatric disorders, not directly related to the disaster. during the last three decades, in the country, there have been many positive developments in the area of disaster mental health. the most important is the way in which mental health care has become an essential part of the disaster interventions. unlike in bhopal, where mental health was thought of after 8 weeks the interventions developed during the marathwada earthquake, (1993), the orissa supercyclone (1999), the gujarat - kutch earthquake (2001), the gujarat riots (2002), tsunami (2004), uttarakhand (2013) in terms of manuals, evaluation reports have been very important contributions. psycho - social support in the context of disasters refers to comprehensive interventions aimed at addressing a wide range of psycho - social problems arising in the aftermath of a disaster. psycho - social support and mental health services should be considered as a continuum of the interventions in disaster situations. psycho - social support will comprise of general interventions related to the larger issues of relief work needs, social relationships and harmony to promote or protect psycho - social well - being of the survivors. mental health services will comprise of interventions aimed at prevention or treatment of psychological symptoms or disorders. these interventions help individuals, families and groups to restore social cohesion and infrastructure along with maintaining their independence and dignity. the bhopal disaster is of importance from mental health point for a number of reasons. first, it is one of the largest man - made disasters in a developing country. second, the disaster effects were a combination of both the substances inhaled and the psychological effects of living through a disaster experience. third, no formal mental health infrastructure was available to provide postdisaster mental health care. fourthly, a number of innovative approaches were developed to provide mental health care, especially suitable for use in developing countries. fifthly, this disaster was the subject of intensive health research in a prospective manner for the first 5 years. this research included mental health aspects of the disaster on the population. during the last three decades, there have been many lapses in the assessment of disability, compensation provided (coverage, amount), and the rehabilitation activities, leaving majority of the population dissatisfied. there has been no continuous research to understand the changing morbidity, adequacy of the care provided and efficacy of the different interventions. in the area of services, inadequacy of the programs to provide longitudinal mental health care to all people with mental disorders, not linking of primary health care with mental health care, lack of rehabilitation, no public mental health education, self - care, use of psycho - social interventions. looking back, it would have met the needs of the bhopal population, if the mental health services were community based and reaching the population, rather than the clinic - based approaches, there was a wide range of services, especially rehabilitation, continuous research into the changing mental health needs of the population and the effectiveness of interventions and most importantly, there was a continuous dialog with the population and sharing of information with the general population. these are the tasks for the immediate future to reorganize the focus of mental health initiatives in bhopal. the following lessons can be drawn from the last three decades of mental health initiatives in bhopal : firstly, disaster is a risk to the surviving population as populations exposed to disasters develop higher rates of mental disorderssecondly, the disaster situation provides an opportunity to enhance the recognition of mental health needs of the population and stimulate mental health servicesthirdly, there is a need for the full range of mental health care, both hospital - based care and community - based mental health care, with good linkages of the two for continuous, coordinated carefourthly, there is a need for integrating mental health care as part of general medical care and specialist medical carefifthly, the complex needs of the survivors of disasters require linkage of mental health care with other sectors like welfare, labor, law to meet the full needs of the populationsixthly, there is a need for continuous dialog with the survivors, to both understand their perceptions and needs, as well as for wide use of self - care measures for mental healthseventhly, there is need for rebuilding of the community, towards strengthening of the community supports and minimize the polarization of the population by compensation, migration and thus decrease the community support to the chronically ill persons, the disabled, the elderly and other vulnerable groupseighthly, there is a need for continuous research to understand the distribution of the psychiatric problems and to evaluate the effectiveness of the interventions. firstly, disaster is a risk to the surviving population as populations exposed to disasters develop higher rates of mental disorders secondly, the disaster situation provides an opportunity to enhance the recognition of mental health needs of the population and stimulate mental health services thirdly, there is a need for the full range of mental health care, both hospital - based care and community - based mental health care, with good linkages of the two for continuous, coordinated care fourthly, there is a need for integrating mental health care as part of general medical care and specialist medical care fifthly, the complex needs of the survivors of disasters require linkage of mental health care with other sectors like welfare, labor, law to meet the full needs of the population sixthly, there is a need for continuous dialog with the survivors, to both understand their perceptions and needs, as well as for wide use of self - care measures for mental health seventhly, there is need for rebuilding of the community, towards strengthening of the community supports and minimize the polarization of the population by compensation, migration and thus decrease the community support to the chronically ill persons, the disabled, the elderly and other vulnerable groups eighthly, there is a need for continuous research to understand the distribution of the psychiatric problems and to evaluate the effectiveness of the interventions. disasters are a challenge in every country, for the affected populations as well as the mental health professionals. the research has shown the high physical and mental morbidity in the general population and the continuing need for longitudinal health studies. using a public health approach in priority setting, identification of interventions and training of existing personnel, utilizing the community resources the needs of the population can be addressed. | bhopal disaster is an important milestone in indian industrial psychiatry. the disaster was not only the biggest industrial disaster but also one in which complex forces have joined hands to demy the mental health needs of the population. though the biggest general population epidemiological study over 5 years was carried out to understand the mental health impact of the disaster, the findings of this study did not get reflected in mental health care for the population. furthermore, the needed longitudinal studies and evaluation of the interventions were not undertaken. there was no sharing of information with the survivors about the impact of the disaster on their health and well - being and sharing of skills for self - care. a result of these factors is the extreme degree of dissatisfaction in the population. looking back, it would have met the needs of the bhopal population, if the mental health services were community based and reaching the population, rather than the clinic - based approaches, there was a wide range of services, especially rehabilitation, continuous research into the changing mental health needs of the population and the effectiveness of interventions and most importantly, there was a continuous dialogue with the population and sharing of information with the general population. these are the tasks for the immediate future to reorganize the focus of mental health initiatives in bhopal. many lessons can be learnt from the bhopal disaster and the continuing tragedy for the population. |
fever, defined as a rectal temperature higher than 38c, is the most common complaint among children admitted to emergency departments (1). infectious and noninfectious triggers cause a release of pyrogenic cytokine such as interleukin-1, interleukin-6, tumor necrosis factor - alpha (tnf-) and interferon - gamma (ifn-). these cytokines in turn act on the thermoregulatory center of the hypothalamus, which leads to an increase in temperature set point, thus causing fever (2, 3). fever, as a part of an effective immune system, improves the immunological response and slows growth and replication of viral and bacterial pathogens (4). increased metabolic reactions, increased oxygen consumption, and increased heart and lung function are some effects of fever, which may lead to dangerous outcomes, especially in children with underlying diseases (5). although fever is mostly self - limited in children and usually the underlying cause of fever should be treated, yet antipyretic therapy is also indicated when there is fever higher than 39c, an underlying disease (such as any cardiac, pulmonary or neurologic problems), discomfort or hemodynamic and electrolyte imbalance (3, 6, 7). paracetamol (acetaminophen) is an antipyretic and analgesic medication, most frequently used in pediatrics for both outpatient and admitted patients. the mechanism of action of this drug is not fully understood, yet it is postulated that the acetaminophen - induced antipyresis occurs via central inhibition of the enzyme cyclooxygenase (cox). acetaminophen does not inhibit cox in peripheral tissues and, thus, has no peripheral anti - inflammatory effects and is not classified as a member of the non - steroidal anti - inflammatory drugs (nsaids) (2, 8). despite its favorable effects, poisoning, kidney failure and hepatotoxicity are the most serious adverse effects of paracetamol, which may occur due to acute exposure (9). diclofenac is a non - steroidal anti - inflammatory drug (nsaid), which has analgesic and antipyretic activities. this drug is readily absorbed from the alimentary tract, and its half time is about one to two hours, and it will quickly dissolve in an environment with a ph higher than five. diclofenac as one of the most potent nsaid has a few side effects when administered rectally (10). antipyretic effects of this drug have received less attention in comparison with its analgesic effects (11, 12). considering the importance of fever and unknown antipyretic properties of diclofenac in the pediatric population, this clinical trial was designed and implemented in order to compare the antipyretic effectiveness of paracetamol and diclofenac, administered rectally. this double - blind controlled clinical trial comprised of 80 children aged one to six years, and was conducted from october 2012 to november 2013, at shahid beheshti hospital of kashan, iran. shahid beheshti hospital has 393 active beds in 16 different specialist units including pediatric, internal medicine, obstetrics and gynecology, infectious diseases, dermatology, cardiology and intensive care. this specialty and subspecialty hospital is a public hospital and is considered a regional referral center. children whose rectal temperatures were higher than 38c and those who had fevers of less than four days were entered in the study. convenience sampling was used and all patients meeting the least required criteria, who had visited our health center since the beginning of the study until completion of the required sample size, were selected. those who had received other antipyretic agents, had undergone non pharmacological interventions to control the fever, had diarrhea, had a history of allergic reactions to paracetamol and/or diclofenac, had any anomalies of the gastrointestinal tract, had been affected by chronic diseases and those whose parents refused to participate, were excluded from the study. however, due to diarrhea (four cases), having had received oral paracetamol prior to the visit (two cases) and having had a history of imperforate anus (one case) seven cases were excluded, thus the final sample size was 80. after being informed of the study objectives, then a questionnaire, which asked for the child s age, gender, weight and possible diagnosis, was completed. patients were randomly allocated to two groups of paracetamol and diclofenac using a permuted - block randomization scheme. for this purpose, 20 blocks with a block size of four were used and the blocks were classified in order of numbers. one of them was responsible for sampling, completing the questionnaires, allocating the patients to different treatment groups and preparation and administration of the drugs. the other one was responsibility for simply measuring the rectal temperature of the child under study before and after the intervention. the parents did not know the type of treatment, either (figure 1). all ethical principles were respected in accordance with resolution 196/96 on research involving human subjects. the ethics committee of kashan university of medical sciences (approval code : 29/5/1/2742/p) approved the study and supervised all stages. this study was also recorded in the iran center of clinical trials registration database (irct2012101711145n1). however, since the antipyretic effects of rectal forms of paracetamol and diclofenac have not been compared so far, to determine the required sample size we used the same methodology as was performed in a previous evaluation to compare the analgesic effects of rectal forms of these two drugs. in their study, the mean pain score one hour after the surgery was 7.8 2.25 in the paracetamol group and 6.4 0.8 in the diclofenac group (11). with a power of 90% and z1-/2 = 1.28, and using the equation following equation, the minimum sample size required in each group was calculated to be 31 cases (equation 1). taking into account our medical facilities and to allow for losses, we finally estimated a sample size of 40 cases in each group. the mt 19f1 digital thermometer, manufactured by microlife company, was used for measuring temperature. this thermometer registers the rectal temperature in degrees celsius within 10 seconds and displays it digitally. the equipment was calibrated by the manufacturing company, and to evaluate the reliability of the measurements, all recordings were compared with the results obtained with a glass mercury thermometer before the study.. supervised by the researcher, the tip of thermometer was gently inserted about 2 cm into the child s rectum by the parents, and then the registration key was pressed. rectal temperatures greater than or equal to 38c were labeled as a fever. the rate of temperature change after treatment in each group was calculated and recorded. three classes of successful treatment were low recovery rate from 0.5 to 0.1, average recovery rate from 1 to 0.6, and high recovery rate of more than 1c temperature decrease. paracetamol and diclofenac suppositories were administered per rectum at 15 mg / kg and 1 mg / kg dose, respectively. one hundred and twenty - five milligrams and 325 mg paracetamol suppositories, and 100 mg and 50 mg diclofenac suppository of aboureihan company (iran) were used for this purpose. to determine the amount of medication required per dose, the suppositories were scraped - off using a very sharp surgical blade, sectioned longitudinally, and weighed by and digital scale (made in japan) with accuracy of 0.001 g. the drug all stages of drug preparation were done by an expert pharmacist. after instructing the parents, the drug was inserted into the child s rectum slowly by the parents under supervision of the researcher. baseline variables, fever reduction grade and fever after treatment were analyzed by the chi - square and fisher s exact tests. the results of the quantitative data analysis were expressed as mean standard deviation. kolmogorov - smirnov was applied to assess the data distribution. according to the normal distribution of age, weight and primary and secondary temperature data, independent t test and paired t test were used to compare the averages. mann - whitney u test was used for comparison of average temperature decrease due to abnormal data distribution. repeated measure analysis of variance (anova) was also used to compare the average body temperature at different study times. this double - blind controlled clinical trial comprised of 80 children aged one to six years, and was conducted from october 2012 to november 2013, at shahid beheshti hospital of kashan, iran. shahid beheshti hospital has 393 active beds in 16 different specialist units including pediatric, internal medicine, obstetrics and gynecology, infectious diseases, dermatology, cardiology and intensive care. this specialty and subspecialty hospital is a public hospital and is considered a regional referral center. children whose rectal temperatures were higher than 38c and those who had fevers of less than four days were entered in the study. convenience sampling was used and all patients meeting the least required criteria, who had visited our health center since the beginning of the study until completion of the required sample size, were selected. those who had received other antipyretic agents, had undergone non pharmacological interventions to control the fever, had diarrhea, had a history of allergic reactions to paracetamol and/or diclofenac, had any anomalies of the gastrointestinal tract, had been affected by chronic diseases and those whose parents refused to participate, were excluded from the study. however, due to diarrhea (four cases), having had received oral paracetamol prior to the visit (two cases) and having had a history of imperforate anus (one case) seven cases were excluded, thus the final sample size was 80. after being informed of the study objectives, then a questionnaire, which asked for the child s age, gender, weight and possible diagnosis, was completed. patients were randomly allocated to two groups of paracetamol and diclofenac using a permuted - block randomization scheme. for this purpose, 20 blocks with a block size of four were used and the blocks were classified in order of numbers. one of them was responsible for sampling, completing the questionnaires, allocating the patients to different treatment groups and preparation and administration of the drugs. the other one was responsibility for simply measuring the rectal temperature of the child under study before and after the intervention. the parents did not know the type of treatment, either (figure 1). all ethical principles were respected in accordance with resolution 196/96 on research involving human subjects. the ethics committee of kashan university of medical sciences (approval code : 29/5/1/2742/p) approved the study and supervised all stages. this study was also recorded in the iran center of clinical trials registration database (irct2012101711145n1). however, since the antipyretic effects of rectal forms of paracetamol and diclofenac have not been compared so far, to determine the required sample size we used the same methodology as was performed in a previous evaluation to compare the analgesic effects of rectal forms of these two drugs. in their study, the mean pain score one hour after the surgery was 7.8 2.25 in the paracetamol group and 6.4 0.8 in the diclofenac group (11). with a power of 90% and z1-/2 = 1.28, and using the equation following equation, the minimum sample size required in each group was calculated to be 31 cases (equation 1). taking into account our medical facilities and to allow for losses, we finally estimated a sample size of 40 cases in each group. the mt 19f1 digital thermometer, manufactured by microlife company, was used for measuring temperature. this thermometer registers the rectal temperature in degrees celsius within 10 seconds and displays it digitally. the equipment was calibrated by the manufacturing company, and to evaluate the reliability of the measurements, all recordings were compared with the results obtained with a glass mercury thermometer before the study.. supervised by the researcher, the tip of thermometer was gently inserted about 2 cm into the child s rectum by the parents, and then the registration key was pressed. rectal temperatures greater than or equal to 38c were labeled as a fever. the rate of temperature change after treatment in each group was calculated and recorded. three classes of successful treatment were low recovery rate from 0.5 to 0.1, average recovery rate from 1 to 0.6, and high recovery rate of more than 1c temperature decrease. paracetamol and diclofenac suppositories were administered per rectum at 15 mg / kg and 1 mg / kg dose, respectively. one hundred and twenty - five milligrams and 325 mg paracetamol suppositories, and 100 mg and 50 mg diclofenac suppository of aboureihan company (iran) were used for this purpose. to determine the amount of medication required per dose, the suppositories were scraped - off using a very sharp surgical blade, sectioned longitudinally, and weighed by and digital scale (made in japan) with accuracy of 0.001 g. the drug was then converted into the usable form. all stages of drug preparation were done by an expert pharmacist. after instructing the parents, the drug was inserted into the child s rectum slowly by the parents under supervision of the researcher. baseline variables, fever reduction grade and fever after treatment were analyzed by the chi - square and fisher s exact tests. the results of the quantitative data analysis were expressed as mean standard deviation. kolmogorov - smirnov was applied to assess the data distribution. according to the normal distribution of age, weight and primary and secondary temperature data, independent t test and paired t test were used to compare the averages. mann - whitney u test was used for comparison of average temperature decrease due to abnormal data distribution. repeated measure analysis of variance (anova) was also used to compare the average body temperature at different study times. a total of eighty children were examined in two treatment groups of paracetamol and diclofenac. all patients completed the study and no drug - related allergic reactions were observed. abbreviations : lrti, lower respiratory tract infection ; uti, urinary tract infection ; urti, upper respiratory tract infection. (%). reduced temperature of at least 0.1c was detected in all patients in both groups. the average temperature one hour after the procedure was 38.39 0.89c in the diclofenac group, which was significantly reduced compared to the initial temperature (p < 0.001). the mean temperature was 38.95 1.09c for the paracetamol group, which had also significantly decreased compared to the initial temperature (p < 0.001). the average temperatures of patients before and after the intervention were compared using the repeated measures anova. despite the significant decrease in the measured temperatures, there were no significant differences between the two groups (p = 0.47) (figure 2). this study was conducted to compare the antipyretic effectiveness of the suppository form of paracetamol versus diclofenac. both diclofenac and paracetamol proved to be successful in reducing temperature, yet diclofenac had a greater antipyretic effect than paracetamol. oral and rectal forms of paracetamol are among the most popular and widely used over - the - counter drugs for the treatment of pain and fever, especially in the pediatric population (13, 14). in previous studies, the antipyretic effect of rectal paracetamol has been well established, yet the antipyretic effect after one hour of administration has not been clarified (15 - 17). to best of our knowledge, only one study has reported a decrease in body temperature one hour following the administration of paracetamol with an average of 1.07 0.16c, which is significantly more than what we detected in our survey (0.65 0.17 c) (17). these differences are hard to explain as the same drugs, dose and methods had been used. however the initial body temperature (39c in the study of karbasi. (17) and 38c in our study) could be the cause of these differences. unlike paracetamol, antipyretic properties of nsaids, especially diclofenac, are somewhat unknown and only a few studies have examined these effects (18 - 22). in one study, that examined the effect of different doses of oral diclofenac in reducing fever and pain, resulting from acute febrile sore throat, diclofenac significantly reduced fever and throat pain. the overall efficacy of all doses of diclofenac (6.25, 12.5 and 25 mg) was rated significantly higher than that of the placebo. also, paracetamol was only slightly better than diclofenac 6.25 mg dose, and the other doses of diclofenac proved to be more effective as compared to paracetamol (22). the study of polman. on the antipyretic effect of suppository diclofenac showed that it significantly decreases fever during the first two hours after administration and the body temperature returns to normal values after two hours in all patients receiving diclofenac (18). litalien. studied the efficacy of nsaids including suppository diclofenac in reduction of fever and pain in children and reported it to be more effective than oral acetaminophen. also, suppository diclofenac was more effective in relieving pain compared to acetaminophen (19). we believe that this is the first attempt that compares the antipyretic effectiveness of the rectal forms of paracetamol and diclofenac. diclofenac is rapidly absorbed in those parts of the gastrointestinal tract that have ph values higher than five (23). paracetamol is also highly absorbed under alkaline conditions ; however, its absorption is less dependent on the environmental ph (24, 25). the alkaline environment of the rectum has a relatively similar impact on the absorption of both drugs (26). so what makes diclofenac more efficient in reducing fever is the variability of the time when maximum concentrations of the drugs are reached, which is one hour for diclofenac and more than two hours for paracetamol (27 - 29). the antipyretic effect during the following hours was not evaluated in this study yet considering the half - life of diclofenac (which is about 80 to 120 minutes) and paracetamol (which is about six to eight hours), it is postulated that over the following hours the degree of reduction in fever becomes similar in both groups (27). the ability of diclofenac to quickly reduce the body temperature makes this drug a suitable choice for children s fever treatment especially in those patients with poor or critical conditions ; however, its short half - life may increase the likelihood of recurrent fever. conducting trials, which simultaneously assess antipyretic and analgesic effect of paracetamol and diclofenac may help identify the advantages of each. as previously stated, to best of our knowledge, this study is the first completed clinical trial comparing the antipyretic properties of rectal forms of diclofenac and paracetamol. one of the strengths of the present study is that the drug administration was based on the patients total body weight achieved through accurate measurement and converting the drugs. this study had some limitations, one of which was the impossibility of assessing the adverse effects of the studied drugs as they were used with other drugs and at a single dose. not measuring the serum levels of the drugs in studied subjects is considered the other limitation, which prevented the evaluation of the inter - individual differences in absorbing the drugs and the time of maximum serum concentrations. the last limitation involves non - cooperative parents, who refused to check the rectal temperature frequently during the following hours. in conclusion, this study showed that suppository diclofenac could significantly decrease rectal temperature as compared with suppository paracetamol during the first hour, yet overall both drugs were found to be equally effective for stopping fever. finally, a similar study with a larger population is warranted to investigate the extent of temperature control at different elapsed times after medication administration in order to better compare the efficacy of these two drugs. | backgroundfever is the most common complaint in pediatric medicine and its treatment is recommended in some situations. paracetamol is the most common antipyretic drug, which has serious side effects such as toxicity along with its positive effects. diclofenac is one of the strongest non - steroidal anti - inflammatory (nsaid) drugs, which has received little attention as an antipyretic drug.objectivesthis study was designed to compare the antipyretic effectiveness of the rectal form of paracetamol and diclofenac.patients and methodsthis double - blind controlled clinical trial was conducted on 80 children aged six months to six years old. one group was treated with rectal paracetamol suppositories at 15 mg / kg dose and the other group received diclofenac at 1 mg / kg by rectal administration (n = 40). rectal temperature was measured before and one hour after the intervention. temperature changes in the two groups were compared.resultsthe average rectal temperature in the paracetamol group was 39.6 1.13c, and 39.82 1.07c in the diclofenac group (p = 0.37). the average rectal temperature, one hour after the intervention, in the paracetamol and the diclofenac group was 38.39 0.89c and 38.95 1.09c, respectively (p = 0.02). average temperature changes were 0.65 0.17c in the paracetamol group and 1.73 0.69c in the diclofenac group (p < 0.001).conclusionsin the first one hour, diclofenac suppository is able to control the fever more efficient than paracetamol suppositories. |
the incidence of vesicoenteric fistulas has been reported to occur in 0.01% to 0.05% of surgical admissions1). in one study of 56 instances of vesicoenteric fistulas, hepatocellular carcinomas (hccs) account for 90% of primary liver cancers ; up to 80% of hccs occur in korea occur in patients with cirrhotic livers. according to our literature review, several malignancies have been reported as causes of ileovesical fistula, though such complications have not yet been reported to be caused by hcc. here, we report a case of ileovesical fistula caused by hcc arising from the noncirrhotic liver. a 27-year - old man was referred to our hospital for the evaluation of hepatic masses. he had consumed 30.0 g alcohol twice weekly for 8 years. on presentation, his vital signs were stable and the physical examination was normal. viral marker study showed that hbs antigen was negative, anti - hbs antibody positive, and anti - hcv negative. -fetoprotein (fp) was over 50,000 iu / ml and carcinoembryonic antigen (cea) was 4.94 ng / ml. abdominal computed tomography (ct) with enhancement revealed two heterogeneous hypodense masses in the right hepatic lobe of about 2.4 2 cm and 3.4 4 cm. no cirrhotic change was observed in the liver. liver magnetic resonance imaging (mri) revealed two relatively well - defined masses in the right hepatic lobe (figure 1). these lesions were homogeneous low signal intensity on t1-weighted image and slightly inhomogeneous high signal change on t2-weighted image. immunohistochemical stains were positive for cytokeratin (ck), epithelial membrane antigen (ema), fp, and hepatocyte - specific antigen (hsa). however, evidence of cirrhosis and chronic injury were absent. under the diagnosis of hcc, two months later, follow - up ct scan showed a newly developed mass in the caudate lobe about 4.5 4.0 cm in size, with several daughter nodules. follow - up ct scan after 3 weeks showed defective lipiodol uptake masses in both the right hepatic lobe and the caudate lobe with remnant tumoral enhancement. several days later, the patient complained of dysuria, fecaluria, and intermittent lower abdominal pain. pelvic ct scan showed a lobulated soft tissue mass about 6 cm in size (figure 3). barium study of the small bowel showed a fistula between the small bowel loop and the urinary bladder (figure 4). there were soft tissue masses between the ileum and the bladder and multiple lymphadenopathies at the mesenteric root. adhesion and fistula were found between the ileum at 10 cm proximal to the ileocecal valve and the dome of the bladder. the distal 65 cm of ileum was segmentally resected and anastomosed end - to - end. several malignancies have been reported to be causes of ileovesical fistula. according to our literature review, these include bladder cancer, lymphoid malignancies, and others1, 3 - 5). although other various malignancies themselves were not direct causes of ileovesical fistula, radiation enteritis complicated by radiation therapy could also cause the ileovesical fistula6). in korea, about 80% of hccs occur in patients with cirrhotic livers. in our case, hcc occurred in the noncirrhotic liver, and the cause remains uncertain. in one study of 403 patients with hcc, 148 patients (37%) with extrahepatic metastases were identified. the lung, abdominal lymph nodes, and bone are the most common sites of extrahepatic metastatic hcc. peritoneal metastases were illustrated at ct scan in 16 (11%) of 148 patients with extrahepatic metastatic hcc7). ho. reported that 3% of patients with hccs in noncirrhotic liver involved the peritoneum8). fecaluria, abdominal pain, and pneumaturia were the most common presenting symptoms of vesicoenteric fistulas. other symptoms were dysuria, gross hematuria, fever and chills, diarrhea, and urine per rectum. the diagnosis of vesicoenteric fistula can be highly indicated by the above mentioned symptoms, using various diagnostic tests to establish diagnosis. in our particular case, cystoscopic and cystographic examinations showed inflammatory mucosal changes on the dome of the urinary bladder, but could not detect a fistula. cystoscopy revealed either the fistula itself or highly suggestive, localized mucosal abnormalities (" herald lesions ") in 86%. ct scan revealed abnormalities, such as pelvic masses or bladder or intestinal wall thickening (87%), and revealed a fistula (24%). it should be possible to establish the diagnosis of a vesicoenteric fistula through a combination of cystoscopy, cystography, and barium study in almost all patients. other diagnostic tests for vesicoenteric fistulas include sigmoidoscopy or colonoscopy, intravenous urography, radioisotope studies, and so on. forms of treatment for vesicoenteric fistula include diverting enterostomy, single stage repair, and multi - staged repair. when secondary to radiation necrosis and recurrent tumor, fistulas have an extremely poor outlook with some palliation afforded by a diverting colostomy2). multi - staged repair is limited to those patients with evidence of intestinal obstruction, uncontrolled local sepsis, and questions of intestinal viability9). in summary, we experienced one case of ileovesical fistula, which was caused by hcc arising from noncirrhotic liver. the combination of cystoscopy, cystography, and barium study of the small bowel confirmed the diagnosis of ileovesical fistula, and single stage repair was performed successfully. | ileovesical fistula is a very rare clinical entity, the most frequent cause of which is crohn 's disease. furthermore, it is an exceptionally rare complication of malignancies. we experienced one case of ileovesical fistula which had been caused by hepatocellular carcinoma (hcc) arising from the noncirrhotic liver.a 27-year - old man was diagnosed with hcc in a noncirrhotic liver. despite treatment with transarterial chemoembolization (tace), the disease status became more aggravated. the patient complained of dysuria, fecaluria, and intermittent lower abdominal pain. pelvic ct scan showed a soft tissue mass of 6 cm abutting on the distal ileum which was downwardly displaced. barium study of the small bowel showed a fistula between the small bowel loop and the urinary bladder. upon operation, adhesion and fistula were found between the ileum and the urinary bladder. the microscopic findings of the surgical specimen were compatible with metastatic hcc. we confirmed that ileovesical fistula had been caused by metastatic hcc. |
primary central nervous system (cns) lymphoma is a relatively uncommon malignancy, accounting for approximately 6% of all malignant cns neoplasms. they typically occur in middle - aged and older adults, although it can be seen more commonly in younger patients with human immunodeficiency virus (hiv) or transplant recipients. more than 90% of primary cns lymphomas are of the diffuse large b - cell variety. most of these lesions appear hyperdense on a non - contrast enhanced computed tomography (ct) scan and tend to demonstrate restricted diffusion on magnetic resonance imaging (mri), given their high cellularity. this is the eighth reported case of cns lymphoma involving the pineal gland in the literature. a 65-year - old male presented with a 2-week history of worsening headache and double vision. initial non - contrast head ct demonstrated a homogeneous hyperdense mass in the pineal gland region with mild hydrocephalus, but no calcification or hemorrhage [figure 1 ]. contrast mri was performed, which demonstrated a homogeneously enhancing mass involving the pineal gland, with increased perfusion with corresponding low apparent diffusion coefficient values [figure 2 ]. the mass was hypointense on t1-weighted images and isointense to mildly hyperintense compared to brain parenchyma on t2-weighted images. leptomeningeal enhancement was identified near the supraoptic recess and along the cerebellar velum [figure 3 ]. 65-year - old male presented with headache and was later diagnosed with pineal gland lymphoma. axial non - contrast ct of the head demonstrates a homogeneously hyperdense mass (arrow) in the pineal gland region. 65-year - old male presented with headache and was later diagnosed with pineal gland lymphoma. (a) axial t1 post - contrast mri image of the head demonstrates a homogeneously enhancing mass (arrow) in the region of the pineal gland. (b) axial mri diffusion - weighted imaging (dwi) sequence demonstrates increased signal (arrow). (c) the corresponding axial apparent diffusion coefficient (adc) map demonstrates low adc values (arrow). 65-year - old male presented with headache and was later diagnosed with pineal gland lymphoma. (a) axial t2 flair mr image demonstrates the pineal gland mass (arrow) as isointense to mildly hyperintense to brain parenchyma. (c) sagittal t1 post - contrast mr image again shows the enhancing pineal gland mass (arrowhead) as well as leptomeningeal enhancement near the supraoptic recess (arrow) and cerebellar velum. the histopathology showed a hypercellular tumor with high nuclear / cytoplasmic ratio, but no pineocytomatous or homer wright rosettes or papillary architecture [figure 4 ]. tumor cells exhibited vimentin, cd45, extensive cd20, cd79a, and nuclear pax 5 reactivity. in situ hybridization showed kappa, but no lambda light chain hybridization. there was focal granular synaptophysin, but no neuron specific enolase (nse), glial fibrillary acidic protein (gfap), beta tubulin, cam 5.2, thyroid transcription factor (ttf-1), cd99, pancytokeratin, myogenin, or neurofilament immunoreactivity. 65-year - old male presented with headache and was later diagnosed with pineal gland lymphoma. (a) hematoxylin and eosin staining at magnification (40) demonstrates malignant lymphoma with coarse chromatin, nucleoli (arrow), and mitoses. (b) diaminobenzidene chromagen at magnification (40) demonstrates tumor cells (arrow) extensively exhibiting cd20 reactivity. (c) diaminobenzidene chromagen at magnification (40) demonstrates tumor cells (arrow) exhibiting extensive cd79a, but no neural, myogenic, or epithelial marker immunoreactivity. at the time of biopsy, staging chest, abdomen, and pelvis ct exams revealed no other areas of lymphomatous involvement. the patient was discharged home in stable condition, but presented to the emergency department 1 week later with worsening hydrocephalus and headache. at this time, the patient underwent ventriculoperitoneal shunt placement for management of his hydrocephalus and received one cycle of high - dose methotrexate. however, he developed severe hypotension and acute kidney injury 2 months after his diagnosis and was re - admitted to the hospital, with repeat non - contrast head ct showing enlargement of the mass. given his multiple medical comorbidities and evidence of disease progression, the patient and his family elected to pursue comfort care measures. they can affect both immunocompetent and immunocompromised patients, although patients who are immunocompromised typically present at a younger age. there is a predilection for the periventricular white matter and basal ganglia, and they can present as either solitary or multiple mass lesions. on non - contrast ct, most masses are hyperdense, given the high cellularity, and surrounding edema is common. contrast enhancement is typically homogeneous, but can have a more variable appearance in immunocompromised patients. on mri, although these lesions can appear hypointense to gray matter on t1-weighted imaging and hypointense to isointense on t2-weighted imaging, the hallmark is restricted diffusion due to the increased cellularity. primary cns lymphoma has rarely been found to involve the pineal gland, with seven cases reported in the literature. headache was one of the most common presenting symptoms in these patients, although symptoms include cranial nerve and cauda equina syndrome, focal neurologic deficits, fever, diplopia, altered mental status, and seizure. the average age at diagnosis was 40 years, and only one female patient has been reported. b - cell lymphoma has been the most common pathologic diagnosis, with cases including large b cell lymphoma, malignant b cell lymphoma, immunoblastic lymphoma, and anaplastic lymphoma kinase positive anaplastic large cell lymphoma (alk-1 positive alcl). there has been a single case of malignant t cell lymphoma and one case providing no additional detail to the diagnosis of lymphoma. imaging features in these cases most commonly included hydrocephalus and relatively homogeneously enhancing lesions identified on mri. the imaging findings in this particular case were typical of highly cellular tumor with leptomeningeal involvement, given the hyperdense mass on non - contrast ct and restricted diffusion with contrast enhancement on mri. a histologic diagnosis is essential prior to beginning invasive treatment, as the imaging characteristics of pineal lymphoma are not necessarily pathognomonic. other differential considerations for a tumor in the pineal region with these imaging characteristics include pineoblastoma, germ cell tumor, and metastatic disease. pineoblastoma is a pediatric diagnosis with masses typically appearing more heterogeneous and with peripheral calcifications. germ cell tumors are also typically diagnosed at a younger age and are associated with calcification. while metastatic disease may fit the imaging pattern and patient age seen in our case, a primary malignancy was not identified. our patient was treated with high - dose methotrexate, which has been shown to increase survival in patients with primary cns lymphoma. the addition of rituximab to this regimen has also been shown to improve remission rates and progression - free survival ; however, our patient decompensated and progressed prior to initiation of this therapy. primary cns lymphoma is an uncommon primary cns malignancy, and rarely involves the pineal gland. however, in the appropriate clinical context and with relevant imaging findings, lymphoma should be in the differential diagnosis of a pineal gland mass. | a 65-year - old male presented to our institution with acute - onset headache. imaging studies demonstrated a mass in the region of the pineal gland, with subsequent histopathology findings being consistent with large b cell lymphoma. the patient was treated with methotrexate, but ultimately did not survive. primary central nervous system (cns) lymphoma rarely involves the pineal gland, but should be considered in the differential diagnosis of pineal gland tumors in the appropriate clinical setting. |
familial hypomagnesaemia with hypercalciuria and nephrocalcinosis (fhhnc, omim # 248250) is a rare autosomal - recessive renal tubular disorder caused by mutations in the cldn16 or cldn19 gene, which, respectively, encode the tight junction - associated proteins, claudin-16 and -19 [13 ]. these transmembrane proteins regulate the paracellular diffusion of selective cations along the thick ascending limb (tal) of henle 's loop (figure 1) [4, 5 ]. fhhnc - associated tubulopathy is thus characterized by massive urinary losses of mg and ca, with subsequent hypomagnesaemia, bilateral nephrocalcinosis and rapid evolution to end - stage renal disease (esrd). a genotype / phenotype correlation regarding the severity of the disease has been proposed upon the impact of cldn16 mutations on protein function. we report on an 18-year - old patient presenting with glomerular proteinuria associated with the typical fhhnc triad. further investigations demonstrated severe tubular atrophy and interstitial fibrosis, as well as secondary glomerulosclerosis. (a) localization of members of the claudin family in a mammalian kidney. claudin-16 and -19 are specifically situated between epithelial cells lining the ascending limb of henle 's loop. adapted from angelow.. (b) schematic view of an epithelial cell lining the tal of henle 's loop. claudin-16 and -19 are positioned at the tight junctions (tj) between adjacent cells, where they selectively regulate the paracellular diffusion of ca and mg. ion transporters implicated in generating and/or modulating the positive trans epithelial electrical gradient, including na / k - atpase, clc - kb / barttin, ca - sensing receptor, romk (renal outer medullary k channel) and nkcc2 (type 2 na / k / cl cotransporter) are depicted. different k channels from four gene families have been identified within the tal basolateral membrane of various mammals. adapted from naderi and reilly, (a) localization of members of the claudin family in a mammalian kidney. claudin-16 and -19 are specifically situated between epithelial cells lining the ascending limb of henle 's loop. adapted from angelow.. (b) schematic view of an epithelial cell lining the tal of henle 's loop. claudin-16 and -19 are positioned at the tight junctions (tj) between adjacent cells, where they selectively regulate the paracellular diffusion of ca and mg. ion transporters implicated in generating and/or modulating the positive trans epithelial electrical gradient, including na / k - atpase, clc - kb / barttin, ca - sensing receptor, romk (renal outer medullary k channel) and nkcc2 (type 2 na / k / cl cotransporter) are depicted. different k channels from four gene families have been identified within the tal basolateral membrane of various mammals. adapted from naderi and reilly, hamilton and devor. the patient was referred for renal investigations after a fortuitous finding of increased serum creatinine levels (1.7 mg / dl), i.e. a glomerular filtration rate (gfr) of 52 ml / min per 1.73 m according to the modification of diet in renal disease (mdrd) equation. his medical history included severe dehydration at birth, as well as persistent polyuria / polydipsia syndrome with nycturia since infanthood. no urinary tract infections or muscular cramps one year prior to consultation, he developed acute kidney injury in a context of rhabdomyolysis and dehydration in a motorcycle crash. at that time note the co - occurrence of hypomagnesaemia, hypermagnesuria and hypercalciuria, with heavy selective proteinuria. pak 's oral ca load test led to a significant decline in parathormone levels, thereby ruling out primary hyperparathyroidism and supporting a renal origin for hypercalciuria. abdominal ultrasound disclosed symmetric 10-cm kidneys, with nephrocalcinosis and multiple millimetric lithiasis as confirmed by computed tomography. a kidney biopsy showed both atrophy and hypertrophy of renal tubules and interstitial fibrosis in association with focal and segmental sclerosis of glomeruli (figure 2a and b). the expression of claudin-16 in tal was lost, whereas the distribution of uromodulin did not appear to be significantly affected (figure 2d e). table 1.analysis of serum and 24-h urine samples at admissionserumsi unitsconventional unitsnormal valuessi unitsconventional unitscreatinine141.41.650110 mol / l0.61.2 mg / dlgfr54>60 ml / min per 1.73 muric acid517.58.7120420 mol / l27 mg / dlmagnesium0.61.40.751 mmol / l1.82.4 mg / dlcalcium2.39.22.12.6 mmol / l8.410.6 mg / dlsodium142142135145 mmol / l135145 meq / lpotassium3.23.23.14.9 mmol / l3.14.9 meq / lbicarbonate33332333 mmol / l2333 meq / lglucose4.4803.35.5 mmol / l60100 mg / dlintact pth2982981258 ng / l1258 pg / ml25-oh vitamin d89.836>80 nmol / l>32 ng / mlurinemagnesium8.51734 mmol / day68.5 meq / daycalcium / creatinine0.290.040.15 g / mgproteins18541.854 t (nonsense) and c.427 + 5g > a (splice - site) mutations of cldn16 gene. eosin colouration (a and b) shows diffuse tubular atrophy and interstitial fibrosis, as well as perihilar segmental sclerosis of some glomeruli (b, arrowhead). immunostaining anti - claudin-16 (d) and anti - uromodulin (e) on serial sections (cfr asterisk,) does not detect claudin-16 (d) in uromodulin - positive tubules lining the tal of henle 's loop (e). analysis of serum and 24-h urine samples at admission gfr, glomerular filtration rate according to mdrd equation ; pth, parathyroid hormone. kidney histology of a patient with c.340c > t (nonsense) and c.427 + 5g > a (splice - site) mutations of cldn16 gene. eosin colouration (a and b) shows diffuse tubular atrophy and interstitial fibrosis, as well as perihilar segmental sclerosis of some glomeruli (b, arrowhead). immunostaining anti - claudin-16 (d) and anti - uromodulin (e) on serial sections (cfr asterisk,) does not detect claudin-16 (d) in uromodulin - positive tubules lining the tal of henle 's loop (e). medical treatment included thiazides and angiotensin - converting enzyme (ace) inhibitors, as well as oral supplementation of mg and active vitamin d. still, despite the complete resolution of proteinuria under treatment, the patient reached esrd at the age of 23. the slope of gfr decline was calculated to be 9 ml / min per 1.73 m / year. the 1-year follow - up showed an uneventful evolution, with a stable gfr 50 ml / min per 1.73 m. the pre - transplant work - up prompted genetic testing, which allowed the identification of two novel mutations in the cldn16 gene : c.340c > t and c.427 + 5g > a. his father, who presented with recurrent nephrolithiasis, could not be tested because of sudden death at the age of 52. hypercalciuria and nephrocalcinosis most probably have a negative impact. still, all inherited tubulopathies characterized by nephrocalcinosis do not uniformly lead to esrd, which suggests that the functional loss of claudin-16 may per se impair kidney architecture and function. previous reports listed 40 missense / nonsense mutations, four splice - site mutations and 5 indels of the cldn16 gene. genotype / phenotype correlation studies postulated an earlier onset of the disease and a more rapid decline of gfr in patients harbouring cldn16 mutations inducing a complete loss of function of the protein in comparison with patients with mutations associated with a partial dysfunction. our patient presented at the age of 18 with stage 3a ckd and significant selective proteinuria. symptoms obviously developed in early infanthood, and the slope of gfr decline was > 5 ml / min per 1.73 m / year, which suggests a complete loss of function of claudin-16. kidney histology further supports an advanced and chronic tubulopathy, with tubular atrophy and ca deposits. in addition, sclerotic lesions were found in some glomeruli, with a particularly perihilar distribution (figure 2). such a location is suggestive of secondary glomerulosclerosis, and accounts for the glomerular proteinuria highly responsive to ace inhibitor therapy. the complete loss of claudin-16, as demonstrated by immunostaining, may thus cause progressive nephron loss with tubular atrophy and interstitial fibrosis and secondary glomerular damage. the c.340c > t (p.r114) mutation is a nonsense mutation located in exon 2, which most likely leads to a premature stop codon and a complete loss of claudin-16 function. this mutation is located in the first extracellular loop of the protein, similar to the large majority of previously reported mutations. the c.427 + 5g > a mutation affects an intronic nucleotide located close to the donor splicing site of cldn16 exon 2. still, given the severity of the disease and the absence of claudin-16 expression in the patient 's tal tubules, we speculate that this mutation also results in a complete loss of claudin-16 function. of note, a mutation with residual expression and function of the protein would predict a milder clinical course [9, 10 ]. in summary in addition to the identification of novel mutations of cldn16 gene, the present case illustrates a late diagnosis of fhhnc in early adulthood and emphasizes the progressive nephron loss associated with this tubulopathy, as well as the glomerular consequences. conservative management of ckd and oral supplementation of mg remain the cornerstones of fhhnc treatment. the efficiency of thiazides, which decrease urinary ca excretion, on gfr decline remains controversial. | familial hypomagnesaemia with hypercalciuria and nephrocalcinosis is an autosomal - recessive disease caused by mutations in the cldn16 or cldn19 genes, which encode tight junction - associated proteins, claudin-16 and -19. the resultant tubulopathy leads to urinary loss of mg2 + and ca2 +, with subsequent nephrocalcinosis and end - stage renal disease (esrd). an 18-year - old boy presented with chronic kidney disease and proteinuria, as well as hypomagnesaemia, hypercalciuria and nephrocalcinosis. a kidney biopsy revealed tubular atrophy, interstitial fibrosis and segmental sclerosis of some glomeruli. two novel mutations in the cldn16 gene were identified : c.340c > t (nonsense) and c.427 + 5g > a (splice site). the patient reached esrd at 23 and benefited from kidney transplantation. |
ischemic stroke and cancer represent a frequent coincidence with large - scale significance on stroke etiology, diagnostics and treatment options. evidence from early autopsy and recent clinical studies suggest a prevalence of ischemic stroke in cancer patients of approximately 3%. considering the annual incidence of cancer in germany published by the robert koch institute in 2012, this number would imply 15,000 affected stroke patients in germany each year. in practice, however, these numbers are much smaller as the association of stroke and cancer often remains unappreciated. this is due to the fact that cancer - related stroke is underestimated and difficult to diagnose, in particular in patients without a known cancer history or unrecognized stroke subtype patterns. owing to the lack of specific diagnostic markers, the diagnosis of cancer - related stroke strongly depends on its phenotype, which is determined by the interrelation of cancer - related hypercoagulation and concomitant arterial embolism. dwi - mri is exquisitely sensitive to detect areas of acute ischemic tissue change and has improved the understanding of distinct pathophysiological mechanisms leading to ischemia in several subgroups of stroke with differences in patterns considering lesion size, localization and distribution. specifying dwi lesions in cancer - related stroke may therefore amplify our understanding of the pathophysiological mechanisms, which take effect in patients with cancer - related stroke and, moreover, may be easily applied to help identifying patients affected by cancer - related stroke mechanisms. on the basis of the contemporary pathophysiological perception of cancer - related stroke, we hypothesized that embolic stroke patterns would be dominant in this selected cohort of stroke patients. patients with acute ischemic stroke, the additional diagnosis of solid and active malignancy, laboratory evidence of strong plasmatic hypercoagulation (d - dimer levels > 3 g / ml) and without competing stroke etiologies according to the ascod (a atherosclerosis, s small vessel disease, c cardiac pathology, o other cause, and d dissection) classification of evidence - rated etiology of stroke subtypes were included in the analysis. participants were recruited from our comprehensive stroke center (department of neurology, university medical centre mannheim, heidelberg university, mannheim, germany) and identified by our prospectively collected stroke data bank for the years 2004 - 2014. active cancer was defined as confirmed malignancy treated or untreated in the last 6 months before stroke. diagnosis of cancer was confirmed by given medical records or, in case of newly diagnosed or recurrent cancer, by histological evidence and oncologist expertise. patients with hematologic malignancies or primary brain tumor were not included in the study because these patients were considered to represent a subgroup with different underlying stroke mechanisms. type of cancer, stroke etiology and d - dimer levels were identified consistently by reviewing each patient. full stroke workup included extra- and intracranial doppler and duplex sonography of brain - supplying arteries, electrocardiography on admission, 72 h of electrocardiographic and vital sign monitoring, transthoracic or transesophageal echocardiography and laboratory tests (routine hematology and biochemistry, including coagulation test) as standard procedures according to stroke unit management recommendations (european stroke organization guidelines, 2008). the mri investigations were performed on 1.5-tesla and 3.0-tesla mri scanners (magnetom sonata, trio or skyra ; siemens, erlangen, germany). the scanning protocol included transversal dwi, t2-weighted fluid - attenuated inversion recovery, t1- and t2-weighted images, obtained with a 5-mm slice thickness, and an mr angiography. the acute lesion pattern on dwi was characterized according to number and localization with regard to the affected vascular territory (fig. the presence of (micro-) embolic lesions was documented, defined as > 5 scattered small dwi lesions (fig. 1). in addition, subacute (dwi hyper- and apparent diffusion coefficient iso- or hyperintense) and chronic ischemic lesions were assessed. images were reviewed independently by two stroke neurologists (c.j.s. and k.s.). in cases of discrepancy, the final pattern classification was reached by mutual agreement of the readers. before inclusion, the patients were phenotypically characterized according to the ascod classification. the prior asco phenotypic classification of stroke has shown good concordance with the most widely used trial of org 10172 in acute stroke treatment classification. additionally, the ascod classification reflects important supplementary information. patients with a competing stroke mechanism, defined by the presence of an ascod phenotype a (for atherothrombosis), s (for small vessel disease), c (for cardiac pathology), o (for other causes) or d (for dissection) graded 1 or 2 were excluded from the analysis. with respect to the suspected cancer - related stroke mechanisms, exceptions were made for patients with no direct cardiac source identified, but multiple brain infarction, repeated either bilateral or in two different arterial territories (e.g. both anterior and posterior circulation), classified c2 according to the ascod classification, and nonbacterial thrombotic the study was approved by the local institutional ethics committee (medizinische ethikkomission ii der medizinischen fakultt mannheim der ruprecht - karls - universitt heidelberg). patients with acute ischemic stroke, the additional diagnosis of solid and active malignancy, laboratory evidence of strong plasmatic hypercoagulation (d - dimer levels > 3 g / ml) and without competing stroke etiologies according to the ascod (a atherosclerosis, s small vessel disease, c cardiac pathology, o other cause, and d dissection) classification of evidence - rated etiology of stroke subtypes were included in the analysis. participants were recruited from our comprehensive stroke center (department of neurology, university medical centre mannheim, heidelberg university, mannheim, germany) and identified by our prospectively collected stroke data bank for the years 2004 - 2014. active cancer was defined as confirmed malignancy treated or untreated in the last 6 months before stroke. diagnosis of cancer was confirmed by given medical records or, in case of newly diagnosed or recurrent cancer, by histological evidence and oncologist expertise. patients with hematologic malignancies or primary brain tumor were not included in the study because these patients were considered to represent a subgroup with different underlying stroke mechanisms. type of cancer, stroke etiology and d - dimer levels were identified consistently by reviewing each patient. full stroke workup included extra- and intracranial doppler and duplex sonography of brain - supplying arteries, electrocardiography on admission, 72 h of electrocardiographic and vital sign monitoring, transthoracic or transesophageal echocardiography and laboratory tests (routine hematology and biochemistry, including coagulation test) as standard procedures according to stroke unit management recommendations (european stroke organization guidelines, 2008). the mri investigations were performed on 1.5-tesla and 3.0-tesla mri scanners (magnetom sonata, trio or skyra ; siemens, erlangen, germany). the scanning protocol included transversal dwi, t2-weighted fluid - attenuated inversion recovery, t1- and t2-weighted images, obtained with a 5-mm slice thickness, and an mr angiography. the acute lesion pattern on dwi was characterized according to number and localization with regard to the affected vascular territory (fig. the presence of (micro-) embolic lesions was documented, defined as > 5 scattered small dwi lesions (fig. 1). in addition, subacute (dwi hyper- and apparent diffusion coefficient iso- or hyperintense) and chronic ischemic lesions were assessed. images were reviewed independently by two stroke neurologists (c.j.s. and k.s.). in cases of discrepancy, the final pattern classification was reached by mutual agreement of the readers. the prior asco phenotypic classification of stroke has shown good concordance with the most widely used trial of org 10172 in acute stroke treatment classification. additionally, the ascod classification reflects important supplementary information. patients with a competing stroke mechanism, defined by the presence of an ascod phenotype a (for atherothrombosis), s (for small vessel disease), c (for cardiac pathology), o (for other causes) or d (for dissection) graded 1 or 2 were excluded from the analysis. with respect to the suspected cancer - related stroke mechanisms, exceptions were made for patients with no direct cardiac source identified, but multiple brain infarction, repeated either bilateral or in two different arterial territories (e.g. both anterior and posterior circulation), classified c2 according to the ascod classification, and nonbacterial thrombotic the study was approved by the local institutional ethics committee (medizinische ethikkomission ii der medizinischen fakultt mannheim der ruprecht - karls - universitt heidelberg). thirty - two patients (16 male, 16 female) met the inclusion criteria and were included in the analysis. lung carcinoma was most frequently observed (n = 13, 40%), followed by colorectal carcinoma (n = 4, 12.5%), gastric carcinoma (n = 4, 12.5%) and cancer of an unknown primary (cup ; n = 4, 12.5%). three patients (9%) suffered from pancreatic carcinoma, 2 (6%) from prostate cancer and 1 patient each from renal (3%) and ovarian (3%) carcinoma. none of the included patients or control subjects showed imaging evidence or clinical syndrome suggestive of cerebral vein thrombosis or primary intracerebral hemorrhage as these patients were not considered in the study per se. metastatic disease, earlier considered as an additional stroke risk factor, was present in 29/32 (90%) patients. the mean d - dimer levels were assessed with a routine coagulation test and were 15.39 g / ml (10.84) after a lower cutoff value of 3 g / ml had been defined for patient inclusion. the time between admittance to hospital and d - dimer assessment was 2.1 days (2.2) on average. mri demonstrated acute ischemic lesions in 2 vascular territories in 27/32 (84%) patients. additionally, (micro-) embolic scattering of infarction, as visualized in figure 1, was present in 25/32 (78%) patients. examples of dwi lesion patterns, characterized according to number and localization, are given in figure 1. the results of acute ischemic lesion patterns are summarized in detail in table 1. as an indication of recurrent strokes, 16/32 (46%) patients presented with additional subacute infarction. the same number of patients (16/32, 46%) also showed evidence of chronic infarction. three patients (8.5%) showed additional evidence of cerebral metastasis. in all of these cases thirty - two patients (16 male, 16 female) met the inclusion criteria and were included in the analysis. lung carcinoma was most frequently observed (n = 13, 40%), followed by colorectal carcinoma (n = 4, 12.5%), gastric carcinoma (n = 4, 12.5%) and cancer of an unknown primary (cup ; n = 4, 12.5%). three patients (9%) suffered from pancreatic carcinoma, 2 (6%) from prostate cancer and 1 patient each from renal (3%) and ovarian (3%) carcinoma. none of the included patients or control subjects showed imaging evidence or clinical syndrome suggestive of cerebral vein thrombosis or primary intracerebral hemorrhage as these patients were not considered in the study per se. metastatic disease, earlier considered as an additional stroke risk factor, was present in 29/32 (90%) patients. the mean d - dimer levels were assessed with a routine coagulation test and were 15.39 g / ml (10.84) after a lower cutoff value of 3 g / ml had been defined for patient inclusion. the time between admittance to hospital and d - dimer assessment was 2.1 days (2.2) on average. mri demonstrated acute ischemic lesions in 2 vascular territories in 27/32 (84%) patients. additionally, (micro-) embolic scattering of infarction, as visualized in figure 1, was present in 25/32 (78%) patients. examples of dwi lesion patterns, characterized according to number and localization, are given in figure 1. the results of acute ischemic lesion patterns are summarized in detail in table 1. as an indication of recurrent strokes, 16/32 (46%) patients presented with additional subacute infarction. the same number of patients (16/32, 46%) also showed evidence of chronic infarction. three patients (8.5%) showed additional evidence of cerebral metastasis. in all of these cases, as the diagnosis of cancer - related stroke is strongly dependent on its phenotype, its specification is of highest importance. especially the lack of recognition of a possibly cancer - related ischemic stroke pattern may prevent the identification of the present cancer - related stroke mechanisms and may be a reason for the undervaluation of this important phenomenon in cancer patients with stroke. currently, evidence of hypercoagulation and the presence of active malignancy in the absence of conventional stroke mechanisms such as atrial fibrillation or large vessel disease dominate the diagnostic criteria of cancer - related ischemic stroke. looking at these diagnostic criteria, dwi lesion patterns have received little attention, even though distinct dwi lesion patterns with involvement of multiple vascular territories have been reported repeatedly in cancer - related stroke. this may be due to the fact that these lesion patterns have never been addressed in detail in a selected cohort of cancer patients with evidence of hypercoagulation before. our data shows that after the exclusion of competing embolic and nonembolic stroke etiologies, dwi lesions in multiple vascular supply territories strongly dominate the phenotype of cancer - related stroke, being observed in 84% of all patients included in this analysis. additionally, (micro-) embolic scattering is a frequent feature of ischemic infarction in this special cohort of patients (being observed in 78% of all patients included in this analysis). this is well in line with previous results showing an elevated prevalence of embolic signal detected by transcranial doppler monitoring in cancer patients in up to 58% and associated with increased d - dimer levels. our results also confirm the common pathophysiological perception of recurrent cerebral (micro-) embolization due to cancer - related hypercoagulation as leading cause of cancer - related ischemic stroke. furthermore, our data shows that signs of recurrent embolization on mri by the detection of infarction at variable stages (acute, subacute or chronic) are also common in cancer patients with evidence of hypercoagulation and without competing stroke etiologies. this is consistent with other data, reporting an increased risk of new dwi lesions in patients with multiple territory lesions at initial presentation. as multiple dwi lesions have been shown to be associated with an increased risk of recurrent stroke, this finding may emphasize the urgency of validated strategies for secondary prevention of ischemic stroke in these patients. this conclusion supports preliminary findings concerning the elevated risk of recurrent stroke in cancer patients. however, these strategies still need to be established by prospective, randomized and controlled data. in the meantime, in selected individuals, the strategy for secondary prevention may be deduced from data concerning secondary prevention of venous thromboembolism in cancer patients. with respect to this data, treatment with low - molecular - weight heparin is currently recommended as first - line therapy by the engaged medical societies. in conclusion, (micro-) embolic scattering of dwi lesions in multiple vascular supply territories and in variable stages appear to be an important aspect of the phenotype of cancer - related stroke and are worth being considered in the diagnostic criteria. these results are not only important for the diagnosis of cancer - related stroke itself but potentially also for identifying cancer - related stroke patients with unknown malignancy at the time of stroke manifestation, which, however, remains to be addressed by a separate prospective approach. | backgrounddue to the lack of specific diagnostic markers, the diagnosis of cancer - related stroke strongly depends on its phenotype. distinct dwi lesion patterns with involvement of multiple vascular territories have been reported repeatedly in cancer - related stroke but have not been addressed in detail in a selected cohort of prospectively recruited cancer patients with emphasis on hypercoagulable conditions.patients and methodsischemic stroke patients with known malignant cancer activity, laboratory evidence of strong plasmatic hypercoagulation (d - dimer levels > 3 g / ml) and without competing stroke etiologies according to the recently introduced ascod (a atherosclerosis, s small vessel disease, c cardiac pathology, o other cause, and d dissection) classification of evidence - rated etiology of stroke subtypes were included in the analysis. cerebral mri on admission was reviewed with respect to ischemic lesion patterns.resultsthirty-two patients met the inclusion criteria. the mean d - dimer levels were 15.39 g / ml (10.84). acute infarction in 2 vascular territories was present in 27/32 (84%) patients. (micro-) embolic scattering of infarction was present in 25/32 (78%) patients. evidence for previous, potentially oligosymptomatic infarction was found in 16 (50%) patients, demonstrated by the additional presence of subacute or chronic ischemic lesions.conclusionwhen excluding competing embolic and nonembolic stroke etiologies, the pattern of scattered dwi lesions in multiple vascular supply territories strongly dominates the phenotype of cancer - related stroke. additionally, evidence of recurrent infarction is frequent in this cohort of patients. this is not only important for the diagnosis of cancer - related stroke itself but may prove helpful for the identification of cancer - related stroke patients with unknown malignancy at the time of stroke manifestation and evaluation of strategies for secondary prevention. |
the reported incidence of ureteral injury during colorectal surgery has ranged from 0.2 to 4.5 %. the use of prophylactic lighted ureteral stent (lus) placement has gained more importance with the advent of laparoscopic colectomy. lighted stents help in the visual identification of the ureters during a laparoscopic procedure. despite the apparent advantages of prophylactic ureteral stent placement, its effectiveness is still controversial. injury to the ureters in spite of stent placement, hematuria, and reflux anuria have also been reported. we report here our experience with prophylactic placement of lighted ureteral stents during laparoscopic colectomy and discuss the value and the outcomes of this procedure. a retrospective analysis was done of 66 patients who underwent laparoscopic colectomy (lco) with preoperative placement of lus at north oakland medical centers, pontiac, michigan, between april 1996 and january 2000. the catheter used was a polyurethane bard 6 lighted ureteral catheter. the parameters evaluated included age and sex of patients, the indication for surgery, the location of the pathology, the presence or absence of preoperative urinary tract symptoms. the urologic operative reports were reviewed for pathology identified during cystoscopy and ureteral catheterization, duration of the procedure, uni - lateral or bilateral placement of the catheters, and the size of the ureteral catheter used. complications related to ureteral catheter insertion, timing of catheter removal, intraoperative ureteral injury, occurrence of postoperative urinary tract infection, hematuria, and reflux anuria were also reviewed. operating room charges were also considered along with a comparison of the complications between unilateral and bilateral stent placement. of the 32 male patients, 3 (9.4%) had a history of urinary frequency and 4 (12.5%) had a history of dysuria. the urologic reports for pathology identification, duration of the procedure, and the side of catheter insertion are described in table 3. urinary tract infection (uti) was assessed with the urine culture reports postoperatively. in spite of the ureteral catheterization, 1 patient suffered an incomplete left ureteral injury during sigmoid colectomy, which was managed conservatively with the reinsertion of a left ureteral stent. the complications following unilateral stenting and bilateral stenting were compared as illustrated in figure 1. the duration of hematuria (days) poststenting between patients who had unilateral (2.5 0.82) and bilateral (3.37 1.05) stent placement was statistically significant with p < 0.001. the cost involved in stent placement included $ 1504.32 with an average increase of 26 minutes in operating room time costing $ 578, the urologist 's charges of $ 452.72, and each stent costing $ 236.80. the total percentage of patients with unilateral and bilateral stenting with complications is depicted in the figure. the usefulness and outcomes of prophylactic ureteral stenting during open colorectal surgery has been well documented in the literature. ureteral catheter placement allows intraoperative tactile localization of the ureters and is helpful in immediate recognition of ureteral injuries. with the introduction of laparoscopic colectomy as an alternative for traditional colorectal surgery, the placement of lus during laparoscopic colectomy has gained more importance because of visual identification of the ureters., we evaluated the value of prophylactic placement of lighted ureteral stents in laparoscopic colectomy. the majority of our patients had bilateral stents placed initially. as the surgeons gain more experience, the number of stents placed depends on the location of the pathology and the surgeon 's preference. ureteral injuries during laparoscopic colectomy have occurred and have also been reported during laparoscopic hysterectomy. ureteral injuries are of 4 types : laceration, ligation, crush, and devascularization. these injuries may be detected either intraoperatively or postoperatively. the placement of prophylactic ureteral stents helps in detecting the ureteral injuries intraoperatively and adequate action to be taken immediately. this injury was recognized on postoperative day 2 with urinary ascites and was managed by reinserting the left ureteral stent temporarily. the placement of ureteral catheters results in postoperative gross hematuria in almost all the patients. overall, this hematuria is of no clinical significance as no blood transfusion is required. on the other hand, anuria is suggested to be the result of neurogenic factors initiated by ureteral manipulation and mediated through the autonomic nervous system. studies have demonstrated that anuria after ureteral catheterization is due to edema which causes mechanical obstruction at the ureterovesical junction. recognition of this symptom, which usually requires repeated ureteral catheterization, is important. in our series, 4 (6.1%) it is possible that the 2 patients who had acute renal failure could have benefited from repeated ureteral stenting. the incidence of urinary tract infections was acceptable and in most cases was not troublesome. it may be attributable to the postoperative foley catheter that these patients received rather than the ureteral stents. we conclude that prophylactic lighted ureteral catheter placement in laparoscopic colectomy is a safe and cost - effective procedure. unilateral stent placement is recommended over bilateral stent placement to reduce operative time and postoperative hematuria. | objective : the placement of indwelling ureteral catheters during colorectal surgery has been recommended for prevention of ureteral injuries. with the advent of laparoscopic colectomy (lco), the role of preoperative placement of lighted ureteral stents (lus) has also become commonplace. we sought to evaluate the value of lighted ureteral stent placement in laparoscopic colectomy.methods:sixty-six patients underwent lco with lus inserted preoperatively. stents were removed in the immediate postoperative period. two surgeons performed all the colectomies ; 32 patients were males and 34 were females. fifty patients underwent sigmoid colectomy, 4 had abdominoperineal resection, 4 had right colectomy, and 1 each had transverse or subtotal colectomy. eighteen patients had a diagnosis of cancer, 34 had diverticular disease, and 14 had neoplastic polyps. forty patients had bilateral and 26 had unilateral stent placement. a review of the incidence of ureteral injuries, hematuria, and anuria as the cause of acute renal failure was accomplished, comparing the unilateral and bilateral stented groups.results:one (1.5%) patient suffered a left ureteral laceration during sigmoid colectomy. this was managed successfully with stent reinsertion. sixty - five (98.4%) patients had gross hematuria lasting 2.93 days (1 to 6 days). the cost of bilateral stent placement was $ 1504.32. a statistically significant difference occurred in the duration of hematuria (days) between patients who had unilateral (2.5 0.82) and bilateral stent placement (3.37 1.05), (p < 0.001). four patients suffered from anuria, 2 required renal support needing hemodialysis for 3 to 6 days, 3 (75%) had bilateral stents, and 1 (25%) had a unilateral stent.conclusions:we recommend the placement of lighted ureteral stents as a valuable adjunct to laparoscopic colectomy to safeguard ureteral integrity. transient hematuria is common but requires no intervention. reflux anuria occurs infrequently and is reversible. |
cine mri, combined with (c)spamm ((c)spamm = (complementary) spatial modulation of magnetization) encoding technology [14 ], admits disambiguation of local tissue motion, thus enabling the extraction of myocardial deformation and strain, which are known to correlate with cardiac pathologies. in particular, gtte. found that strain is more accurate than geometry in discriminating dysfunctional from functional myocardium. deformation and strain can be operationalized in various ways, either without explicit a priori regularization, or through exploitation of sparse constraints combined with interpolation and/or regularization [5, 733 ].. aletras. [2), and (harp) harmonic phase, cf. tagging - based methods using harp technology form our point of departure, compare with figure 1 for an illustration. given a dense motion field within the myocardium, our aim is to devise an operational procedure for direct extraction of myocardial deformation and strain. by direct we mean that we seek to obviate sophisticated preprocessing steps, such as segmentation of, or interpolation between tag lines, and finite element methods explicitly coupled to the tagging pattern. although such sophisticated indirect procedures exist and have been proven powerful, they require specific algorithmics that is neither trivially implemented nor readily available. instead we aim for a multiscale, optimally conditioned, intrinsically parallelizable, linear algorithm for obtaining deformation (and thus strain) in analytically closed form. optimal conditioning is achieved by exploitation of the scale degree of freedom in the definition of spatiotemporal differential image structure. we believe that the parsimony of our method facilitates applicability and optimization, since only off - the - shelf algorithms (linear filtering and inversion of linear systems) are needed in our computation of myocardial deformation and strain. our approach is as follows. to begin with, the velocity gradient tensor field, which is the prerequisite for the model we present in this paper, is obtained using the multiscale motion extraction algorithm for scalar image sequences proposed by florack.. this algorithm is adapted to the situation of tagging mri data [10, 13 ]. slick encoding and/or linear filtering yields an n - tuple of independent scalar phase images of some fixed spatiotemporal region of interest, in which n is the dimension of space (n = 2,3, as may be the case). optic flow constraint equation can, by construction, be applied to each of these n phase images, yielding an unambiguous system of equations for the underlying dense motion field to any desired differential order, and obviating tikhonov regularization (which would bring in at least one additional control parameter) as a disambiguating prior (as is typically the case in optic flow applications due to the aperture problem). it has been verified that the first - order scheme indeed produces a plausible, dense, and robust velocity gradient tensor field within the myocardium. details of its construction as well as the underlying automatic scale selection mechanism can be found in the cited literature (for an example of automatic scale selection, cf. in view of the cited work we will de - emphasize multiscale motion extraction and concentrate on subsequent deformation and strain analysis. in section 2 we outline in detail how to arrive at a closed - form analytical solution for the deformation tensor field (section 2.1) and hence the strain tensor field (section 2.2), given the velocity gradient tensor field. the novelty of this approach is that we circumvent numerical approximations in all intermediate steps, and introduce numerics only at the ultimate stage where we sample the resulting analytical tensor field expressions. besides avoiding in this way numerical errors that may be difficult to quantify, this procedure has the advantage of being mathematically transparent, computationally trivial, and intrinsically parallellizable. (these properties, in fact, also hold for the multiscale motion extraction algorithm used to provide the input velocity gradient tensor field, loc. some experimental results are given in section 3 to demonstrate the feasibility of our approach. section 4 concludes our work with a summary and briefly sketches work in progress. data acquisition details for the scans used in this paper are given in the appendix. the velocity gradient tensor, with components l relative to a coordinate frame, relates the rate of change of a momentary infinitesimal line element dx to the line element dx itself. infinitesimal vector attached to a fiducial material point in euclidean space and representing a directed material line element, that is, an infinitesimal number or as the th element of a local covector frame, depending on engineering or mathematical mind set.) from dx=dv it follows, using the chain rule, that (1)dx=l dx with l=vx(,=1, the numbers l constitute a matrix l with row and column index,. to get an estimate of this tensor field we have applied the algorithm of florack. and van assen., solving a linear system of algebraic equations enforcing optimal conditioning through scale selection. the reader is referred to the literature for details [10, 12, 13 ]. tissue deformation can be described by a map f : m n : x(x, t0) x(x, t), in which x(x, t) denotes the spatial position of a material point at time t, with reference position x = x(x, t0) at time t0 (lagrange picture). domain m and codomain n are copies of the deformable tissue medium (a subset of) at times t0, respectively, t t0. the associated differential map, (2)fdf : tmxtnx : vf(v)=vi fi f, relative to a basis { f } of tnx, provides a local linearization of tissue deformation, that is, the deformation tensor. here tmx is the tangent space of m at the fiducial point x = x(x, t0), and tnx that of n at the f - mapped point x = x(x, t). the matrix f(t, t0) of this linear map relative to the local coordinate charts { x, n } and { x, m } is given by the jacobian (3)fi=xxi. by virtue of the chain rule, the relation between deformation and velocity gradient tensors, (1) and (3) is given by the first - order ode (4)f=l f, subject to an initial condition, namely, f(t = t0, t0) = i. equivalently, (5)f(t, t0)=i+t0 t l(s) f(s, t0) ds. only for stationary flows, that is, l(t) = l0 pointwise constant, this initial value problem admits a trivial solution f(t, t0) = exp ((t t0)l0). (for the sake of simplicity the spatial dependency of all field entities is suppressed in the notation.) however, we do not have stationarity, and so we must proceed more carefully. equation (5) induces an expansion known as the matricant, compare with gantmacher : (6)f(t, t0)=i+t0tl()d+t0tl()t0l()d d+. the matricant has the following property : (7)f(t, t0)=f(t, t1)f(t1,t0) (t0<t1<t). it follows that, if we split the interval [t0, t ] into n parts by using intermediate points t1,, tn1 separated by tk = tk tk1 (k = 1,, n, with tn = t), (8)f(t, t0)=f(t, tn1)f(t1,t0),with t0<t1<<tn1<tn = t. for an infinitesimally narrow time interval [tk1, tk ] we have by approximation (9)f(tk, tk1)=i+l(tk) tk+higher order terms in tk, in which tk is any point satisfying tk1 tk tk. equations (8) and (9) give rise to a representation in terms of a so - called multiplicative integral : (10)f(t, t0)=~t0t(i+l()d)=deflim tk0(i+l(tn)tn)(i+l(t1)t1). one recognizes the multiplicative counterpart of the riemann sum approximation for ordinary (additive) integrals. one can show that this is identical to (11)f(t, t0)=~t0texp (l() d)=deflim tk0exp (l(tn)tn)exp (l(t1) t1). for instance, for square matrices a, b, one has det ab = det adet b, det (i + a) = 1 + tr a + (), det exp a = exp tr a. (12)det f(t, t0)=~t0t(1+tr l()d)=~t0texp (tr l()d). in particular, a divergence free velocity field (div v = tr l = 0) preserves volumes : det f(t, t0) = 1. furthermore, exp aexp b = exp (a + b) if [a, b ] = 0, whence for a stationary velocity field one obtains f(t, t0) = exp ((t t0)l0), as we already noticed. finally, the multiplicative integral suggests a straightforward numerical approximation, namely by using either (10) or (11) without limiting procedure. in this case the two representations are of course no longer identical. for computational efficiency we have chosen the former, with tk = t corresponding to the (constant) frame interval of our tagging mri sequence, and tk = kt. on the basis of the differential map f, (2), and its transpose f, one defines an intrinsic mapping on tmx, known as the lagrangian strain tensor, (13)e12(ftfidtmx):tmxtmx : ve(v)=vieijej, relative to a basis { ej } of tmx, with mixed tensor components (14)eij=12(gjfhfiij). here g are the components of the dual euclidean metric tensor in domain coordinates { x, m }, and h are those pertaining to codomain coordinates { x, n}. e vanishes identically if f is an isometry, thus e captures genuinely nonrigid deformations. the general coordinate convention, see (14), which admits different bases for deformed and undeformed configurations, is instructive. although one would normally prefer identical bases, let us consider the case in which the metric components h are those induced from gij by the deformation map itself (carry - along). that is, we assume that the coordinate frame for the deformed configuration is just the deformed coordinate frame of the reference configuration. one then expects the lagrangian strain tensor to be nullified, because everything, including the local reference frames, is intrinsically deformed in a consistent manner. indeed, if we write the infinitesimal material line element as (15)ds2=gijdxidxj = gijxixxjxdxdxhdxdx, we recognize the deformation tensor, see (3), and it follows that (16)gij = hfifj. as anticipated for the coordinate frames employed, (14) indeed reduces to (17)eij=12(gjhffi(16)ij)=12(gjgiij)=0. in practice one of course carries out computations relative to fixed (deformation independent) coordinate frames for undeformed and deformed configurations, but there is no reason to assume input and output frames to coincide a priori, nor to restrict oneself to cartesian coordinate frames.. this may be beneficial in certain situations, for example, those that suggest spherical, cylindrical or other coordinate systems depending on the configuration of acquisition data, compare with the phantom study by young., and the desired output representation. below we consider a single cartesian coordinate system for both domain and codomain. (in such a system the representations of the various metric tensors, gij and h and their duals, g and h, all simplify to identity matrices.) our analysis is confined to a single short - axis plane, whence n = 2, that is, we only account for in - plane motion components. figure 2 illustrates various scalar fields extracted from the strain tensor field, (13) and (14), by contraction with a pair of local unit vectors, namely radial (r = radial) and azimuthal (c = circumferential) basis vectors of the polar coordinate system centered at the midpoint of the region of interest, and those defining the strain tensor 's eigensystem. if u, v tmx are two such unit vectors, then the local scalar quantity derived from the strain tensor is given by the inner product (u, e(v)) = (e(u), v) = gjkueiv. figure 3 illustrates temporal evolutions of these quantities spatially averaged over the respective regions of interest, together with their standard deviations. we have proposed a novel, simple and robust linear model for extracting myocardial deformation and strain. material motion and gradient velocity are determined by a multiscale linear system of algebraic equations for the phase images extracted from the tagging mri data. these phase images are scalars, not densities, and are not hampered by tag fading due to t1 relaxation. we have analytically solved the linear matrix - ode governing myocardial deformation. by discretizing the closed - form solution we have subsequently solved for the induced lagrangian strain tensor field, yielding results that are typical for healthy volunteers, compare with garot., and atypical for a patient with a medical history of small infarcts on either side of a deviatory region and confirmed by late - enhancement mri. an advantage of our method is that only off - the - shelf algorithms are needed. the method is applicable in any spatial dimension, does not require conversion to a cartesian sampling grid, is optimally conditioned due to local scale selection, and is readily adapted to other modalities such as velocity encoding mri. our quantitative results in this feasibility study demand further evaluation so as to reveal their statistical significance and clinical value. furthermore, experiments based on carefully controlled synthetic or phantom data will allow us to disclose the physical significance and numerical tolerance of the mathematical framework in terms of ground truth deformation properties. moreover, by virtue of the transparent mathematical theory, it is also possible to assess tolerances on the basis of theoretical error propagation models, which can then be compared to those established experimentally for synthetic and phantom data. finally, although the local scale selection criterion exploited in our approach guarantees optimal conditioning, it does not necessarily yield optimal results due to the intrinsically parallel and thus spatiotemporally uncorrelated nature of the selected neighbouring scales. the effect of spatiotemporal regularization on the pattern of locally selected scales needs to be investigated. the short - axis mr tagging image data used in this study were acquired with a philips intera 1.5 t scanner from three volunteers (table 1) and a patient in basal, midventricular, and apical slices. the patient (figure 4) had a history of severe stenoses, and small infarction areas confirmed with late - enhancement mri 14 minutes after gadolinium contrast injection. a 2d multishot gradient - echo with echo planar imaging (epi factor 9) with breath - holding in end - expiration was used. scan parameters were : te 4.4 ms, tr 19 ms, flip angle 10, field - of - view 300 mm, scan matrix 128, acquisition voxel size 2.34 2.68 8 mm reconstructed into 1.17 1.17 8 mm. | myocardial deformation and strain can be investigated using suitably encoded cine mri that admits disambiguation of material motion. practical limitations currently restrict the analysis to in - plane motion in cross - sections of the heart (2d + time), but the proposed method readily generalizes to 3d + time. we propose a new, promising methodology, which departs from a multiscale algorithm that exploits local scale selection so as to obtain a robust estimate for the velocity gradient tensor field. time evolution of the deformation tensor is governed by a first - order ordinary differential equation, which is completely determined by this velocity gradient tensor field. we solve this matrix - ode analytically and present results obtained from healthy volunteers as well as from patient data. the proposed method requires only off - the - shelf algorithms and is readily applicable to planar or volumetric tagging mri sampled on arbitrary coordinate grids. |
skull base defects can derive from both traumatic and nontraumatic causes. in the traumatic group, nonsurgical trauma is the most common overall, and surgical (iatrogenic) damage is a minor cause. in the nontraumatic group, skull base erosion can be caused by high intracranial pressure due to tumor obstruction or from malignant neoplasms. spontaneous cerebrospinal fluid (csf) leaks (idiopathic) can also occur. as resections for skull base pathology become more complex, the resultant defects require more difficult and extensive reconstructions. this principle has become the pillar for most skull base reconstruction techniques, from traditional open to novel endoscopic endonasal or other minimally invasive approaches. the approach for reconstruction of skull base defects should be guided by the size and location of the defect, flow of the csf leak after resection, history of radiation or previous sinonasal surgery, and the experience of the surgical team. over the past 15 years, significant advances in surgical and reconstructive techniques have evolved in the treatment of multiple extradural and intradural skull base lesions via expanded endoscopic endonasal approaches (eeas), adding complexity to the reconstructive paradigm. the presence of a wide variety of available surgical techniques poses the question if it is better to access skull base lesions via traditional transcranial routes or via minimally invasive expanded eeas. historically, transcranial resection has been considered the gold standard for surgical removal of numerous suprasellar lesions.1 in 1907, herman schloffer performed the first transsphenoidal surgery.2 in 1916, cushing reported the first successful removal of a tuberculum sellae meningioma via a unilateral subfrontal approach. in 1950, norman dott introduced the lighted nasal speculum retractor. continuing the trend, gerard guiot introduced the x - ray film intensifier and fluoroscopy and pioneered image guidance surgery in 1956. in 1965, jules hardy introduced the use of the microscope in skull base surgeries, and in 1971, donald wilson introduced keyhole surgery, transitioning the trend to minimally invasive surgery.3 resection of skull base tumors has evolved to integrate modified skull base techniques such as supraorbital, orbitozygomatic, and orbitopterional approaches. regardless of the surgical technique, the primary objectives of skull base tumor resection remain the same : gross total tumor resection with adequate decompression and preservation of surrounding structures, prevention of future recurrence, support of the brain and orbit, complete separation of the cranial cavity from the sinonasal tract, elimination of dead space, and a watertight seal to avoid the consequences of csf leaks, such as meningitis and pneumocephalus. the disadvantages of the supraorbital keyhole approach include narrower viewing angles with limited maneuvering of instrumentation, especially in patients with optic canal involvement. although the bilateral frontal and unilateral frontal approaches provide excellent views of critical structures, the bilateral subfrontal approach has been noted to carry a greater risk of csf leak, olfactory nerve damage, and postoperative brain edema.4 5 additionally, with the pterional approach there can be significant frontal lobe retraction. the introduction of the endoscope to skull base surgery eliminated many of the previous problems associated with microsurgical techniques. casiano described the first pure eea for resection of an esthesioneuroblastoma.6 since its introduction, this approach has been internationally utilized for the resection of a variety of skull base lesions. important advantages of eea compared with classical surgical techniques and approaches are the better access to deeply seated lesions, a more direct exposure of the midline, reduced trauma to brain parenchyma, less manipulation of the neurovascular structures, rapid decompression of the optical structures, and more efficient devascularization of neoplasms from their surroundings.7 8 9 it is important to acknowledge that when important neurovascular structures are above or surrounding the capsule of the tumor, the eea is an ideal approach ; however, when a major vessel is to be encountered before reaching the surgical target, then open approaches are favored. for reconstructing skull base defects, it is important to understand the indications and limitations of each approach. preferably, the same route used for tumor removal should be used to repair the skull base defect, thus avoiding the comorbidity of a second incisional approach. in specific scenarios, gross tumor removal and avoidance of skull base defects can be achieved using the endoscope. endoscopic closure of csf leaks was first described by wigand in 1981 using free mucosal grafts, and to date, it continues to be the preferred method of csf leak closure because of its high success rate (90 to 97%).10 repairs can be achieved by mucosal grafting or a pedicled flap based on branches of the sphenopalatine, anterior ethmoidal, and facial arteries.11 also, collagen matrix materials, fascia, or fat can be used as inlay grafts to help seal these defects. a free graft is a tissue cut from one site and transplanted to another site. a pedicled flap is tissue that is left attached to its donor site and transposed to a new location keeping its pedicle intact. prior to the routine use of vascularized tissue flaps for skull base reconstruction, free grafts of biologic or synthetic material were used primarily in a multilayer approach. reconstructive allografts include duragen (integra lifescience corporation, plainsboro, new jersey, united states) and alloderm (lifecell corporation, branchburg, new jersey, united states), dural sealants such as duraseal (confluent surgical, inc., waltham, massachusetts, united states), and fibrin glue. the middle turbinate is the most common donor site for the harvesting of intranasal free grafts. other non - nasal free grafts, such as temporalis fascia, fascia lata, and palatal mucosa, can be used. it is recommended that the free graft be 25% larger than the defect, because there is a 20% reduction in size.12 free grafts have the advantage of easy harvest with low donor site morbidity. among pedicled vascular flaps, the posteriorly pedicled nasoseptal flap or hadad - bassagaisteguy flap (nsf) is the tip of the spear of most endoscopic skull base defect repairs (figs. 1 to 3).13 however, there are other intranasal pedicled flaps such as inferior turbinate flaps11 14 and middle turbinate flaps,15 each with its own advantages and disadvantages. (a) unilateral anterior cranial base defect following the resection of a sinonasal malignancy. (b) inlay placement of duragen (integra lifescience corporation, plainsboro, new jersey, united states). the nsf is a vascular flap composed of mucoperiosteum and mucoperichondrium from the nasal septum, pedicled on the posterior nasoseptal artery. it can cover a wide area of the skull base, from the posterior wall of the frontal sinus to the sella turcica, and from orbit to orbit. the flap should be separated from any nonabsorbable packing with nonadherent material so that the graft is not disrupted upon removal of the packing. the size of the nsf can be compromised by lesions that involve the cropped area or septal spurs and in patients under 10 years of age where the septum is not fully developed.16 the nsf is limited in its ability to reach extremely anterior defects, such as those involving the posterior frontal table, frontal break, and anterior cribriform plate. posteriorly17 or anteriorly18 pedicled inferior turbinate flaps or middle turbinate flaps can be considered as a second option for smaller defects when an nsf is not available or not feasible. hadad described a modification of the anteriorly pedicle inferior turbinate flaps where dissection of the lateral nasal wall was added (hadad - bassagaisteguy flap 2 or hb2).11 the hb2 has the capacity to cover combined defects (transcribriform and transplanum). it pedicle includes the territory of the facial (angular) artery and the anterior ethmoidal artery. this flap can be modified to have a posteriorly based pedicle to cover defects of the planum sphenoidale, sella, clivus, and nasopharynx.19 these lateral nasal wall flaps require special surgical endoscopic skills, as they can be difficult to dissect. significant crusting can occur, as with other nasal flaps, which usually continues until total remucosalization occurs. such flaps are harvested from regional areas including the palatal flap, the pericranial flap, facial buccinators flap, and temporoparietal fascial flap. the palatal flap has a vascular supply derived from the greater palatine artery, providing a 3-cm pedicle that could potentially reach any area of the skull base. nevertheless, this technique has mainly been described in cadaveric studies, making it a resource of last choice.20 21 the pericranial flap was historically used as the first option before the use of endoscopic repairs.21 its pedicle is based upon the supraorbital and supratrochlear arteries, and it is transposed through a small nasionectomy into the endoscopic field. it can cover from the anterior cranial fossa as far as the sella, without reaching the posterior cranial base defects. in the past, this flap was considered to have a negative impact on cosmetics ; however, with the use of the endoscope, better visualization and minimally invasive surgery have avoided this problem.22 the facial buccinator flap is pedicled upon the facial artery after it branches off the external carotid artery.23 it can be a solely muscular or a combined myomucosal flap. it has a coverage area that fluctuates from 2 cm to 20.76 cm, with an average of 15.90 cm, and it is able to reach the anterior skull base and planum sphenoidale.24 this flap has only been described in cadavers, so the drawbacks are so far theoretical. these include the introduction of oral flora to the surgical field, vascular and nerve injuries, and the most important of them all, variability in flap extensions due to gravitational forces and retraction ability. however, it is important to acknowledge this flap 's advantages, such as its extension, the axis of rotation, and the absence of external scars. the temporoparietal flap is a good reconstructive option for defects of the sella, parasellar area, and clivus (fig. the pterygopalatine fossa is dissected, the vidian nerve is sectioned, and the anterior aspects of the pterygoid plates are drilled. externally, a hemicoronal incision is carried down to the level of the hair follicles, a wide tunnel beneath the superficial layer of the deep temporalis fascia is created, and a lateral canthotomy incision is used to expose and separate the temporalis muscle from the lateral orbital wall and pterygomaxillary fissure, creating a tunnel that communicates the temporal, infratemporal fossa, and endoscopic transpterygoid approach. a portion of the lateral wall of the maxillary sinus is removed to open a wide communication with the infratemporal fossa, thus establishing communication between the flap and the skull base. (a) defect following an endoscopic nasopharyngectomy. (b) reconstruction of the defect with the nasoseptal flap providing coverage for the upper cervical spine (black arrow) and the temporoparietal fascia flap covering the parapharyngeal space and ica (white arrow). it is helpful in separating the intracranial / extracranial spaces and provides sufficient bulk to obliterate the dead space created by the resection of infratemporal fossa / nasopharyngeal pathology. for those lateral skull base defects that require skin as well as soft tissue bulk, the pectoralis major myocutaneous flap still remains a viable option (fig. 5). (a) lateral skull base defect following the excision of a parotid malignancy extending to the jugular foramen. (b) reconstruction of the defect with a pectoralis myocutaneous rotational flap. success rates of pedicled flaps approximate 95%, which makes them a reliable reconstruction option for skull base defects.25 however, when the size and location of the skull base defect to be repaired exceeds the excursion limits of the pedicled flap, free flaps can be considered. the major disadvantage of this flap is it bulkiness due to the width of the subcutaneous tissue of the abdominal wall. the radial forearm flap is a fasciocutaneous flap supplied by the radial artery and vein. it is very pliable, which makes it versatile for skull base defect reconstruction (fig. the flap has a very long pedicle, which allows for usage of neck vessels as recipient vessels. although it is very rare, hand ischemia can occur because of radial artery sacrifice. in our experience, when the physical exam suggests poor hand perfusion during occlusion of the radial artery, doppler ultrasonography is helpful in determining the patency of the palmar arch. the anterolateral thigh has become the preferred free flap by many reconstructive surgeons ; however, in our practice we still favor the forearm free flap due to pedicle length. (a) lateral skull base defect following the excision of a cutaneous malignancy with extension to the temporal bone and middle cranial fossa. (b) dural reconstruction was performed with suturable duragen (not shown ; integra lifescience corporation, plainsboro, new jersey, united states). the residual craniotomy bone flap was repositioned and the soft tissue and skin defect were reconstructed with a radial forearm free flap. osteocutaneous free flaps can be used in cases where significant orbital reconstruction is required. in our experience, 7).26 other osteocutaneous options include the scapula and hip bones with respective overlying skin. (a) maxillectomy and orbital rim defect following the excision of a maxillary sinus sarcoma. (b) inset of a stacked fibula free flap for reconstruction of the orbit and maxilla. patients are followed in the office every 2 to 4 weeks for removal of crusting and disruption of any synechiae and to spot signs of possible csf leaks. complications arising from skull base reconstruction can be direct or indirect, or perioperative and late postoperative. the nasoseptal flap can lead to displacement of the olfactory epithelium, persistent nasal crusting, and sphenoid sinus obstruction due to the pedicle 's orientation. special areas of skull base reconstruction like the frontal sinus, the orbit, or major neurovascular structures must have special consideration. complications from external incisions like alopecia, pain, hypesthesia, and/or infections can arise.27 crusting is the most common symptom after skull base reconstruction (3 to 4 weeks of duration),28 followed by nasal discharge, which is associated with more complex dissections.29 resections of large portions of mucosa, such as the inferior turbinate, could lead to atrophic rhinitis. improvement in nasal quality of life is usually achieved 4 months after surgery in most cases and in 6 months for more complex cases.30 the use of endoscopic skull base techniques usually does not significantly contribute negatively to nasal quality of life scores ; however, careful perioperative planning must be preformed to avoid such complications. early complications (28 days postoperatively) include palatal fistula, wound infection, meningitis, intracranial abscess, ectropion, enophthalmos, orbital dystopia, persistent diplopia, and facial cellulitis. prior radiotherapy is a statistically significant predictor of wound complications, and the existence of medical comorbidities is the only independent risk factor for death.31 the incidence of local and/or systemic postoperative complications following microvascular reconstruction of the skull base ranges between 30 and 40%.32 33 34 35 36 mortality rates are close to 4.7% following craniofacial resection.31 the presence of a wide variety of available surgical techniques poses the question if it is better to access skull base lesions via traditional transcranial routes or via minimally invasive expanded eeas. historically, transcranial resection has been considered the gold standard for surgical removal of numerous suprasellar lesions.1 in 1907, herman schloffer performed the first transsphenoidal surgery.2 in 1916, cushing reported the first successful removal of a tuberculum sellae meningioma via a unilateral subfrontal approach. in 1950, norman dott introduced the lighted nasal speculum retractor. continuing the trend, gerard guiot introduced the x - ray film intensifier and fluoroscopy and pioneered image guidance surgery in 1956. in 1965, jules hardy introduced the use of the microscope in skull base surgeries, and in 1971, donald wilson introduced keyhole surgery, transitioning the trend to minimally invasive surgery.3 resection of skull base tumors has evolved to integrate modified skull base techniques such as supraorbital, orbitozygomatic, and orbitopterional approaches. regardless of the surgical technique, the primary objectives of skull base tumor resection remain the same : gross total tumor resection with adequate decompression and preservation of surrounding structures, prevention of future recurrence, support of the brain and orbit, complete separation of the cranial cavity from the sinonasal tract, elimination of dead space, and a watertight seal to avoid the consequences of csf leaks, such as meningitis and pneumocephalus. the disadvantages of the supraorbital keyhole approach include narrower viewing angles with limited maneuvering of instrumentation, especially in patients with optic canal involvement. although the bilateral frontal and unilateral frontal approaches provide excellent views of critical structures, the bilateral subfrontal approach has been noted to carry a greater risk of csf leak, olfactory nerve damage, and postoperative brain edema.4 5 additionally, with the pterional approach there can be significant frontal lobe retraction. the introduction of the endoscope to skull base surgery eliminated many of the previous problems associated with microsurgical techniques. casiano described the first pure eea for resection of an esthesioneuroblastoma.6 since its introduction, this approach has been internationally utilized for the resection of a variety of skull base lesions. important advantages of eea compared with classical surgical techniques and approaches are the better access to deeply seated lesions, a more direct exposure of the midline, reduced trauma to brain parenchyma, less manipulation of the neurovascular structures, rapid decompression of the optical structures, and more efficient devascularization of neoplasms from their surroundings.7 8 9 it is important to acknowledge that when important neurovascular structures are above or surrounding the capsule of the tumor, the eea is an ideal approach ; however, when a major vessel is to be encountered before reaching the surgical target, then open approaches are favored. for reconstructing skull base defects, it is important to understand the indications and limitations of each approach. preferably, the same route used for tumor removal should be used to repair the skull base defect, thus avoiding the comorbidity of a second incisional approach. in specific scenarios, gross tumor removal and avoidance of skull base defects can be achieved using the endoscope. endoscopic closure of csf leaks was first described by wigand in 1981 using free mucosal grafts, and to date, it continues to be the preferred method of csf leak closure because of its high success rate (90 to 97%).10 repairs can be achieved by mucosal grafting or a pedicled flap based on branches of the sphenopalatine, anterior ethmoidal, and facial arteries.11 also, collagen matrix materials, fascia, or fat can be used as inlay grafts to help seal these defects. a free graft is a tissue cut from one site and transplanted to another site. a pedicled flap is tissue that is left attached to its donor site and transposed to a new location keeping its pedicle intact. prior to the routine use of vascularized tissue flaps for skull base reconstruction, free grafts of biologic or synthetic material were used primarily in a multilayer approach. reconstructive allografts include duragen (integra lifescience corporation, plainsboro, new jersey, united states) and alloderm (lifecell corporation, branchburg, new jersey, united states), dural sealants such as duraseal (confluent surgical, inc., waltham, massachusetts, united states), and fibrin glue. the middle turbinate is the most common donor site for the harvesting of intranasal free grafts. other non - nasal free grafts, such as temporalis fascia, fascia lata, and palatal mucosa, can be used. it is recommended that the free graft be 25% larger than the defect, because there is a 20% reduction in size.12 free grafts have the advantage of easy harvest with low donor site morbidity. among pedicled vascular flaps, the posteriorly pedicled nasoseptal flap or hadad - bassagaisteguy flap (nsf) is the tip of the spear of most endoscopic skull base defect repairs (figs. 1 to 3).13 however, there are other intranasal pedicled flaps such as inferior turbinate flaps11 14 and middle turbinate flaps,15 each with its own advantages and disadvantages. (a) unilateral anterior cranial base defect following the resection of a sinonasal malignancy. (b) inlay placement of duragen (integra lifescience corporation, plainsboro, new jersey, united states). the nsf is a vascular flap composed of mucoperiosteum and mucoperichondrium from the nasal septum, pedicled on the posterior nasoseptal artery. it can cover a wide area of the skull base, from the posterior wall of the frontal sinus to the sella turcica, and from orbit to orbit. the flap should be separated from any nonabsorbable packing with nonadherent material so that the graft is not disrupted upon removal of the packing. the size of the nsf can be compromised by lesions that involve the cropped area or septal spurs and in patients under 10 years of age where the septum is not fully developed.16 the nsf is limited in its ability to reach extremely anterior defects, such as those involving the posterior frontal table, frontal break, and anterior cribriform plate. posteriorly17 or anteriorly18 pedicled inferior turbinate flaps or middle turbinate flaps can be considered as a second option for smaller defects when an nsf is not available or not feasible. hadad described a modification of the anteriorly pedicle inferior turbinate flaps where dissection of the lateral nasal wall was added (hadad - bassagaisteguy flap 2 or hb2).11 the hb2 has the capacity to cover combined defects (transcribriform and transplanum). it pedicle includes the territory of the facial (angular) artery and the anterior ethmoidal artery. this flap can be modified to have a posteriorly based pedicle to cover defects of the planum sphenoidale, sella, clivus, and nasopharynx.19 these lateral nasal wall flaps require special surgical endoscopic skills, as they can be difficult to dissect. significant crusting can occur, as with other nasal flaps, which usually continues until total remucosalization occurs. such flaps are harvested from regional areas including the palatal flap, the pericranial flap, facial buccinators flap, and temporoparietal fascial flap. the palatal flap has a vascular supply derived from the greater palatine artery, providing a 3-cm pedicle that could potentially reach any area of the skull base. nevertheless, this technique has mainly been described in cadaveric studies, making it a resource of last choice.20 21 the pericranial flap was historically used as the first option before the use of endoscopic repairs.21 its pedicle is based upon the supraorbital and supratrochlear arteries, and it is transposed through a small nasionectomy into the endoscopic field. it can cover from the anterior cranial fossa as far as the sella, without reaching the posterior cranial base defects. in the past, this flap was considered to have a negative impact on cosmetics ; however, with the use of the endoscope, better visualization and minimally invasive surgery have avoided this problem.22 the facial buccinator flap is pedicled upon the facial artery after it branches off the external carotid artery.23 it can be a solely muscular or a combined myomucosal flap. it has a coverage area that fluctuates from 2 cm to 20.76 cm, with an average of 15.90 cm, and it is able to reach the anterior skull base and planum sphenoidale.24 this flap has only been described in cadavers, so the drawbacks are so far theoretical. these include the introduction of oral flora to the surgical field, vascular and nerve injuries, and the most important of them all, variability in flap extensions due to gravitational forces and retraction ability. however, it is important to acknowledge this flap 's advantages, such as its extension, the axis of rotation, and the absence of external scars. the temporoparietal flap is a good reconstructive option for defects of the sella, parasellar area, and clivus (fig. the pterygopalatine fossa is dissected, the vidian nerve is sectioned, and the anterior aspects of the pterygoid plates are drilled. externally, a hemicoronal incision is carried down to the level of the hair follicles, a wide tunnel beneath the superficial layer of the deep temporalis fascia is created, and a lateral canthotomy incision is used to expose and separate the temporalis muscle from the lateral orbital wall and pterygomaxillary fissure, creating a tunnel that communicates the temporal, infratemporal fossa, and endoscopic transpterygoid approach. a portion of the lateral wall of the maxillary sinus is removed to open a wide communication with the infratemporal fossa, thus establishing communication between the flap and the skull base. (b) reconstruction of the defect with the nasoseptal flap providing coverage for the upper cervical spine (black arrow) and the temporoparietal fascia flap covering the parapharyngeal space and ica (white arrow). it is helpful in separating the intracranial / extracranial spaces and provides sufficient bulk to obliterate the dead space created by the resection of infratemporal fossa / nasopharyngeal pathology. for those lateral skull base defects that require skin as well as soft tissue bulk (a) lateral skull base defect following the excision of a parotid malignancy extending to the jugular foramen. (b) reconstruction of the defect with a pectoralis myocutaneous rotational flap. success rates of pedicled flaps approximate 95%, which makes them a reliable reconstruction option for skull base defects.25 however, when the size and location of the skull base defect to be repaired exceeds the excursion limits of the pedicled flap, free flaps can be considered. the major disadvantage of this flap is it bulkiness due to the width of the subcutaneous tissue of the abdominal wall. also, there is risk of a ventral hernia owed to abdominal wall weakness. the radial forearm flap is a fasciocutaneous flap supplied by the radial artery and vein. it is very pliable, which makes it versatile for skull base defect reconstruction (fig. the flap has a very long pedicle, which allows for usage of neck vessels as recipient vessels. although it is very rare, hand ischemia can occur because of radial artery sacrifice. in our experience, when the physical exam suggests poor hand perfusion during occlusion of the radial artery, doppler ultrasonography is helpful in determining the patency of the palmar arch. the anterolateral thigh has become the preferred free flap by many reconstructive surgeons ; however, in our practice we still favor the forearm free flap due to pedicle length. (a) lateral skull base defect following the excision of a cutaneous malignancy with extension to the temporal bone and middle cranial fossa. (b) dural reconstruction was performed with suturable duragen (not shown ; integra lifescience corporation, plainsboro, new jersey, united states). the residual craniotomy bone flap was repositioned and the soft tissue and skin defect were reconstructed with a radial forearm free flap. osteocutaneous free flaps can be used in cases where significant orbital reconstruction is required. in our experience, 7).26 other osteocutaneous options include the scapula and hip bones with respective overlying skin. (a) maxillectomy and orbital rim defect following the excision of a maxillary sinus sarcoma. (b) inset of a stacked fibula free flap for reconstruction of the orbit and maxilla. patients are followed in the office every 2 to 4 weeks for removal of crusting and disruption of any synechiae and to spot signs of possible csf leaks. complications arising from skull base reconstruction can be direct or indirect, or perioperative and late postoperative. the nasoseptal flap can lead to displacement of the olfactory epithelium, persistent nasal crusting, and sphenoid sinus obstruction due to the pedicle 's orientation. special areas of skull base reconstruction like the frontal sinus, the orbit, or major neurovascular structures must have special consideration. complications from external incisions like alopecia, pain, hypesthesia, and/or infections can arise.27 crusting is the most common symptom after skull base reconstruction (3 to 4 weeks of duration),28 followed by nasal discharge, which is associated with more complex dissections.29 resections of large portions of mucosa, such as the inferior turbinate, could lead to atrophic rhinitis. improvement in nasal quality of life is usually achieved 4 months after surgery in most cases and in 6 months for more complex cases.30 the use of endoscopic skull base techniques usually does not significantly contribute negatively to nasal quality of life scores ; however, careful perioperative planning must be preformed to avoid such complications. early complications (28 days postoperatively) include palatal fistula, wound infection, meningitis, intracranial abscess, ectropion, enophthalmos, orbital dystopia, persistent diplopia, and facial cellulitis. prior radiotherapy is a statistically significant predictor of wound complications, and the existence of medical comorbidities is the only independent risk factor for death.31 the incidence of local and/or systemic postoperative complications following microvascular reconstruction of the skull base ranges between 30 and 40%.32 33 34 35 36 mortality rates are close to 4.7% following craniofacial resection.31 the reconstructive technique largely depends on the approach used for resection and the nature of the resection. intradural tumor surgery can be extra - arachnoidal (pituitary surgery) or intra - arachnoidal surgery (where an intraoperative csf leak can always be expected). intra - arachnoidal surgery can be further divided into high - flow and low - flow leaks depending on whether a cistern was directly opened. the risk for postoperative csf leak often depends on the location and size of the defect. it is always important to have in mind factors that increase the risk of postoperative csf leaks : obesity (associated with high ventricular pressure), pathology being treated (craniopharyngiomas), cushing disease, and history of radiotherapy (poor state of tissue healing) or prior surgery with compromise of local vascularized tissue reconstructive options. location of the skull base defect is the key factor in determining the type of reconstruction to be used. in a recent systematic review,37 the authors suggested that anterior fossa lesions can be repaired with inlay grafts, because the pressure from the brain could hold the material in position and avoid its migration ; defects of the tuberculum sellae or the clivus are best reconstructed with pedicled flaps due to their proximity to the anterior brain cisterns and ventricles. in general, low - flow csf leaks or small defects (90%.37 38 39 vascularized skull base reconstructions for large dural defects (> 3 cm) involving wide dural and arachnoid dissection, high - flow csf leaks, and poorly vascularized beds (defects) have a clear and significant advantage over free grafting in the prevention of postoperative csf leaks.37 38 40 when open techniques are utilized, the extent of injury to the entry point needs to be assessed and reconstructed accordingly. with free flaps being more reliable in recent years, they should be considered and utilized whenever possible as they provide excellent functional and cosmetic results. | introduction a substantial body of literature has been devoted to the distinct characteristics and surgical options to repair the skull base. however, the skull base is an anatomically challenging location that requires a three - dimensional reconstruction approach. furthermore, advances in endoscopic skull base surgery encompass a wide range of surgical pathology, from benign tumors to sinonasal cancer. this has resulted in the creation of wide defects that yield a new challenge in skull base reconstruction. progress in technology and imaging has made this approach an internationally accepted method to repair these defects. objectives discuss historical developments and flaps available for skull base reconstruction. data synthesis free grafts in skull base reconstruction are a viable option in small defects and low - flow leaks. vascularized flaps pose a distinct advantage in large defects and high - flow leaks. when open techniques are used, free flap reconstruction techniques are often necessary to repair large entry wound defects. conclusions reconstruction of skull base defects requires a thorough knowledge of surgical anatomy, disease, and patient risk factors associated with high - flow cerebrospinal fluid leaks. various reconstruction techniques are available, from free tissue grafting to vascularized flaps. possible complications that can befall after these procedures need to be considered. although endonasal techniques are being used with increasing frequency, open techniques are still necessary in selected cases. |
a 38-year - old male was admitted to the local hospital with pneumonia. despite medical treatment, his symptoms did not show any signs of improvement, and he was therefore referred to our hospital. when he arrived at the emergency department, initial arterial blood gas analysis (abga) showed a ph of 7.451, a pco2 of 29.3, a po2 of 43.6, an hco3 level of 20.6 mmol / l, and an o2 saturation value of 82.3%, and bilateral lung infiltration was noted on a simple chest x - ray. his heart rate was 12l beats per minute, his respiratory rate was 40 breaths per minute, his blood pressure was 162/94 mmhg, and his body temperature was 37.7c. he was diagnosed with acute respiratory failure and therefore, after endotracheal intubation, he was supported with mechanical ventilation. on the day that the patient was admitted, despite the 100% oxygen supplied by the ventilator, his abga results were as follows : a ph of 7.295, a pco2 of 54.6, a po2 of 69.7, a hco3 value of 26.8 mmol / l, and an o2 saturation value of 91.3%. therefore, we decided to initiate venovenous extracorporeal membrane oxygenation (vv ecmo) for systemic oxygenation and pulmonary support. vv ecmo (pls ; maquet, rastatt, germany) was established via bilateral femoral cannulation under local anesthesia (biomedicus 21 fr venous cannula ; medtronic biomedicus inc., anaheim, ca, usa). a right cannula was inserted into the superior vena cava - right atrium junction and a left cannula was inserted at the diaphragm level (fig. ecmo flow was initially started with a flow rate of 5.0 l / min, considering the patient s body weight of 103 kg. after the ecmo insertion, the following abga results were obtained : a ph of 7.429, a pco2 of 29.5, a po2 of 82.9, an hco3 value of 19.4 mmol / l, and a saturated o2 value of 96.6%. the patient was finally diagnosed with influenza (h1n1)-induced acute respiratory distress syndrome (ards) and was treated with intravenous antiviral drugs. however, 20 days after admission, despite treatment with intravenous inotropics and high - fractionated oxygen (100%) using ecmo and a mechanical ventilator, his blood pressure decreased to < 90 mmhg and his o2 saturation value fluctuated between 80%85%. on follow - up echocardiography, a severely dilated right ventricle (rv) with diminished systolic function was noted. a flattened interventricular septum was noted during systole, which suggested pressure overloading in the rv. additionally, pulmonary hypertension with an estimated pulmonary arterial pressure of 40 mmhg (fig. 2a) and a central venous pressure of 21.5 mmhg were noted, which indicated the existence of right ventricular dysfunction. laboratory findings showed worsening of end organ function : the ratio of aspartate transanimase to alanine transaminase was 151:96, his bilirubin level (total / direct) was 14.8/8.4 mg / dl, his d - dimer level was 3.34 g / ml, and his fibrinogen level was 468 mg / dl. we decided to add adjunctive cardiac support and changed the ecmo mode from vv mode to venoarteriovenous (vav) mode. we inserted an additional 15 fr biomedicus arterial cannula into the right femoral artery (fig. 1b), changed the oxygenator, and created a y - shaped inflow line. in order to unload the rv pressure, we partially clamped the venous input cannula at approximately 90% of its diameter and maintained an ecmo flow rate of 5.56.0 l / min. approximately 5 l of blood flow was infused into the femoral artery through the new arterial cannula, and the remnants were infused into the right atrium through the venous input cannula. after changing the ecmo mode, the rv size decreased to close to normal, and the interventricular septal flattening disappeared. improved rv systolic function we changed the degree of clamping of the venous input cannula according to the changing conditions of the patient. the patient s condition gradually improved as his pneumonia resolved, and we eventually decided to wean him from ecmo based on his lung function status. after 37 days of hospitalization, we removed the right femoral venous inflow line, and on the next day we successfully weaned the patient from ecmo. on day 42, the patient was weaned from the mechanical ventilator, and on day 54, he was transferred from the intensive care unit to the general ward. after a long period of rehabilitation therapy, his tracheostomy cannula when avian flu was widespread worldwide in the early 2000s, cases of ards due to influenza also increased abruptly. when severe respiratory failure is refractory to medical and conventional mechanical ventilator therapy, vv ecmo is the last line of treatment. vv ecmo can supply oxygenated blood to the right atrium and reduce the burden of the mechanical ventilator. thus, it provides time for the patient s lungs to rest and recover from respiratory failure and shock. hill. reported the first case of a patient who recovered from trauma - induced adult respiratory failure through the application of vv ecmo treatment. he and his colleagues showed the possibilities of ecmo to be an effective treatment for respiratory failure and cardiogenic shock. in the cesar (conventional ventilation or ecmo for severe adult respiratory failure) trial, which was a multicenter randomized controlled trial comparing conventional ventilation to ecmo for severe respiratory failure, ecmo showed a survival benefit of six months. the results of ecmo in that group were excellent, with reported survival rates of 68%77%. reported a survival rate of 50% in adults with severe respiratory failure who were treated with ecmo support. the main reason for the development of rvf in ards is the increase in the pulmonary vascular resistance (pvr). during the treatment of ards patients with a mechanical ventilator, the elevation of pvr is caused by hypoxic pulmonary vascular constriction, endothelial hyperplasia, and myointimal proliferation. mechanical ventilation with positive end - expiratory pressure itself is also a cause of pvr elevation [710 ]. the incidence of rvf related to ards has been reported to be 9.6%25% [1012 ]. rvf is known to develop from rv pressure overload, reduced rv contractility, and volume overload. rvf with pulmonary hypertension has poor outcomes in the intensive care unit. in studies addressing hemodynamic variables and survival with pulmonary arterial hypertension, a low cardiac index and high mean right atrial pressure reported a mode change from vv to venoarterial (va) in 3.3% of ards cases in the extracorporeal life support organization registry between 1986 and 2006. they hypothesized that right heart failure could be one of the causes of mode change. in our case, the patient s cardiac function was found to be good on the echocardiography imaging that was performed on admission. however, with continuing hypoxia and bilateral lung infiltration that lasted for longer than 20 days despite vv ecmo, his rv function probably decreased due to the increase in pvr caused by the abovementioned factors. this situation was verified by the high central venous pressure, pulmonary arterial pressure, and d - shaped left ventricle found on echocardiography (fig. another reason that vv ecmo may have led to rvf was the long duration of high flow from ecmo. non - pulsatile flow has been reported to show disadvantages in tissue perfusion and ventricular recoil function when compared to physiological pulsatile flow. non - pulsatile ecmo flow may sustain rv overloading despite normal rv contraction, which can induce a decrease in rv recoil function. with this hypothesis in mind, we are planning to carry out additional research on this topic in the near future. ecmo consists of long tubing systems, a membrane oxygenator, and a high - speed rotating centrifugal pump. it can infuse highly oxygenated blood into the venous or arterial circulation, but can also induce a systemic inflammatory reaction, hemolysis, bleeding, or thromboembolism. as occurred in this case our patient, when rv failure develops, vv - mode ecmo can be changed to va or vav mode for unloading the rv volume and rv shear stress. va or vav mode infuses the oxygenated blood into the left heart in order to elevate the cardiac output. however, in cases where pulmonary function has decreased significantly, va mode alone may not deliver enough oxygen to the tissues. if the cardiac function of an ards patient is good, va ecmo flow may not reach the aortic root, because the retrograde ecmo outflow may collide with the strong outflow from the patient s heart. in such cases, when unsaturated blood is supplied to the proximal aorta (coronary arteries or brachiocephalic arteries), which is a well - known complication of femoral va ecmo, vav ecmo can be utilized to overcome the upper body hypoxemia. if rvf occurs in a patient with severe ards who is being treated with peripheral vv ecmo, adding an arterial cannula to the femoral artery and partially clamping the inflow venous cannula may be an easy solution for overcoming rvf. in this case, rvf during vv ecmo support was successfully treated by changing the mode to vav ecmo. | a 38-year - old male was admitted with symptoms of upper respiratory infection. despite medical treatment, his symptoms of dyspnea and anxiety became aggravated, and bilateral lung infiltration was noted on radiological imaging studies. his hypoxemia failed to improve even after the application of endotracheal intubation with mechanical ventilator care, and we therefore decided to initiate venovenous extracorporeal membrane oxygenation (vv ecmo) for additional pulmonary support. on his twentieth day of hospitalization, hypotension and desaturation (arterial saturated oxygen < 85%) developed, and right ventricular failure was confirmed by two - dimensional echocardiography. therefore, we changed from vv ecmo to venoarteriovenous (vav) ecmo, and the patient ultimately recovered. in this case, right ventricular dysfunction and volume overloading were induced by long - term vv ecmo therapy, and we successfully treated these conditions by changing to vav ecmo. |
milk as an excretion of the mammary gland can carry numerous xenobiotic substances, which constitute a technological risk factor for dairy products and, above all, for the health of the consumer. determination of the residual concentrations of metals in milk could be an important direct indicator of the hygienic status of the milk, as well as an indirect indicator of the degree of pollution of the environment in which the milk was produced (licata. trace metals are a general collective term applying to the group of metals and metalloids with an atomic density greater than 6 g / cm. this term is widely recognized and usually applied to the elements such as cadmium (cd), cu, fe, lead (pb), and zn which are commonly associated with pollution and toxicity problems (malhat. metal residues in milk are of particular concern because milk is largely consumed by infants and children (tripathi. the food chain is an important source of cd and pb accumulation, especially for plants grown on polluted soils. significant amounts of cd and pb can be transferred from contaminated soil to plants and grass, causing accumulation of these potentially toxic metals in grazing ruminants, particularly in cattle (lpez alonso. accumulation of cd and pb in ruminants causes toxic effects in cattle, but also in humans consuming meat and milk contaminated with toxic metals (gonzlez - weller. cd and pb are amongst the elements that have caused the most concern in terms of adverse effects on human health. this is because they are readily transferred through food chains and are not known to serve any essential biological function. the poisoning is more common in farm ruminants, which are considered most susceptible to the toxic effects of lead (swarup. for that reason, the concentration of cd and pb in cow 's milk should be monitored to ensure the consumers ' health (jen. the concentrations of toxic heavy metals and trace elements were determined in the milk obtained most frequently from holstein - friesian cows. it seems justified to compare the concentration and the relationships between the levels of individual elements in the milk of simmental and holstein - friesian cows, kept in the same environment and fed identically, which allows finding breed differences in the concentration of elements and assimilability of heavy metals. therefore, the aim of this study was the comparison of the concentrations of toxic heavy metals and trace elements in the milk of simmental and holstein - friesian cows kept on an organic farm. the research material comprised milk obtained from 20 simmental dairy cows and 20 holstein - friesian cows kept on an organic farm with a total area of 4,000 ha of arable land including 3,000 ha of grassland, located in the northwestern part of the lubuskie province in poland (fig. 1). approximately 1,000 ha is located in the mouth of warta national park. this farm, i.e., specializing in milk production, as the only one in this area maintained the holstein - friesian and simmental cows under the same conditions, grazing them in the park area and its buffer zone.fig. 1location of the province and the farm location of the province and the farm the animals were kept in a loose barn with a free access to the stockyard located along the long walls of the building. winter feeding of cows was based on the preserved feeds obtained from the farm (maize silage, wilted silage, ensilaged sugar beet pulp, hay, and concentrates), whereas during the summer feeding, cows additionally used the pasture. milk samples from a total of 40 cows (20 samples from each breed) at the amount of 50 ml from one cow were taken in september 2008. each group, within the evaluated breed, consisted of cows of the similar milk yield (3,8004,000 kg), similar age (37 years old), and similar lactation stage (100150 day post - calving). elements (cd, pb, ca, mg, p, cu, fe, mn, se, zn) were determined using inductively coupled plasma emission atomic spectrometry by means of an optima 2000 dv instrument (perkinelmer inc.), after mineralization in a microwave system using the anton paar microwave oven. the analyzed elements were quantified using calibration curves plotted from analytical standards (merck, darmstadt, germany). the limit of detection was as follows : cd 0.1 g / l ; pb, 1.0 g / l ; ca, 0.05 g / l ; p, 4 g / l ; mg, 0.04 g / l ; cu, 0.4 g / l ; fe, 0.1 g / l ; mn, 0.1 g / l ; se, 4 g / l ; and zn, 0.2 g / l. the correctness of the analytical procedure was tested by determining the analyzed elements in reference material seronorm (trace elements serum, sero as) (n = 3) together with the samples. blank digests (n = 4) were run with a series of milk samples, and no major interferences were found in the quantitative element analysis. statistical analysis of the data was performed using statistica software (statsoft inc., version 10.0). the concentrations of elements were log transformed to attain or approach a normal distribution of the data. concentration of the analyzed elements in cow 's milk between simmental and holstein - friesian breeds was compared by the student 's t test. differences were considered significant at the level of p < 0.05, p < 0.01, and p < 0.001. the relationships between the levels of individual elements in the milk of examined animals were calculated using spearman rank correlation analysis. statistical significance of coefficients of correlation was tested at the levels of p < 0.05, p < 0.01, and p < 0.001. all data are expressed throughout as an arithmetic mean, geometric mean, minimum values, maximum values, quartile deviation, and also standard error mean. the concentration of toxic heavy metals and trace elements in milk samples is given in table 1. the milk of simmental cows had significantly lower concentration of pb and cd (p < 0.001) and cu (p < 0.05) and significantly higher concentration of fe and mg (p < 0.05) than did the milk of holstein - friesian cows. in the milk of simmental cows, higher concentrations of mn, se, and ca and lower concentrations of zn and p were also found ; however, these were not statistically significant differences.table 1content of mineral elements and toxic heavy metals in milk of simmental and holstein - friesian cowselementsconcentration, g / ml p valuesimmentalholstein-friesianmeangmmin.max.semqdgmmin.max.semqdca1,701.331,689.151,409.862,156.4547.332323.3011,606.671,600.371,273.211,949.8132.430149.9850.11mg130.87129.8496.66161.323.72620.498119.72119.1893.66149.992.62210.6660.02p980.90975.61719.931,186.5522.90486.6581,008.901,004.25813.251,216.5522.076116.6550.38cu0.03770.03290.01470.14200.005920.015720.04530.04260.02210.09930.003770.018160.02fe0.25760.23400.12830.70660.031080.070830.19840.18840.14000.49660.017850.068330.04mn0.02150.02100.01160.03250.001030.005920.02010.01910.00980.04070.001560.006550.46se0.01980.01600.01000.11870.005230.003350.01620.01600.01150.02090.000580.004020.49zn3.0272.9672.0264.4330.14030.926573.2773.2092.0434.8000.15100.82660.23pb0.03660.03640.03160.04400.000820.006000.04120.04110.03630.04770.000810.00667<0.001cd0.00350.00350.00280.00410.000070.000490.00400.00400.00330.00470.000070.00052<0.001 gm geometric mean, min. maximum values, qd quartile deviation, sem standard error mean content of mineral elements and toxic heavy metals in milk of simmental and holstein - friesian cows gm geometric mean, min. minimum values, max. maximum values, qd quartile deviation, sem standard error mean concentration of mg, p, fe, mn, se, and zn in the milk of cows of both breeds was within the standards proposed by gaucheron (2005) and hunt and nielsen (2009), that of ca was higher than the given range 1,0431,283 g / ml, whereas that of cu was at least 50 % too low in relation to the suggested normal concentration of 0.10.35 g / ml. unfortunately, the pb content in the milk of cows of both breeds was two times higher than the permissible concentration of 0.02 g / ml in the raw milk given by the standards of the european commission regulation (2006) establishing the highest permissible levels of some pollutants in foodstuffs. fisher. (1970) conducted a study in which ayrshires and holsteins were treated identically. the magnesium and calcium content of the milk from ayrshires was higher than that from holsteins. in an experiment by hermansen. (2005), a total of 480 samples of milk from 10 organically and 10 conventionally producing dairy farms in denmark were analyzed for 45 trace elements and 6 major elements. the dairy cattle breeds were danish - holstein or jersey. compared with the holsteins, jerseys produced milk with higher concentrations of ba, ca, cu, fe, mg, mn, mo, p, rh, and zn and with a lower concentration of bi. in the study by barowska. (2006) on five polish dairy cattle breeds (simmental, polish red, whitebacks, polish holstein - friesian of black - and - white and red - and - white variety), the milk from simmental cows was characterized by a significantly higher fe, mg, and zn content and a lower mn content compared with the milk from polish holstein - friesian cows of black - and - white variety. it was found that the ca, mg, and zn content was higher in the milk from simmental, polish red, and whiteback cows in comparison with polish holstein - friesian cows of black - and - white and red - and - white varieties. the cu content in the milk from simmental cows was also very low (only 0.03 g / ml), two times lower than that in the milk from polish holstein - friesian cows of black - and - white and red - and - white varieties as well as polish red cows (0.06 g / ml) and three times lower compared with polish holstein - friesians of red - and - white variety and whitebacks (0.09 and 0.10 g / ml, respectively). in the milk of simmental and holstein - friesian cows from the organic farm, particularly low cu concentrations amounting to 0.0377 and 0.0453 g / ml, respectively, were recorded, indicating the deficiency of this element in animals and thus in environment and feed either. copper deficiency is a common nutritional problem in ruminants, though cu excess is also commonly encountered, especially in sheep. it is considered that cu concentrations between 0.1 and 0.9 g / ml are the breed effects for efficiency in metabolizing cu are well documented (smart and christensen 1985 ; littledike. 1995 ; ward. (1996), holstein and jersey primiparous cows and growing heifers were supplemented with either 5 or 80 mg of copper per kilogram dry matter. at the end of the 60-day experiment, the hepatic cu concentration, plasma cu concentration, and ceruloplasmin oxidase activity clearly showed a genetic difference in cu absorption and post - absorption metabolism between holstein and jersey breeds. furthermore, liver copper concentrations increased more rapidly and were higher in the jerseys compared to holsteins supplemented with 80 mg of copper per kilogram dry matter. overall serum ceruloplasmin oxidase activity was higher in jerseys than holsteins. additionally, jersey cows and heifers had higher liver fe and lower liver zn concentrations than did holstein cows and heifers at the end of the experiment. these data indicate that jerseys and holsteins metabolize cu, zn, and fe differently. the genetic difference may be related to the efficiency of dietary cu absorption, the excretion of endogenous cu, or the amount of feed intake. (1994) suggested that the differences in endogenous cu excretion also contributed to the genetic differences in the retention of hepatic cu. cu deficiency occurs more frequently in simmental than in other breeds of cattle (gooneratne. bile cu concentration and bile cu excretion were higher in simmental cattle than in angus cattle (gooneratne. 1994). (1995) conducted a study in which angus and simmental steers were placed in metabolism crates to monitor apparent absorption and retention of copper. at the end of the experiment, plasma copper concentrations, apparent absorption, and retention of copper were higher in angus steers. the authors indicate that simmental cattle may have a higher copper requirement than angus cattle and that these different requirements may be related to the differences in copper absorption from the gastrointestinal tract between breeds. furthermore, it has also been suggested that these breed differences in copper metabolism may not be due solely to differences in absorption, but also to the manner in which copper is utilized or metabolized post - absorption. simmental steers had also lower serum and liver cu concentrations and serum ceruloplasmin activity than angus throughout the study by mullis. smart and christensen (1985) reported that hereford - sired cows had greater plasma cu concentrations during gestation than did simmental - sired cows. others have reported differences in serum mg, ca, and p. wiener (1980) found a difference in blood cu, ca, p, and mg concentrations between friesian and jersey cattle. angus had higher serum mg than did hereford cows in a study by greene. (1989) and true digestibility of mg in angus cows of this herd was higher than that of hereford. however, chirase. (1988) found that serum mg, ca, and p were similar in angus, angus hereford, angus jersey, brahman hereford, brahman jersey, and hereford jersey cows grazing oat pastures. (1995) compared the mineral status of angus, braunvieh, charolais, gelbvieh, hereford, limousin, red poll, pinzgauer, and simmental breeds consuming similar diets. in adult cattle, liver cu was higher for the limousin breed than for all others, except for angus. liver zn concentrations were higher for limousin than for pinzgauer, but no other breed differences were observed. serum ca concentrations were higher for angus, red poll, and limousin than for simmental, and red poll had higher concentrations of serum ca than did braunvieh. concentrations of serum ca were positively correlated with serum concentrations of cu, zn, and mg, but negatively correlated with liver fe. in few studies, an effect of breed on the efficiency of se metabolism was also shown. in an experiment by sprinkle. (2006), brahman cross (brahman salers or brahman hereford) cows were more efficient in metabolizing se, having greater whole blood se than either composite cows (25 % hereford, angus, gelbevieh, and senepol or barzona) or hereford cows. langlands. (1980) reported that brahman cattle in australia had greater blood se than brahman cross, africander, africander cross, brahman - africander hereford - shorthorn cross, or hereford shorthorn cross cattle. in evaluating specific sire breeds, they also reported that brahman hereford crosses had greater se than hereford hereford, friesan hereford, and simmental hereford genotypes. cd and pb are environmental pollutants toxic to humans and animals (cai. cd and pb are nonbiodegradable, and their accumulation in the environment raises agricultural and public health concerns (olsson. pb and cd concentrations in the milk of simmental cows were significantly lower (p < 0.001) compared to holstein - frisian cows. the pb concentration in the milk of both breeds exceeded, however, permissible eu standards, amounting to 0.0366 and 0.0412 g / ml for simmental and holstein - friesian cows, respectively. in the study by gabryszuk. (2010), the pb concentration in the milk of cows from organic farms was much lower and ranged from 0.0041 to 0.0062 g / ml. heavy metal contamination in milk has been reported also in different countries and regions (simsek. (2011), mean lead concentrations exceeded the maximum residue levels in the north and the south regions of croatia (0.0587 and 0.0362 g / ml, respectively). levels above 0.020 g / ml were measured in 35.5 % of samples from the north and 28.3 % of samples from the south regions. in the study by pavlovic (2004), the pb level ranged from 0.028 to 0.036 g / ml, but that of cd was between 0.005 and 0.006 g / ml for a majority of 15 farms in croatia. (2003) reported even higher pb concentration in milk, which amounted to 0.0230.070 g / ml. the interesting aspect in the present study is the interaction between toxic heavy metals (pb and cd) and major nutritional and trace elements (ca, mg, p, cu, fe, mn, se, zn) in milk because the nutritional function of milk is important for health. in the liver or kidney, the interactions of zn, cu, se, fe, ca, and pb or cd are well known from the earlier literature, but their relation in milk is not clearly reported (isaac. significant very high or high positive correlations (table 2) were found between the concentrations : pb cd (r = 0.86 vs. r = 0.87), pb se (r = 0.68 vs. r = 0.83), cd se (r = 0.62 vs. r = 0.70), cd mn (r = 0.49 vs. r = 0.61), zn cu (r = 0.57 vs. r = 0.46), zn p (r = 0.46 vs. r = 0.57), ca p (r = 0.64 vs. r = 0.81), mg p (r = 0.55 vs. r = 0.66), and ca mg (r = 0.89 vs. r = 0.62).table 2spearman rank correlations between milk concentration of different mineral elementselementsmgpcufemnseznpbcdsimmentalca0.890.640.220.260.080.220.380.410.36mg0.55 0.130.130.080.260.360.400.46p0.280.320.200.090.460.320.29cu0.290.190.130.570.060.03fe0.240.420.030.570.28mn0.420.150.430.49se0.100.680.62zn0.300.35pb0.86holstein - friesianca0.620.810.260.280.55 0.330.350.170.17mg0.660.090.240.370.170.370.270.27p0.200.400.650.54 0.570.47 0.49cu0.250.390.120.46 0.240.15fe0.330.140.350.020.05mn0.48 0.580.570.61se0.330.830.70zn0.49 0.55pb0.87p < 0.001, p < 0.01, p < 0.05, statistically significant coefficient of correlation spearman rank correlations between milk concentration of different mineral elements p < 0.001, p < 0.01, p < 0.05, statistically significant coefficient of correlation moreover, in the milk of simmental cows, statistically significant correlations were observed between milk concentrations of : cd and mg (r = 0.46), pb and ca (r = 0.41), and pb and fe (r = 0.57). in the milk of holstein - friesian cows, the concentration of pb and cd was positively and significantly correlated with p (r = 0.47 and r = 0.49, respectively), mn (r = 0.57 and r = 0.67, respectively), se (r = 0.83 and r = 0.70, respectively), and zn (r = 0.49 and r = 0.55 respectively). in addition, mn concentration significantly correlated with ca (r = 0.55), p (r = 0.65), se (r = 0.48), and zn (r = 0.58), concentration of zn with cu (r = 0.57) and p (r = 0.57), and concentration of se with p (r = 0.54). a very high, significant, and positive correlation between ca and mg (r = 0.873) in the milk of cows was reported also by rodrguez rodrguez. however, contrary to our results, they found significant negative correlations between cu and zn (r = 0.377) and not high positive correlations between fe and cu as well as fe and zn. in the study by sikri. (2003), the cu concentration in milk was significantly positively correlated with the concentrations of fe (r = 0.613) and zn (r = 0.629) and that of zn with mn (r = 0.731), which was proved in our work. in the milk of both analyzed breeds, the pb concentration was very highly correlated with cd concentration (r = 0.85 vs. r = 0.87), whereas in the study by pavlovic. (2007) reported strong positive correlations between cd and ca (r = 0.220), cd and mg (r = 0.201), cd and zn (r = 0.279), and cu and ca (r = 0.347) and negative correlation between pb and ca (r = 0.295) in breast milk. there is a paucity of earlier literature, particularly concerning animals, to compare the present finding. the correlations between the elements of milk were rarely analyzed (rodrguez rodrguez. 1999). the available literature describes relationships between different elements, principally in the liver, kidneys, and muscles of animals (lpez alonso. 2006), in the limited number in blood or serum (lpez alonso. the largest number of significant correlations between toxic and essential elements is found in the kidneys followed by liver (tomza - marciniak. (2004) is a reflection that these organs play the main role in trace element metabolism. experimental exposure of rats to either lead or cadmium or both concomitantly has been demonstrated to influence the metabolism and tissue concentration of divalent cations like zinc, copper, and iron and tissue - specific changes in the distribution of iron, zinc, copper, cobalt, and manganese have been documented after experimental administration of lead and cadmium in cattle and rats (patra and swarup 1998 ; oishi. pb and cd are thought to be transported from maternal plasma to mammary gland and secreted into milk along with cu and zn. however, the interaction between toxic heavy metals and trace elements (ca, mg, p, cu, fe, mn, se, zn) has not been understood clearly, particularly in milk. there is a good level of understanding of the role of major nutritional elements like ca, mg, p, na, and k in milk, but the effects of pb and cd on their metabolism have not been sufficiently investigated (isaac. cd and pb mainly distribute in the liver and kidney, where cd is bound to metallothionein (mt), a small, cysteine - rich metal - binding protein (klaassen. 1999). several studies have suggested that interactions between cd or pb and zn in the organism result in a high degree from an affinity of both metals to mt and their ability to induce its synthesis. they can induce mt synthesis in various tissues, especially in the intestine, liver, and kidney (brzska and moniuszko - jakoniuk 2001 ; cai. metallothionein, which is synthesized in response to cadmium, lead, zinc, copper, or mercury exposure (mt inducers), contributes to the accumulation of metals by eliminating them from metabolism. olsson. (2010) found a significant relationship between kidney levels of cd and metallothionein. another way of eliminating metals from metabolism is through the formation of neutral complexes (e.g., se cd and se pb) by selenium, which are then bound by proteins similar to metallothionein. this is possible because of the high affinity of selenium for these elements (nehru and iyer 1994 ; tomza - marciniak. the interaction between zn and cu has been extensively reported (blanco - penedo. 2006 ; bremner and beattie 1995) and is a consequence of the ability of these metals to induce synthesis of metallothioneins and of their competition for metallothionein - binding sites. interaction between toxic heavy metals (pb and cd) and major nutritional and trace elements was also found in humans, most frequently in blood and serum (brny. 2002) and in the milk of nursing mothers (perrone. wang. (2012) reported correlations among the toxic (cd, pb) and nutritionally essential (zn, cu, fe, mn, se) elements in the blood, also urine and feces in the male. in the case of the toxic metals, a significant positive correlation was found for cd pb in blood and a moderate correlation in urine. cd was positively correlated with most of the essential elements in both urinary and fecal excretion. moreover, significant direct correlations were found between cd and either zn or se concentration in both urine and feces, whereas a significant negative correlation was found between cd and se in blood. at present, we have no biological explanations to several others of the reported correlations and more studies are needed in this area. the correlations between pb and zn as well as cd and zn are noteworthy. in the milk from holstein - friesians (hf) cows, very high positive and significant correlations between these elements were recorded (0.83 and 0.70, respectively), whereas in the milk from simmental cows, these correlations were negative, weak, and statistically nonsignificant (0.30 and 0.35, respectively). in simmental cows, negative correlations were also found between pb and cd and ca, mg, and p ; however, only those between pb and ca (0.41) and cd and mg (0.46) were significant. in hf cows, only correlations between these elements and p were positive and significant (0.47 and 0.49 for pb was also observed in simmental cows, whereas it was low in hf cows. in the milk from hf cows, high positive and significant correlations between mn and ca, p, se, zn, and pb as well as between se and p were additionally found, whereas in simmental cows, these correlations were nonsignificant and the obtained correlation coefficients much smaller. the present research showed that the milk of simmental cows had more favorable mineral composition and lower concentration of toxic heavy metals compared to the milk of holstein - friesian cows. in the milk of simmental cows, significantly lower concentrations of pb, cd, and cu, significantly higher concentrations of fe and mg, as well as nonsignificantly higher concentrations of ca, mn, and se were found. in the milk of both breeds, particularly low cu concentrations (0.0377 vs. 0.0453 g / ml), at least two times lower than the recommended standards, were recorded, which indicates the deficiency of this element in animals as well as in environment and feed. also, the pb concentration in milk that was higher than the recommended standards was found. simmental and holstein - friesian cows remained in the same environment and were identically fed, which allows us to suppose that the differences obtained between these breeds in the content of the examined elements and heavy metals in milk are caused by differences of metabolic background. | concentrations of toxic heavy metals (cadmium (cd), lead (pb)) and major nutritional and trace elements (ca, mg, p, cu, fe, mn, se, zn) were analyzed in the milk of simmental (n = 20) and holstein - friesian (n = 20) cows from an organic farm. elements were determined using inductively coupled plasma emission atomic spectrometry. the conducted research showed that the milk of simmental cows was characterized by the more advantageous mineral composition and lower concentration of noxious heavy metals compared to the milk of holstein - friesian cows. in the milk of simmental cows, significantly lower concentrations of pb and cd (p < 0.001) and cu (p < 0.05) and significantly higher concentrations of fe and mg (p < 0.05) as well as nonsignificantly higher concentrations of ca, mn, and se were found. in the milk of both breeds, very low cu concentrations were recorded. the higher - than - recommended concentration of pb in milk was also found. in the milk of both breeds, the significant positive correlations between concentrations of the following elements were observed : pb cd, pb se, cd se, cd mn, zn cu, zn p, ca p, ca mg, and mg p. the correlations between other elements within each of the analyzed breeds separately were also found. |
multiple sclerosis, a chronic and debilitating inflammatory disease of the cns is characterized by myelin damage. in multiple sclerosis, although resident oligodendrocyte progenitors are found around the lesions, they remain in a quiescent state and remyelination is incomplete. thus, therapeutic strategies that promote remyelination are likely to have significant beneficial effects. the idea that sex steroids are involved in ms and that they might be a therapeutic option also originates from retrospective studies revealing that pregnancy has a favorable trend for the course of ms on a short- and long - term basis. remission of symptoms is seen during pregnancy, in particular during the last trimester when estrogens and progesterone plasma levels are at their maximum. recent reports, however, have shown that progesterone decreases neuropathology when given at the time of demyelination induction, but its therapeutic role in promoting myelin repair after induced demyelination is far less investigated. this is due to the conflicting results obtained in the experimental autoimmune encephalomyelitis (eae) model, which indicated negative as well as positive effects for progesterone on disease progression. in vivo studies on animal models of ms have drawn the attention on immune modulatory functions of sex steroids and thus eae model has been used. to be able to distinguish between the immunomodulating effects of progesterone and direct effects on repair capacity of the brain, we used the potential of non - immune driven cuprizone intoxication. demyelination in the cuprizone model is an early observable event without damaging other cns cell types ; besides oligodendrocytes and without provoking the breakdown of the blood brain barrier and subsequently causing the invasion of lymphocytes. in addition, the cuprizone model shares common features with the earliest phases of ms lesion development as described by barnett and prineas. in this study, remyelinating potential of progesterone in corpus callosum was evaluated after cuprizone - induced demyelination as an experimental model of ms. a total of 20 male c57bl/6 mice (pasteur institute of iran) were fed with 0.2% (w / w) cuprizone (sigma, usa) in ground breeder chow ad libitum for 6 weeks. this diet leads to selective oligodendrocyte death followed by demyelination of axons mainly in the corpus callosum. on day zero after cuprizone removal, animals were randomly divided into two groups : (a) placebo group, which received saline pellet implant (n=10), (b) progesterone group, which received progesterone pellet implant (n=10). some mice of the same age were fed with their normal diet to serve as healthy control group (n=10). on day zero after cuprizone removal, progesterone treated mice were implanted with 14-day release pellets containing 200 mg progesterone (innovative research of america, sarasota, usa). pellets were implanted subcutaneously in the scapular region behind the neck with a 12-gauge trocar as described by the manufacturer. placebo mice were implanted with saline pellets (innovative research of america, sarasota, usa). the animal experiments were approved by the ethics committee of tehran university of medical sciences, tehran, iran. for histological and immunohistochemical studies, mice were intracardially perfused with 4% paraformaldehyde (merck, germany) containing picric acid. after overnight postfixation, brains were dissected and processed routinely for paraffin (merck, germany) embedding. then, 10 m serial, sagital sections were prepared from corpus callosum. among these serial sections, four representative sections at least 240 m apart from each other were subject to immunostaining with anti olig2 antibody. to stain for myelin content, tissue sections from mice were treated with luxol fast blue (lfb, sigma, usa). sections were stained overnight in lfp at 56c and washed in 95% ethanol and distilled water to remove excess blue stain. the color was then differentiated (until white matter was easily distinguishable from gray matter) in lithium carbonate solution (merck, germany) for 15 sec, followed by distilled water and three washes of 80% alcohol. slides were passed through fresh xylene (merck, germany) twice, mounted with entellan (merck, germany) and cover slipped. in order to evaluate remyelination in lfb stained sections of cuprizone demyelination of the corpus callosum in the mice that received progesterone, three blinded readers scored lfb stained sections between zero and three. a score of three is equivalent to totally myelinated corpus callosum whereas zero is equivalent to totally demyelinated corpus callosum. a score of one or two corresponds to one - third or two - third fiber myelination of the corpus callosum, respectively. protein extract from corpus callosum were centrifuged at 560 g for 5 min ; then stored at -20c. to ensure loading of equal amounts during western blotting, protein concentration of each sample equivalent amounts of total protein extract from each sample were mixed with sample buffer, boiled, and loaded onto sds polyacrylamide gels. the proteins were then transferred to polyvinylidene (pvdf) (gibco, ca) and probed with mbp antibody (1:3000 in blocking buffer) (abcam, usa) and plp antibody (1:1000 in blocking buffer) (abcam, usa) for an overnight on shaker in 4c. membranes were subsequently rinsed in blocking buffer (4 times/3 min each time) and stained with secondary antibody (1:1000 in blocking buffer) (abcam, usa) for 1 hour. then, membranes were washed with tbs tween-20 blocking buffer (3 times) and the proteins were visualized with diaminobenzidine (dab) kit (sigma, usa). ten m sections from body of corpus callosum were incubated with 10% goat serum (sigma, usa) for 45 min, and then were incubated overnight with rabbit anti - olig2 polyclonal antibody (1:2000 ; millipore, ca, usa). the next day, sections were incubated with biotinylated secondary antibody (sigma, usa). this was followed by 1 h incubation at rt with avidin - biotin - peroxidise complex. for visualization, the dab was used. finally, the sections were dehydrated in a graded series of ethanol, cleared in xylene, and coverslipped with entellan (merck, germany) and examined with a nikon eclipse 55i (nikon, germany) microscope. quantification of cell numbers was performed by manual counting the number of positive cells using image j software. the complete corpus callosum was microdissected from pbs - perfused mice 14 days after progesterone pellet implantation. the tissue was placed into a petri dish containing 2 ml of digestion buffer, 1 mg / ml of collagenase d (roche, germany), 1 mg / ml of neutral protease (worthington, uk), dnase i (qiagen, uk), and diced into small pieces with a razor blade before incubation at 37c for 30 min. following the incubation, pbs was added to stop the enzymatic digestion and cells washed through a 70 m filter with facs buffer and centrifuged at 2,000 rpm for 5 min at 4c. isolated cells were incubated on ice for 5 min with anti - olig2 antibody (1:100, millipore, ca, usa) or anti - o4 antibody (1:50, millipore, ca, usa). for the secondary antibody, goat anti - mouse antibody igg fluorescein isothiocyanate (fitc) statistical analysis was performed by one - way analysis of variance (anova) followed by tukey s post - hoc test. a total of 20 male c57bl/6 mice (pasteur institute of iran) were fed with 0.2% (w / w) cuprizone (sigma, usa) in ground breeder chow ad libitum for 6 weeks. this diet leads to selective oligodendrocyte death followed by demyelination of axons mainly in the corpus callosum. on day zero after cuprizone removal, animals were randomly divided into two groups : (a) placebo group, which received saline pellet implant (n=10), (b) progesterone group, which received progesterone pellet implant (n=10). some mice of the same age were fed with their normal diet to serve as healthy control group (n=10). on day zero after cuprizone removal, progesterone treated mice were implanted with 14-day release pellets containing 200 mg progesterone (innovative research of america, sarasota, usa). pellets were implanted subcutaneously in the scapular region behind the neck with a 12-gauge trocar as described by the manufacturer. placebo mice were implanted with saline pellets (innovative research of america, sarasota, usa). the animal experiments were approved by the ethics committee of tehran university of medical sciences, tehran, iran. for histological and immunohistochemical studies, mice were intracardially perfused with 4% paraformaldehyde (merck, germany) containing picric acid. after overnight postfixation, brains were dissected and processed routinely for paraffin (merck, germany) embedding. then, 10 m serial, sagital sections were prepared from corpus callosum. among these serial sections, four representative sections at least 240 m apart from each other were subject to immunostaining with anti olig2 antibody. to stain for myelin content, tissue sections from mice were treated with luxol fast blue (lfb, sigma, usa). sections were stained overnight in lfp at 56c and washed in 95% ethanol and distilled water to remove excess blue stain. the color was then differentiated (until white matter was easily distinguishable from gray matter) in lithium carbonate solution (merck, germany) for 15 sec, followed by distilled water and three washes of 80% alcohol. slides were passed through fresh xylene (merck, germany) twice, mounted with entellan (merck, germany) and cover slipped. in order to evaluate remyelination in lfb stained sections of cuprizone demyelination of the corpus callosum in the mice that received progesterone, three blinded readers scored lfb stained sections between zero and three. a score of three is equivalent to totally myelinated corpus callosum whereas zero is equivalent to totally demyelinated corpus callosum. a score of one or two corresponds to one - third or two - third fiber myelination of the corpus callosum, respectively. protein extract from corpus callosum were centrifuged at 560 g for 5 min ; then stored at -20c. to ensure loading of equal amounts during western blotting, protein concentration of each sample equivalent amounts of total protein extract from each sample were mixed with sample buffer, boiled, and loaded onto sds polyacrylamide gels. the proteins were then transferred to polyvinylidene (pvdf) (gibco, ca) and probed with mbp antibody (1:3000 in blocking buffer) (abcam, usa) and plp antibody (1:1000 in blocking buffer) (abcam, usa) for an overnight on shaker in 4c. membranes were subsequently rinsed in blocking buffer (4 times/3 min each time) and stained with secondary antibody (1:1000 in blocking buffer) (abcam, usa) for 1 hour. then, membranes were washed with tbs tween-20 blocking buffer (3 times) and the proteins were visualized with diaminobenzidine (dab) kit (sigma, usa). ten m sections from body of corpus callosum were incubated with 10% goat serum (sigma, usa) for 45 min, and then were incubated overnight with rabbit anti - olig2 polyclonal antibody (1:2000 ; millipore, ca, usa). the next day, sections were incubated with biotinylated secondary antibody (sigma, usa). this was followed by 1 h incubation at rt with avidin - biotin - peroxidise complex. for visualization, the dab was used. finally, the sections were dehydrated in a graded series of ethanol, cleared in xylene, and coverslipped with entellan (merck, germany) and examined with a nikon eclipse 55i (nikon, germany) microscope. quantification of cell numbers was performed by manual counting the number of positive cells using image j software. the complete corpus callosum was microdissected from pbs - perfused mice 14 days after progesterone pellet implantation. the tissue was placed into a petri dish containing 2 ml of digestion buffer, 1 mg / ml of collagenase d (roche, germany), 1 mg / ml of neutral protease (worthington, uk), dnase i (qiagen, uk), and diced into small pieces with a razor blade before incubation at 37c for 30 min. following the incubation, pbs was added to stop the enzymatic digestion and cells washed through a 70 m filter with facs buffer and centrifuged at 2,000 rpm for 5 min at 4c. isolated cells were incubated on ice for 5 min with anti - olig2 antibody (1:100, millipore, ca, usa) or anti - o4 antibody (1:50, millipore, ca, usa). for the secondary antibody, goat anti - mouse antibody igg fluorescein isothiocyanate (fitc) (1:100, invitrogen, ca statistical analysis was performed by one - way analysis of variance (anova) followed by tukey s post - hoc test. luxol fast blue histologic stain was used to assess the extent of remyelination in corpus callosum after implantation of progesterone pellet. two weeks after progesterone implantation, an obvious remyelination was seen (figure 1c), while less remyelination was seen in the placebo group (figure 1b). the photomicrographs were taken from sagital sections of body of corpus callosum two weeks after treatment. corpus callosum (cc) of a healthy control group (a), placebo group (b), and progesterone implantation group (c). quantification of myelin content in the body of corpus callosum (d) shows that in progesterone pellet implanted mice there was a further significant increase in the remyelination score compared with saline pellet implanted (placebo) mice. quantification of myelin content in the body of corpus callosum (figure 1d) shows that in progesterone pellet implanted mice there was a further significant increase in the remyelination score (1.650.1 ; p0.05), compared with saline pellet implanted (placebo) mice (0.250.05 ; p0.05). densitometric measurements of immunoblots demonstrated that mbp and plp contents were significantly lower in all treated groups compared with the healthy control group (figure 2a). in the progesterone pellet implantation group, after two weeks of hormone administration, there were significant increase in mbp (0.720.1 ; p0.05) contents compared with the placebo group (0.240.1 ; p0.05) but less than the healthy control group (0.950.06 ; p0.05) (figure 2b). the plp contents were significantly increased in progesterone receiving group (0.80.09 ; p0.05) compared with the placebo group (0.10.03 ; p0.05) but less than the healthy control group (0.990.08 ; p0.05). protein expression of myelin basic protein (mbp) and proteolipid protein (plp) were measured by western blot and actin was used as housekeeping control. a : the representative western blot pictures of mbp and plp protein in corpus callosum of the healthy control, placebo and progesterone administration mice. b : bar chart showing the relative quantities of mbp and plp measured densitometrically from the western blots in different groups. the value represented here as meansem of 3 mice in each group (p<0.05). as shown in figure 3, progesterone treatment during 2 weeks after cuprizone - induced demyelination, significantly increased the number of olig2+cells, an oligodendroglial progenitor cell marker, in body of corpus callosum of mice (figures 3a, b, and c). the number of olig2+cells displayed a tendency to be higher in the placebo group (13115 ; p0.05) when compared with the control health group (11614 ; p0.05) without reaching statistical significant values (figure 3d). in contrast, the number of olig2+cells was significantly higher in progesterone receiving group (32717 ; p0.05) when compared with the placebo and control healthy groups (figure 3d). in addition, olig2 and o4 expression was quantified by flow cytometry (figure 4a). according to figure 4c, the mean percentages of expression of olig2 decreased in the placebo group (4.271.2 ; p0.05) when compared with the control health group (5.540.64 ; p0.05) without reaching statistical significant values. such decrease was significantly reversed in animals that received progesterone (40.061.4 ; p0.05). effects of progesterone treatment on the number of olig2 positive cells (oligodendroglial progenitor cell) in the corpus callosum of cuprizone induced demyelination mice after two weeks of injury. light photomicrographs of immunohistochemistry with anti - olig2 antibody (arrows) in healthy control group (a), placebo group (b), and progesterone implantation group (c). the cell counting of olig2 positive cells in body of the corpus callosum (d) shows that in progesterone pellet implanted mice there was a further significant increase in the number of olig2 + cells, compared with the healthy control and saline pellet implanted (placebo) mice. scale bars : 100. oligodendrocyte and oligodendroglial progenitor cell numbers are markedly increased in the corpus callosum of progesterone receiving mice. mononuclear cells were isolated from corpus callosum and the frequencies of olig2 positive cells (oligodendroglial progenitor cell) and o4 positive cells (oligodendrocyte) determined using flow cytometry two weeks after treatment. data shown in the figure a are representative of the three performed independent experiments. the total number of o4 positive cells, shown in b, and the total number of olig2 positive cells, shown in c of the healthy control, placebo, and progesterone - receiving mice are shown. the data are presented as the average of the cell counts from three mice per group sem in b and c (p<0.05). likewise, the mean percentages of expression of o4, an oligodendrocyte marker, was significantly increased in the progesterone group (24.40.6 ; p0.05) compared with the placebo group (1.520.2 ; p0.05), but less than the healthy control group (54.20.8;p0.05) (figure 4b). luxol fast blue histologic stain was used to assess the extent of remyelination in corpus callosum after implantation of progesterone pellet. two weeks after progesterone implantation, an obvious remyelination was seen (figure 1c), while less remyelination was seen in the placebo group (figure 1b). the photomicrographs were taken from sagital sections of body of corpus callosum two weeks after treatment. corpus callosum (cc) of a healthy control group (a), placebo group (b), and progesterone implantation group (c). quantification of myelin content in the body of corpus callosum (d) shows that in progesterone pellet implanted mice there was a further significant increase in the remyelination score compared with saline pellet implanted (placebo) mice. quantification of myelin content in the body of corpus callosum (figure 1d) shows that in progesterone pellet implanted mice there was a further significant increase in the remyelination score (1.650.1 ; p0.05), compared with saline pellet implanted (placebo) mice (0.250.05 ; p0.05). densitometric measurements of immunoblots demonstrated that mbp and plp contents were significantly lower in all treated groups compared with the healthy control group (figure 2a). in the progesterone pellet implantation group, after two weeks of hormone administration, there were significant increase in mbp (0.720.1 ; p0.05) contents compared with the placebo group (0.240.1 ; p0.05) but less than the healthy control group (0.950.06 ; p0.05) (figure 2b). the plp contents were significantly increased in progesterone receiving group (0.80.09 ; p0.05) compared with the placebo group (0.10.03 ; p0.05) but less than the healthy control group (0.990.08 ; p0.05). protein expression of myelin basic protein (mbp) and proteolipid protein (plp) were measured by western blot and actin was used as housekeeping control. a : the representative western blot pictures of mbp and plp protein in corpus callosum of the healthy control, placebo and progesterone administration mice. b : bar chart showing the relative quantities of mbp and plp measured densitometrically from the western blots in different groups. the value represented here as meansem of 3 mice in each group (p<0.05). as shown in figure 3, progesterone treatment during 2 weeks after cuprizone - induced demyelination, significantly increased the number of olig2+cells, an oligodendroglial progenitor cell marker, in body of corpus callosum of mice (figures 3a, b, and c). the number of olig2+cells displayed a tendency to be higher in the placebo group (13115 ; p0.05) when compared with the control health group (11614 ; p0.05) without reaching statistical significant values (figure 3d). in contrast, the number of olig2+cells was significantly higher in progesterone receiving group (32717 ; p0.05) when compared with the placebo and control healthy groups (figure 3d). in addition, olig2 and o4 expression was quantified by flow cytometry (figure 4a). according to figure 4c, the mean percentages of expression of olig2 decreased in the placebo group (4.271.2 ; p0.05) when compared with the control health group (5.540.64 ; p0.05) without reaching statistical significant values. such decrease was significantly reversed in animals that received progesterone (40.061.4 ; p0.05). effects of progesterone treatment on the number of olig2 positive cells (oligodendroglial progenitor cell) in the corpus callosum of cuprizone induced demyelination mice after two weeks of injury. light photomicrographs of immunohistochemistry with anti - olig2 antibody (arrows) in healthy control group (a), placebo group (b), and progesterone implantation group (c). the cell counting of olig2 positive cells in body of the corpus callosum (d) shows that in progesterone pellet implanted mice there was a further significant increase in the number of olig2 + cells, compared with the healthy control and saline pellet implanted (placebo) mice. the value represented here as meansem of 3 mice in each group (p<0.05). scale bars : 100. oligodendrocyte and oligodendroglial progenitor cell numbers are markedly increased in the corpus callosum of progesterone receiving mice. mononuclear cells were isolated from corpus callosum and the frequencies of olig2 positive cells (oligodendroglial progenitor cell) and o4 positive cells (oligodendrocyte) determined using flow cytometry two weeks after treatment. data shown in the figure a are representative of the three performed independent experiments. the total number of o4 positive cells, shown in b, and the total number of olig2 positive cells, shown in c of the healthy control, placebo, and progesterone - receiving mice are shown. the data are presented as the average of the cell counts from three mice per group sem in b and c (p<0.05). likewise, the mean percentages of expression of o4, an oligodendrocyte marker, was significantly increased in the progesterone group (24.40.6 ; p0.05) compared with the placebo group (1.520.2 ; p0.05), but less than the healthy control group (54.20.8;p0.05) (figure 4b). patients suffering from ms experience a significant decline in the rate of relapse during the third trimester of pregnancy and a significant increase during the first 3 months post - partum. thus, relapse decrease when levels of many hormones are elevated, and in particular those of progesterone, and increase when hormone levels decline to pre - pregnancy levels following delivery. our study attempted to investigate the role of progesterone after experimentally induced demyelination in the male corpus callosum using cuprizone as toxic agent. to induce the demyelination, cuprizone feeding for several weeks causes massive demyelination of distinct white and grey matter brain region, including the corpus callosum. continued ingestion of the copper chelator cuprizone results in a white matter pathology that is similar to pattern iii ms lesions. our results show that progesterone increase remyelination in corpus callosum after a demyelinating insult with cuprizone. a numerous experimental studies, have demonstrated the multiple actions of progesterone and its metabolites throughout the nervous system and their considerable influence on the functioning of the glial cells. the influence of progesterone administration on the course of demyelination in experimental models for ms is well documented. for instance, some authors reported that administration of progesterone could attenuate demyelination in the lysolecithin - injured spinal cord. others have shown the application of progesterone possesses the capacity of inhibiting mature oligodendrocyte damage and reduced demyelination in cuprizone provoked demyelination of corpus callosum. a particular asset of progesterone is that it not only could attenuate demyelination, but also promotes myelin formation either during development or during the remyelination of axons in the adult. demonstrated that short prog treatment increased the number of opc, increased the expression of mbp, and enhanced the expression of the olig2 and nkx2.2 transcription factors involved in specification and differentiation of the oligodendrocyte lineage. in another study, mice were induced with eae and treated with progesterone had a decreased clinical severity and enhanced expression of transcription factors essential for oligodendrocyte and myelin protein transcripts. showed that progesterone strongly increased the reappearance of cells of the oligodendroglial lineage in the demyelinated area. although the cellular mechanisms responsible for the therapeutic effects of progesterone on remyelination remain unclear, however two hypotheses need to be considered. progesterone as a neurosteroid hormone may participate to remyelination by either stimulating the proliferation and maturation of oligodendrocyte progenitor cells into mature oligodendrocytes able to form new myelin or indirectly by modulating autoimmune and inflammatory processes. however, in cuprizone - induced demyelination, an invasion of t or b cells is not observed. therefore, our study clearly shows that progesterone treatment is able to enhance remyelination of injured corpus callosum also independent of a b and t cell initiated autoimmune reaction. in this study, results of immunohistochemistry and flow cytometry analysis show that progesterone treatment after cuprizone induced demyelination significantly increased the number of olig2 + cells (an oligodendroglial progenitor cell marker) and o4 + cells (an oligodendrocyte marker) in corpus callosum of mice. in addition, western blot analysis shows that progesterone increased expression of mbp and plp proteins. the remyelination of corpus callosum involves initially the recruitment of oligodendrocyte progenitors from the surrounding intact white matter into the area of demyelination and their subsequent differentiation into remyelinating oligodendrocytes. the improvements of the remyelination rate that we find in progesterone treated animals may result from an enhancement in either or both of the recruitment and differentiation phases of remyelination. however, the signaling mechanisms involved in the myelinating actions of progesterone are not well defined. this is an important question since progesterone acts on multiple targets within the nervous system. although the molecular mechanisms employed by progesterone to improve remyelination after cuprizone demyelination induction were beyond the scope of our current study, some possibilities are worth mentioning. it has been shown that oligodendrocytes generation is a multiple - step process in which oligodendroglial progenitor cells proliferate, migrate, and differentiate into mature oligodendrocytes. in a chemically induced multiple sclerosis model, remyelination is due to differentiation and maturation of adult oligodendroglial progenitor cells instead of pre - existing oligodendrocytes that suffer apoptotic cell death. progesterone was shown to stimulate the proliferation and maturation of oligodendrocyte progenitor cells in these slices. recently, it was reported that progesterone increased the number of new oligodendrocytes and this results supports the idea that the progesterone imposes a milieu favoring differentiation of proliferating progenitors into mature forms. the results obtained in the present study indicate that progesterone therapy enhanced remyelination of demyelinated corpus callosum axons in cuprizone model of ms. | background : progesterone as a sex steroid hormone is thought to affect and prevent demyelination, but its role in promoting myelin repair is far less investigated. in this study, remyelinating potential of progesterone in corpus callosum was evaluated on an experimental model of ms.methods:in this experimental study, adult male c57bl/6 mice were fed with 0.2% (w / w) cuprizone in ground breeder chow ad libitum for 6 weeks. at day zero, after cuprizone removal, mice were divided randomly into two groups : (a) placebo group, which received saline pellet implant, (b) progesterone group, which received progesterone pellet implant. some mice of the same age were fed with their normal diet to serve as the healthy control group. two weeks after progesterone administration, myelin content was assessed by luxol - fast blue staining. the myelin basic protein (mbp) and proteolipid protein (plp) expression were assessed using western blot analysis and the changes in the number of oligodendrocytes and oligodendroglial progenitor cells were assessed by immunohistochemistry (ihc) and flow cytometry.results:luxol-fast blue staining revealed enhanced remyelination in the progesterone group when compared with the placebo group. densitometry measurements of immunoblots demonstrated that mbp and plp proteins contents were significantly increased in the progesterone group compared with the placebo group. flow cytometry and ihc analysis showed increases in olig2 and o4 cells in the progesterone group compared with the placebo group.conclusion:overall, our results indicate that progesterone treatment can stimulate myelin production and that it may provide a feasible and practical way for remyelination in diseases such as multiple sclerosis. |
biosurfactants are amphiphilic molecules which have the ability to depict a wide variety of surface activity. they comprise both a hydrophobic and a hydrophilic group that aid in its accumulation between fluid phases. because of this property, they allow easy accessibility to nonpolar hydrocarbons so that microorganisms in oil - rich ecological niches can easily degrade them. a number of high molecular weight biosurfactants and bioemulsifiers are produced by both bacteria and fungi. biosurfactants of bacterial origin belong to most classes of compounds including polysaccharides, proteins, lipopolysaccharides, lipoproteins and combinations of many of these structural types. almost all classes of microorganisms (table 1) produce biosurfactants (finnerty and healy.). owing to their xenobiotic nature, synthetic surfactants have the potential disadvantage of persisting in the environment, long after they are applied for a remedial measure. also, some of the synthetic surfactants are comparatively more toxic to human health (dehghan - noudeh.). as biosurfactants are of microbial origin, they have been under the active scrutiny of researchers for more than a decade. biosurfactants have the potential to be considered as a viable alternative to the chemically synthesized surfactants for environmental cleanup. according to habe and omori, the biological treatment of pah - contaminated soil should be an economically viable and efficient process. the biological approach has a lot of advantages including complete degradation of the pollutants, lower treatment cost, greater safety, and lesser soil disturbance. kosaric enlisted an exhaustive list of advantages in favour of biosurfactants : biodegradability, low toxicity, biocompatibility and digestibility, availability of raw materials for production, acceptable production economics, environmental control, specificity, and effectiveness. biodegradation of the hydrocarbons at a specific contaminated site depends on the indigenous soil microbial population, type and concentration of hydrocarbons present, soil characteristics, and availability of nutrient and oxygen. the genera of soil microorganisms that are known to degrade hydrocarbons include pseudomonas, flavobacterium, achromobacter, arthrobacter, micrococcus, and acinetobacter (kosaric). the present work attempts to investigate the use of a novel biosurfactant - producing bacterial strain, isolated from oil - contaminated sites in manipal (karnataka, india) for enhanced oil recovery operations. oil - soaked soil samples were collected from a local automobile workshop in manipal (karnataka, india). the samples were enriched by inoculating 1 g of the soil sample into 50 ml of sterile bushnell haas broth (himedia, mumbai), taken in a 250 ml conical flask at 30c in a shaker incubator (rotek, india), & set at 150 rpm. the medium was also constituted with filter - sterilized 1% (v / v) n - hexadecane as the sole carbon source. serial dilution of the sample was performed after 48 h of incubation and plated onto sterile bushnell haas agar plates. after incubation for 48 h at 30c, morphologically distinct colonies were reisolated by transfer to fresh agar plates thrice to obtain pure cultures. the chosen isolates were further screened for the production of biosurfactants using multiple screening methods. 48-hour - old cultures of the isolates grown in bushnell haas broth were taken to perform the screening tests. all the screening tests were performed in triplicate. in the oil spreading technique developed by morikawa., 30 ml of distilled water was taken in a petri dish to which 1 ml of coconut / sesame oil was added to the centre. 20 l of the culture supernatant from the broth was added on top of the oil layer. the petri dishes were closely observed for a zone of displacement in the oil, and the diameter of displacement was measured. blood agar hemolysis test was performed to check the hemolytic activity of the microbial isolates, as described by mulligan.. the isolates were streaked onto sheep blood agar plates (himedia, mumbai) in a sterile environment. the bacterial colonies were visually examined for the presence of clear zones around the streaks. the extent of clearing was classified into 4 categories, as described by rodrigues. : no hemolysis ; incomplete to partial hemolysis with a clearing 1 cm but 3 cm. sterile glass slides were coated with commercially available engine oil (sg sae 20w-40 grade) and fully covered to allow equilibration for 24 hours at room temperature. 0.01 ml of the culture supernatant was dropped on the surface of the equilibrated glass slides. the shape of the drops was observed for activity of the culture supernatant on the oil after an hour. depending on the concentration of the crude biosurfactant, the drop collapses to varying degrees. the extent of drop collapse was assessed as follows : no collapse ; partial collapse if diameter after collapse 1 cm and 1.5 cm. ctab agar plate test, developed by siegmund and wagner, was performed for detection of anionic surfactants. mineral salts agar (himedia, mumbai) was supplemented with 2% (w / v) glucose as carbon source, 0.5 mg / ml cetyltrimethylammonium bromide (himedia, mumbai), and 0.2 mg / ml methylene blue (himedia, mumbai) as performed by satpute.. a well was punctured into the plate using a sterile cork borer and filled with 50 l of the culture supernatant. the plates were incubated for 4872 hours at 30c and observed for the appearance of bluish / greenish halos around the wells to imply biosurfactant production. in the tilting glass slide test, developed by persson and molin, a single colony is picked up from the bushnell haas agar plate and transferred on the surface of a sterile glass slide near one of the edges. the slide is gradually tilted to the other side and was examined for flow of a water droplet over its surface. the surface tension of the 48-hour - old culture broth was measured using a digital surface tensiometer (described in section 2.7.2). the microbial isolate rt10 was identified based on its morphological and biochemical characteristics as per bergey 's manual of determinative bacteriology. gene sequencing (16s rrna method) was performed at agharkar research institute (pune, india) to identify the bacterial strain. genomic dna was isolated from the culture by using a commercial kit (genelute bacterial genomic dna kit, sigma, usa). a polymerase chain reaction was carried out using the universal primers for 1.5 kb fragment amplification for eubacteria. the 20 l master mix for the pcr was composed of 3 l of template dna (10 ng), 2 l each of 200 m dntp mix and 10x pcr buffer, 0.4 l each of forward and reverse primers, and 0.2 l of taq dna polymerase (bangalore genei, bangalore) in 12 l of double distilled water. the pcr was performed using gradient mastercycler system (eppendorf, germany) with the following cycle program : 94c for 5 min ; 30 cycles of 94c, 60c, and 72c for 1 min each, and final extension at 72c for 10 min followed by a final sample hold at 4c. the pcr product was precipitated using 8.5% polyethylene glycol6000, washed thrice using 70% ethanol, and dissolved in 10 mm tris - hcl (ph 8). the abi prism bigdye terminator cycle sequencing ready reaction kit (applied biosystems, usa) was used for the sequencing reaction. the sequencing output was analyzed using the dna sequence analyzer software (applied biosystems). the sequence was compared with national center for biotechnology information (ncbi) genbank entries by using the blast algorithm. samples of the identified microbial isolate were obtained both from bushnell haas broth and nutrient broth (himedia, mumbai). the extracted plasmid dna samples and the control were subjected to agarose gel electrophoresis (0.7% agarose) as described by sambrouk and russell. the bacterial isolate rt10 was inoculated in 25 ml sterile nutrient broth in a shaking incubator set at 30c and 150 rpm until od600 nm reaches 0.8 - 0.9. this seed culture was used in the production medium at 2% (v / v). as formulated by makkar and cameotra, biosurfactant production was carried out in 250 ml conical flasks containing 50 ml of a mineral salt medium with the following composition : kno3 (0.3%), na2hpo4 (0.22%), kh2po4 (0.014%), nacl (0.001%), mgso4 (0.06%), cacl2 (0.004%), feso4 (0.002%), and 0.1 ml of trace element solution containing (g / l) : 2.32 g znso47h2o, 1.78 g mnso44h2o, 0.56 g h3bo3, 1.0 g cuso45h2o, 0.39 g na2moo42h2o, 0.42 g cocl26h2o, 1.0 g edta, 0.004 g nicl26h2o, and 0.66 g ki (all chemicals were of analytical grade from merck, usa). crude oil (from a local refinery) was used as the sole source of carbon at 2% (v / v) concentration. the temperature of the medium was maintained at 30c with shaking at 150 rpm. culture medium samples were drawn for estimation of biomass, biosurfactant production, and surface tension, once every 24 hours for five days. bacterial cell growth was monitored by measuring the dry cell weight, as described by cooper and goldenberg. the culture broth was centrifuged in a refrigerated centrifuge (plastocraft model superspin r - v / fm, mumbai) at 10000 rpm for 20 min at 4c to obtain a cell - free supernatant. the ph of the supernatant was adjusted to 2 using 6n hcl and was subjected to acid precipitation by placing it at 4c overnight. the off - white precipitate was separated by centrifugation at 10000 rpm for 30 min at 4c. the precipitate was extracted thrice with a 2 : 1 chloroform - ethanol mixture. the organic phase was removed, and the biosurfactant was concentrated using a rotary evaporator (superfit model supervac, mumbai) at 40c. the solvents were evaporated leaving behind relatively pure biosurfactant as a viscous light brown matter. at periodic time intervals, 1 ml samples of culture broth were collected in a sterile manner and centrifuged at 10000 rpm for 20 min. the paste was dried by heating in a hot air oven set at 50c70c until constant weight was attained, without allowing the cells to be charred. the surface tension property was studied by taking a sample of the culture broth and centrifuging at 10000 rpm for 20 min. the cell pellet was discarded, and the surface tension of the supernatant was measured by the wilhelmy plate method using a sigma model 702 digital surface tensiometer (ksv instruments ltd., helsinki, finland). initially, the plate and glassware were cleaned with chromic acid, milli - q water, and acetone. all the measurements were taken in triplicate. the ability of the biosurfactant to emulsify hydrocarbons was determined by the addition of 2 ml sample of the culture supernatant and 2 ml of a hydrocarbon (hexadecane), taken in a glass test tube. the emulsification activity was checked after being allowed to settle for 24 h, and the emulsification index (e24) was calculated by measuring the emulsion layer, expressed as a percentage of the total height of the mixture in the tube, as described by cooper and goldenberg. the emulsification power of a mixture of equal volumes of 1 mg / ml sds and the hydrocarbon was used as the control. the biosurfactant sample was spotted on precoated silica gel 60 f254 plate (merck, usa) and subjected to thin layer chromatography (tlc), as described by das.. the plate was developed with a solvent system consisting of chloroform, methanol, and water (65 : 25 : 4). further, the plate was sprayed with 0.2% ninhydrin (in absolute alcohol) and heated to 110c. to understand the overall chemical nature of the extracted biosurfactant, fourier transform infrared spectroscopy (ftir) was employed. the technique helps to explore the functional groups and the chemical bonds present in the crude extract. samples were prepared by homogeneous dispersal of 1 mg of the biosurfactant sample in pellets of potassium bromide (merck, usa). ir spectra were collected over the range of 4504500 cm with a resolution of 4 cm. the spectral data were the average of 50 scans over the entire range covered by the instrument. the stability studies were carried out with respect to the effect of temperature, ph, and salinity on surface tension and emulsification capacity of the biosurfactant. the analysis was done using the 24-hour cell - free culture broth obtained by centrifuging the culture sample at 10000 rpm for 15 minutes., 10 ml of the cell - free broth was incubated at temperatures ranging from 4c to 121c for 30 minutes. the effect of ph was determined by estimating the variation of surface activity by adjusting the ph of 10 ml of cell - free broth from 2 to 12 with 6n hcl or 6n naoh solutions. the effect of salinity was checked by varying the concentration of sodium chloride (0% to 20% w / v), added to 10 ml samples of cell - free broth. in all the three studies, the samples were allowed to stand at room temperature for 6 hours after the respective treatments, before making the measurements of surface tension and emulsification index. microbial enhanced oil recovery (meor) is a unique residual oil extraction technology making use of microorganisms. the suitability of the biosurfactant for meor was investigated by employing a jacketed glass column as described by abu - ruwaida.. the performance of the biosurfactant in terms of oil recovery was also compared against sodium dodecyl sulphate (sds) (merck, usa), an amphiphilic surfactant and a common ingredient of many commercial detergents. 75 mg of sand was pretreated by washing with 1n hcl in a conical flask, rotated at 150 rpm for 1 hour. it was dried completely in a hot air oven (rotek, india) set at 100c for 12 hours. a glass column (45 cm 2 cm i.d.) the column was saturated with 50 ml of commercially available engine oil (sl 20w-40 ; jaso m 345 grade). an aqueous solution of 25 mg biosurfactant in 50 ml distilled water was applied to the column. the temperature of the column was maintained at 30c by passing water through the jacket using a peristaltic pump coupled to a water bath maintained at 30c. the leachate from the column was collected over a 36-hour period, and the volume of engine oil released from the column was measured. the impact of temperature on biosurfactant - mediated oil recovery was observed by conducting similar runs at 50c and 70c. in a separate set of studies, an aqueous solution of 25 mg sds in 50 ml sterile water was poured onto the column and the oil recovery was determined at 30c, 50c, and 70c. a total of 11 different bacterial specimens were isolated from the oil contaminated soil samples. the isolates were chosen based on their distinct colony morphology, obtained by serial dilution and streak plating techniques. the eleven isolates were then subjected to screening for biosurfactant production by multiple methods like oil spreading technique, blood agar haemolysis, drop collapse test, ctab agar plate test, and tilting glass slide test (table 2). in the oil spreading technique, morikawa. showed that the extent of oil displacement is directly proportional to the concentration of the biosurfactant produced. of the eleven isolates, four samples significantly displaced the oil layer and started to spread in the water, showing a zone of displacement. in the blood agar technique, mulligan. however, it is not necessary that all biosurfactants have a hemolytic activity (carrillo.). accordingly, in the present study, three of the isolates displayed excellent hemolytic activity. described the drop collapse test according to which the degree of collapse of the culture supernatant describes the surfactant concentration. of the eleven isolates, three strains showed near - complete collapse, while for two other samples the drops turned absolutely flat. in the ctab test designed by siegmund and wagner, two isolates showed greenish halos around the colonies on ctab methylene blue agar medium. the tilted glass slide test, developed by persson and molin, was positive for four isolates. but in the case of rt10, the rate of drop collapse was very rapid and exhibited the lowest surface tension of 29.8 mn / m. it satisfied four of the six tests, and hence, this isolate was picked up as a potential candidate for further studies. the current report, henceforth, discusses the potential use of the strain rt10 for enhanced oil recovery. the results of screening procedures consistently showed the biosurfactant - producing property of the isolate. the results of almost all the tests led to the selection of the bacterial isolate, rt10. the reduction of surface tension was the greatest in case of rt10. based on morphological and biochemical analysis, in accordance with bergey 's manual of determinative bacteriology, the best isolate belonged to the genus bacillus (tables 3 and 4). the 16s rrna analysis revealed that the isolate rt10 showed 99.8% similarity to bacillus siamensis. the neighbor - joining tree based on the 16s rrna sequence for the strain has been shown in figure 1. the 16s rrna sequence alignment shows that the strain rt10 was closely related to the species in genus bacillus. the results of the plasmid extraction process did not reveal the presence of any plasmid in the isolate, irrespective of the media in which the cells were cultured. the study infers that the ability of the isolate to produce biosurfactant was not conferred upon due to the presence of any plasmid. the property should be the result of a chromosomally - mediated mechanism of the bacterium. since the isolate was enriched from an oil - contaminated site, preliminary batch fermentation studies were performed in mineral salt medium, makkar and cameotra, supplemented with 2% (v / v) crude oil as the sole carbon substrate. 24-hour cultures were periodically sampled out to monitor the biomass growth, biosurfactant production, and surface tension. as shown in figure 2, at 96 h of fermentation maximum biosurfactant yield of 0.64 g / l, maximum biomass yield of 3.2 g / l, and the lowest surface tension of 36.1 mn / m were obtained. the emulsification index (e24) of the culture supernatant against hexadecane reached a maximum of 70% at 72 h. when the plate was sprayed with 0.2% ninhydrin, the biosurfactant component was observed as a single spot on the tlc plate. the observation implied the presence of amino acids in the sample. as a result of c h stretching vibrations and n h stretching vibrations, a broad absorbance peak (centred around 3433 cm) with wave numbers ranging from 3600 cm to 3100 cm was observed (figure 3). sharp absorbance peaks are observed at 1463 cm, 1379 cm, 2955 cm, and 2854 cm and are indicative of aliphatic chains (ch3 and ch2). a strong band was also observed at 1741 cm, 1726 cm, and 1713 cm. the presence of c = o bonds causing c = o stretching vibrations leads to absorbance peaks in these regions. the stability of the biosurfactant was checked by subjecting the fermentation broth to conditions of high stress that included temperature, ph, and salinity. when the temperature was varied from 0c to 121c, both surface tension and emulsification index (e24) showed little variation and remained nearly constant at around 39 - 40 mn / m (figure 4(a)). with respect to ph variation from 2 to 12, the values of surface tension were centred around 39 mn / m without large deviations (figure 4(b)). the emulsification index (e24) dipped to lower values (60%), when the ph was increased. variability in surface tension and emulsification index was not profoundly observed in various studies involving changes in broth ph which is in accordance with the literature [26, 27 ]. surface tension was similar to the effect of ph with largely no changes (figure 4(c)). the literature study in the past also confirms that many biosurfactants have stable surface activity even at high levels of salinity [27, 28 ]. the applicability of the biosurfactant was verified using the sand pack column test, while maintaining the column at 30c, 50c, and 70c. with rise in temperature, the recovery also increased to 55% and 60% at 50c and 70c, respectively. bordoloi and konwar performed the study at room temperature, 70c, and 90c using a sand pack column. they reported oil recovery in the range from 35% to 60% across the temperatures, for various bacillus and pseudomonas strains. in the present study, biosurfactant - producing organisms were enriched and isolated from an oil - contaminated site. the morphological, biochemical, and 16s rrna analysis were performed to identify the organism. this is the first report describing the isolation and use of bacillus siamensis as a biosurfactant producer. during fermentation studies, the isolate was able to produce a lipopeptide biosurfactant, using crude oil as the sole carbon source. the biosurfactant was extracted and partially characterized by using tlc and ftir spectroscopy to confirm its chemical nature. a maximum biosurfactant yield of 0.64 g / l was obtained at 96 h. the biosurfactant had good surface tension reducing capability, reducing the surface tension to 36.1 mn / m. stability studies were performed to investigate the effect of extreme variations in temperature, ph, and salinity levels on the surface tension and emulsification capacities of the biosurfactant. in order to examine the suitability of the biosurfactant in enhanced oil recovery processes, the column studies were performed at higher temperatures, and the percentage of oil recovery was estimated to be 60%. the organism and its product present a huge potential for use in environmental remediation strategies. | biosurfactants are surface - active compounds derived from varied microbial sources including bacteria and fungi. they are secreted extracellularly and have a wide range of exciting properties for bioremediation purposes. they also have vast applications in the food and medicine industry. with an objective of isolating microorganisms for enhanced oil recovery (eor) operations, the study involved screening of organisms from an oil - contaminated site. morphological, biochemical, and 16s rrna analysis of the most promising candidate revealed it to be bacillus siamensis, which has been associated with biosurfactant production, for the first time. initial fermentation studies using mineral salt medium supplemented with crude oil resulted in a maximum biosurfactant yield of 0.64 g / l and reduction of surface tension to 36.1 mn / m at 96 h. characterization studies were done using thin layer chromatography and fourier transform infrared spectroscopy. ftir spectra indicated the presence of carbonyl groups, alkyl bonds, and c h and n h stretching vibrations, typical of peptides. the extracted biosurfactant was stable at extreme temperatures, ph, and salinity. its applicability to eor was further verified by conducting sand pack column studies that yielded up to 60% oil recovery. |
the first patient was a 31-year - old caucasian woman admitted to our facility after sustaining 65% tbsa thermal injuries in a residential fire. the patient received lactated ringers during initial resuscitation titrated to a combined endpoint of urine output and tissue perfusion according to clinical judgment supported by a computer - based clinical decision support system. given circumferential burns and decreased pulses, escharotomies were performed on her right upper extremity and bilateral lower extremities. albumin was started at 8 hours post - burn at 0.4 ml / kg/%tbsa/24 hours when she was transiently hypotensive (mean arterial pressure [map ] 4050 for 30 minutes). mg / kg / hr was initiated 11 hours post - burn as a rescue therapy to reduce oxidative stress and overall fluid requirements. she received a total of 101 g of ascorbic acid in 18 hours (table 1). before it could be initiated, she became progressively hypotensive and developed heart block leading to pulseless electrical activity. despite cardiopulmonary resuscitative efforts, the patient died on hospital day 2. at autopsy, there was mild cerebral edema, and birefringent calcium oxalate crystals were identified in her intratubular spaces in both kidneys. patient demographics and vitamin c dosages the second patient was a 20-year - old man with 67% tbsa thermal injuries sustained from a reported industrial accident at a steel plant. on arrival, he was awake with a glasgow coma scale of 15. vitamin c infusion at 66 mg / kg / hr was initiated at 8 hours post - burn to help reduce oxidative stress and total resuscitative volume. in addition, he received an additional 200 mg of ascorbic acid in his total parenteral nutrition. his hospital course was complicated by bilateral lower extremity and right upper extremity escharotomies for circumferential burns. he then developed primary metabolic acidosis, refractory shock, and aki requiring continuous venovenous hemofiltration. he ultimately required a left above - the - knee amputation for progressively necrotic tissue. with worsening lactic acidosis and fever despite broad spectrum antibiotics (started 24 hours post - burn), an exploratory laparotomy was performed to identify necrotic bowel, and none was identified. on hospital day 3, his pupils were fixed and dilated with brain imaging showing cerebral edema and tonsillar herniation. autopsy showed evidence of early cerebellar herniation with ischemic necrosis of the brainstem, cerebellum, and upper cervical spinal cord. calcium oxalate crystals were identified in the intratubular space in both kidneys (figure 1). high power preclinical studies have demonstrated the role of free radicals in the development of edema and increased vascular permeability after thermal injury. other mediators of vascular permeability in burn patients include histamine, prostaglandins, catecholamines, and thromboxane. histamine released from mast cells in injured tissue results in upregulation of xanthine oxidase activity and free radical formation. local antioxidant activity is altered in the injured tissue and damaged neutrophils contribute to the formation of more free radicals. for this reason, antioxidants, such as ascorbic acid, were investigated to decrease the amount of resuscitative volumes and secondary injury caused by free radicals. ascorbic acid was shown to have free radical scavenging effects, an antihistamine effect, and helped to regulate collagen denaturation. more recent studies have shown high - dose vitamin c infusions to reduce post - burn lipid peroxidation, vascular permeability, edema, and fluid resuscitative volumes. animal studies were performed to analyze the effect of high - dose vitamin c on resuscitative volume and edema formation. the water content of the burned skin in the vitamin c group was markedly decreased, suggesting reduced post - burn capillary permeability. another animal study was performed to test the hypothesis that there is evidence of increased negative interstitial hydrostatic pressure in burn injured tissue. this is suggested as a major pathophysiological mechanism necessary to cause such a rapid and massive edema formation after thermal injury. tanaka investigated high - dose vitamin c and its effect on counteracting the increased negativity of interstitial pressure in rats. they showed a marked attenuation of post - burn interstitial pressure by high - dose vitamin c with moderate decrease in the total body weight. in sheep, there was a significant decrease in the resuscitative volume in those sustaining a 40% tbsa after infusion of high - dose vitamin c. even in delayed initiation of high - dose vitamin c (2 and 6 hours post - burn), there was still a decrease in the fluid volume required in thermally injury guinea pigs. despite the delayed initiation of high - dose vitamin c and decrease in resuscitation volume, they found that the 24-hour fluid requirement to be reduced to 32.5% of the parkland formula. a randomized, prospective study was performed utilizing a continuous ascorbic acid (66 mg / kg / hr) infusion on burn patients with 30% tbsa for 24 hours. they found a 45% decrease in the volume required for resuscitation in the high - dose vitamin c treatment group (5.5 vs 2.1 ml / kg for control p < 0.01). there was a 3-fold weight gain in the control group compared with the ascorbic acid group. ultimately, the vitamin c treatment group required fewer days on mechanical ventilation (p < 0.05). of note in the retrospective review by kahn, their institute did not give the high - dose vitamin c if there was a delay in transfer or if there was baseline renal impairment. careful attention was also paid to the patients volume status as ascorbic acid is hyperosmolar and can cause osmotic diuresis. they carefully followed hematocrit, decreased central venous pressure, decreased urine output, or maps. ultimately, they found infusion of high - dose vitamin c safe and efficacious. in the above - mentioned studies, vitamin c supplementation is recommended in burn patients with typical doses from 500 to 1500 mg / d. this is for increased requirements because of stress and need for wound healing. in the average person, one review of the literature reports that ingestion of up to 10 g / d of ascorbic acid orally does not pose a health risk to humans and another stated up to 2 g / d can be tolerated. given the positive effects of high - dose vitamin c infusions in animal and human studies, literature proposes a continuous ascorbic acid infusion at 66 mg / kg / hr for the initial 24 hours of burn resuscitation. vitamin c supplementation, both high and low doses, contributing to renal failure secondary to calcium oxalate deposits has been reported in the literature. in addition to being part of a daily multivitamin, vitamin c is also being used as an alternative medicine in cancer, amyloidosis, and nephropathy. ascorbic acid can induce oxalate nephropathy, worsen renal injury, and delay kidney recovery. oxalate nephropathy, or aki as a result of calcium oxalate accumulation, can occur in both primary and secondary hyperoxaluria. primary hyperoxaluria is because of a group of autosomal recessive inheritance, whereas secondary hyperoxaluria is because of increased oxalate intake, increased absorption of oxalate, or increased production of oxalate. increased production of oxalate is typically because of increased ingestion of oxalate precursors, such as ethylene glycol, and more rarely, vitamin c. two patients identified in the literature were given 45 and 60 g intravenously of ascorbic acid as an alternative therapy in amyloidosis and cancer, respectively. both patients subsequently developed acute renal failure and showed birefringent crystals on polarized light microscopy consistent with calcium oxalate nephropathy. these patients had normal native renal function before the administration of vitamin c. oxalate nephropathy has been described in non - burn patients even at low doses. of note, these patients received anywhere from 500 mg to 6.5 g orally. all of the patients reported taking the dosages of vitamin c for months and had normal renal function prior. review of the literature identified one other case report of vitamin c - associated nephropathy in a burn patient. the patient sustained 40% tbsa and was given vitamin c supplementation of 1 g / d intravenously. renal biopsy showed extensive calcium oxalate deposits within his tubules. only after decreasing the vitamin c dosage to 0.2 g / day and increasing the dialyzate flow the author proposed that the aki was initially caused by volume loss from his burn injury and potentially exacerbated by amikacin. he argues that the vitamin c supplementation either potentiated his renal injury or delayed its resolution. overall, this shows oxalate nephropathy, and calcium oxalate crystals can form in the presence of normal renal function and by taking low doses. the finding of calcium oxalate crystals in renal tubules of burn patients has not been previously reported. others have reported patients developing aki following vitamin c infusion for adjuvant treatment of amyloidosis and cancer and subsequently found calcium oxalate nephropathy on renal biopsy. all of these patients had normal renal function before the start of vitamin c. in these patients, calcium oxalate nephropathy was described as a clinically significant morbidity. second, our patients had significant burns and were at risk for renal failure. in regard to its delayed initiation, both tanaka and sakurai described a beneficial effect of high - dose vitamin c in guinea pigs, even after its infusion was postponed by 6 hours. in addition, in these studies, no lab abnormalities were noted despite their high - dose vitamin c infusion delay. for this reason, we used high - dose vitamin c as a rescue therapy to help attenuate the total volume of resuscitation and possible reduce the systemic inflammatory response. of note, the initial studies in utilizing vitamin c were in accordance with the parkland formula. however, in 1991, matsuda showed that with high - dose vitamin c the total 24-hour resuscitation was able to be reduced from 4 to 1 ml / kg/%tbsa. in the article by sakurai, they resuscitated with lactated ringer s solution according to the parkland formula for only the first 6 hours, and then reduced the volume by 25% of the parkland formula once the high - dose vitamin c was initiated. tanaka initiated resuscitation with the parkland formula for 0.5 to 2 hours post - injury and then reduced the volume to 25% of the parkland formula. despite the delayed initiation of high - dose vitamin c and decrease in resuscitation volume, they found the 24-hour fluid requirement to be reduced to 32.5% of the parkland formula. although our patients did receive a lower volume of resuscitation compared to the parkland formula (table 1), they did not have profound diuresis, and their laboratory findings did not show evidence of hemoconcentration after the initiation of vitamin c (table 1). our resuscitation practices are guided by a computerized clinical decision support system with initial crystalloid rate determined by the isr rule of 10s formula. as expected, the initiation of vitamin c resulted in increased urine output (table 1). lactate was also monitored serially and rose in both patients consistent with worsening tissue perfusion, despite apparent adequacy of volume resuscitation and oxygen delivery evidence by elevated scvo2 (table 1). the first patient had normal renal function (cr 0.8 mg / dl) at the time of high - dose vitamin c infusion. ten hours after high - dose vitamin c was started, her cr increased to 1.40 mg / dl and was associated with an increase in urine output. our second patient had renal impairment before high - dose vitamin c infusion, and it only worsened during his hospital course. it is difficult to ascertain the exact cause for the renal impairment in our patients. both are likely secondary to the extent of burns ; however, given the presence of calcium oxalate crystals in their renal tubules, oxalate nephropathy can not be ruled out. it is expected that the aki in thermally injured patients is related to hypotension and shock leading to acute tubular necrosis. however, at autopsy, microscopic evaluation of acute tubular necrosis was hindered by diffuse postmortem autolysis of renal tubules. therefore, with the presence of calcium oxalate crystals on postmortem analysis, the crystals either caused the aki or were a possible contributor. knowing oxalate nephropathy can occur when high - dose vitamin c is used as an adjunct to burn resuscitation should be a cause for legitimate concern. it is not our intent to put a moratorium on high - dose vitamin c therapy as burn centers with lots of experience using this resuscitative adjunct have reported overall success without any reports of this particular complication. it may be that the benefits of high - dose vitamin c outweigh potential complications. only a well - designed prospective clinical trial could answer this question while keeping in mind to deliberately monitor for this complication given our findings. this report identifies a known complication of vitamin c therapy in 2 patients receiving high - dose vitamin c as a rescue therapy in complicated burn resuscitations. currently, there is much enthusiasm in the burn community about high - dose vitamin c infusions, and there are data supporting its use as an adjunct to complicated burn resuscitations. in other patient populations, high - dose vitamin c becomes a standard of care in burn units, further prospective research is necessary to determine the prevalence of adverse - effects, the optimal dose, timing, and the appropriate patient population for this therapy. | fluid resuscitation is the foundation of management in burn patients and is the topic of considerable research. one adjunct in burn resuscitation is continuous, high - dose vitamin c (ascorbic acid) infusion, which may reduce fluid requirements and thus decrease the risk for over resuscitation. research in preclinical studies and clinical trials has shown continuous infusions of high - dose vitamin c to be beneficial with decrease in resuscitative volumes and limited adverse effects. however, high - dose and low - dose vitamin c supplementation has been shown to cause secondary calcium oxalate nephropathy, worsen acute kidney injury, and delay renal recovery in non - burn patients. to the best of our knowledge, the authors present the first case series in burn patients in whom calcium oxalate nephropathy has been identified after high - dose vitamin c therapy. |
a 39-year - old woman 161 cm tall weighing 51 kg was diagnosed with uterine myoma and an abdominal wall mass and scheduled for laparoscopic myomectomy. she was premedicated with 2 mg of midazolam and a 0.5 mg atropine intramuscular injection 30 minutes before arrival to the operation room. she was monitored intraoperatively by electrocardiography, non - invasive blood pressure, pulse oximetry, end - tidal capnography, sevoflurane concentration, and bispectral index (bis ; a-2000, aspect medical system, norwood, massachusetts, usa). carbon dioxide was injected into the peritoneal cavity via a veress needle until peritoneal pressure reach 12 mmhg. two hours after the operation was initiated, the anesthesiologist notified the surgeon of gaseous distention of the urinary bag which is filled with 150 ml of urine (fig. 1). the surgeon tried to detect a perforation site by sight but failed to detect a bladder perforation. subsequently, the bladder was distended with 250 ml of saline under cystoscopy after which a 0.7 cm hole in the dome of the bladder was discovered. the vital signs of the patient were stable and the urine volume after bladder repair was 100 ml. the foley catheter was removed after there was no more urine leakage as determined by cystoscopy on postoperative day 15. a 45-year - old woman 159 cm tall and weighing 59 kg preoperative blood tests, chest radiography, electrocardiogram, vital signs, and physical examination were normal. premedication, anesthesia induction, maintenance, and monitoring were the same as case 1. after the patient was placed in the lithotomy position, carbon dioxide was injected into the peritoneal cavity via a veress needle until peritoneal pressure reach 12 mmhg. marked gaseous distention of the urinary bag which was filled with 350 ml was noticed 30 minutes after the operation started. laparoscopy revealed a 1.5 cm hole in the dome of the bladder including the mucosa layer. total hysterectomy and bladder repair was performed. the urine volume after bladder repair was 150 ml. the foley catheter was removed after there was no more urine leakage as determined by cystoscopy on postoperative day 13. a 39-year - old woman 161 cm tall weighing 51 kg was diagnosed with uterine myoma and an abdominal wall mass and scheduled for laparoscopic myomectomy. she was premedicated with 2 mg of midazolam and a 0.5 mg atropine intramuscular injection 30 minutes before arrival to the operation room. she was monitored intraoperatively by electrocardiography, non - invasive blood pressure, pulse oximetry, end - tidal capnography, sevoflurane concentration, and bispectral index (bis ; a-2000, aspect medical system, norwood, massachusetts, usa). carbon dioxide was injected into the peritoneal cavity via a veress needle until peritoneal pressure reach 12 mmhg. two hours after the operation was initiated, the anesthesiologist notified the surgeon of gaseous distention of the urinary bag which is filled with 150 ml of urine (fig. 1). the surgeon tried to detect a perforation site by sight but failed to detect a bladder perforation. subsequently, the bladder was distended with 250 ml of saline under cystoscopy after which a 0.7 cm hole in the dome of the bladder was discovered. the vital signs of the patient were stable and the urine volume after bladder repair was 100 ml. the foley catheter was removed after there was no more urine leakage as determined by cystoscopy on postoperative day 15. a 45-year - old woman 159 cm tall and weighing 59 kg was diagnosed with endometrioma and scheduled for laparoscopic - assisted vaginal hysterectomy. preoperative blood tests, chest radiography, electrocardiogram, vital signs, and physical examination were normal. premedication, anesthesia induction, maintenance, and monitoring were the same as case 1. after the patient was placed in the lithotomy position, carbon dioxide was injected into the peritoneal cavity via a veress needle until peritoneal pressure reach 12 mmhg.. marked gaseous distention of the urinary bag which was filled with 350 ml was noticed 30 minutes after the operation started. laparoscopy revealed a 1.5 cm hole in the dome of the bladder including the mucosa layer. total hysterectomy and bladder repair was performed. the urine volume after bladder repair was 150 ml. the foley catheter was removed after there was no more urine leakage as determined by cystoscopy on postoperative day 13. since it was first reported, it has been noted to occur during procedures involving a verres needle, trocars (primary or secondary), and electrocautry regardless of improvements in surgical techniques and safety instructions. predisposing risk factors for complications during laparoscopic procedures include operation history of the patient, pelvic adhesion, endometriosis, obesity, urachal anomaly, and unskilled surgeons. bladder perforation during laparoscopic surgery may be detected by intraperitoneal bleeding, clear liquid in the operation field, hematuria, and gaseous distention of the urinary bag. among these, hematuria and gaseous distention of the urinary bag if hematuria is found intraoperatively or postoperatively, cystoscopy or cystoradiography can be used to confirm bladder perforation. hematuria may not occur in all bladder perforation cases and there are difficulties in diagnosing intraoperatively. sia - kho and kelly reported that they detected a gaseous distention of the urinary bag at the conclusion of laparoscopic adhesiolysis but ignored this, and that the patient complained of abdominal tenderness, nausea, and vomiting 4 hours after operation. they emphasized that gaseous distention of urinary bag is an initial sign of bladder perforation, and anesthesiologists should monitor the urinary bag. subsequent cases have verified that observing the urinary bag is a reliable way to monitor bladder perforation [6 - 9 ]. gaseous distention of the urinary bag due to bladder perforation indicates communication between bladder and intraperitoneal space. because the volume of urinary bag commonly used in korea is 2,000 ml, gaseous distention could reveal within 2,000 ml of total amount including urine and gas. when a urinary bag with air outlet is used, gaseous distention can occur because the diameter of the inlet for urine and gas is larger than that of the outlet for air. however, gaseous distention would not appear in an inelastic urinary bag, so carbon dioxide gas could be measured at the air outlet by mass spectroscope or infrared spectrometer when bladder perforation is suspected. detected 10% carbon dioxide at a urinary bag. in our cases, it is thought that bladder perforation occured through laparoscopic dissection of severe adhesion of uterus with bladder despite no previous abdominal surgery. in case 1, the urinary bag seemed to become distended gradually because the size of the bladder injury was small. in case 2, the urinary bag was distended instantly because the size of the bladder injury was large. therefore, the size of injury is likely to affect the time of gaseous distention presentation. further studies about cause - and - effect relationship between peritoneal pressure, the size of injury, and gaseous distention of the urinary bag are needed. early detection during laparoscopic surgery allows immediate bladder repair via laparoscopy or laparotomy and decreases postoperative morbidity. a small undetected tear may heal if bladder decompression is maintained, but may cause oliguria, peritonitis, and fistula formation. insertion of a foley catheter is associated with a risk of infection ; on the other hand, this decreases the risk of bladder injury because it permits continuous decompression of bladder. it also allows early detection of bladder injury which is not found in the operation field, so insertion of indwelling foley catheter is recommended. in conclusion, continuous monitoring of gaseous distention of the urinary bag should aid in the early detection of bladder perforation in patients with predisposing factors of bladder injury. | bladder perforation during laparoscopy is a recognized, uncommon complication. we present two cases of bladder perforation during laparoscopic gynecologic operations that were detected by gaseous distention of the urinary bag. bladder perforation occurred through laparoscopic division of adhesion. one bladder perforation was repaired laparoscopically, and the other case was repaired by laparotomy during the same general anesthesia. in this report, we present evidence that monitoring a gas - distended urinary bag during a laparoscopic procedure can help detect intraoperative bladder perforation. |
it is responsible for the death of 1 in 10 adults, with 4.9 million deaths occurring worldwide each year. prisoners are also more likely to have comorbid conditions (psychiatric disorders) and substance dependence, and there is less likelihood of smoking cessation among prisoners in the absence of intensive interventions. referring to butler. tobacco smoking is an integral part of prison life and an established part of the culture. prevalence rates of smoking in prison are at least double or even triple compared to the general population. the various treatment approaches for smoking cessation include cognitive behavioral strategies (self - monitoring and coping skills), motivational strategies (techniques to clarify desire for change and reduce ambivalence toward change), and social influence strategies (addressing the social influences that serve to promote or maintain smoking). motivational interviewing (mi) is a directive, patient - centered style of counseling, designed to help people to explore and resolve ambivalence about behavior change. the concept of mi evolved from experience in treating alcohol abuse, and was first described by miller in 1983. the aim of this study was to assess the effectiveness of smoking cessation intervention by mi in male prisoners at central jail, parappana agrahara, bangalore. a randomized controlled study was conducted to assess the effectiveness of smoking cessation intervention among male prisoners at central jail in bangalore city. ethical clearance was obtained from the ethical committee of the oxford dental college, hospital and research centre, bangalore, india. the inspector general officer of the central jail was approached, and the details of the study were explained to him and his approval was obtained to proceed with the study. there were 4600 prisoners in the central jail, of which 1600 were convicted male prisoners, 2700 were undertrial prisoners, and 300 were female prisoners. a self - administered questionnaire was given to the prisoners to assess their smoking behavior by which the prevalence of tobacco smoking was found. prevalence of tobacco use that included both chewable and smoking tobacco was 84.5% and prevalence of smoking among prisoners was 92.60%. inclusion criteria were current adult smokers who smoked any tobacco product either daily or occasionally at the time of the study, convicted male prisoners with at least 1 year left to serve, and prisoners giving informed consent to quit smoking. exclusion criteria were inmates with acute mental illness (current suicidal ideation / actively psychotic) or mental retardation such that they could not provide informed consent and medically compromised inmates (like those with respiratory disorders). smokerlyzer, the micro co monitor, was used to measure alveolar carbon monoxide in ppm concentrations and the percentage carboxyhemoglobin (cohb). before commencing the study, to the 1600 convicted prisoners, a self - administered questionnaire was given to assess their smoking behavior by which the prevalence of tobacco was found. there were 1352 tobacco users in the present study. among these, there were 1252 smokers. based on inclusion criteria, a sample of 600 was chosen for the study by systematic random sampling. among 600 prisoners, 300 were selected for each group (study and control) by simple random sampling. the topics for the intervention included : introduction to tobacco, prevalence of tobacco use, effects of tobacco use on general health and dental health, psychosocial factors influencing tobacco use, healthy diet and behavioral intervention for prevention of tobacco use. follow - up was done for both study and control groups at the end of the 6 month using the same proforma, and fagerstrom test was done by using fagerstrom questionnaire and carbon monoxide grade was estimated by using smokerlyzer. results on continuous measurements are presented on mean (sd) (min.max.) and the results on categorical measurements are presented as number (%). significance was assessed at 5% level of significance. chi - square / fisher 's exact test were used to find the significance of study parameters on a categorical scale between two or more groups. the total study population was 600 : 300 in the study group and 300 in the control group. also, 5% prisoners in the study group and 3% prisoners in the control group were aged between 18 and 20 years, 44% prisoners in the study group and 48.66% prisoners in the control group were between 21 and 30 years, 32.33% prisoners in the study group and 29.33% prisoners in the control group were between 31 and 40 years, 11.66% prisoners in the study group and 11.66% prisoners in the control group were between 41 and 50 years, and 7% prisoners in the study group and 7.33% prisoners in the control group were 5160 years of age. among the tobacco users, 1252 (92.60%) were smokers [table 1 ]. age of onset of tobacco usage showed that it was 1520 years in 45.33% in the study group and 39.66% in the control group, 2130 years in 47.66% in the study group and 53% in the control group, and more than 30 years in 7.0% in the study group and 7.33% in the control group. 48.33% prisoners in the study group and 52% prisoners in the control group started using tobacco due to stress. about 90.66% in the study group gave prison stress as the reason that increased the need for tobacco use. prisoners who were willing to quit tobacco use formed 88.66% in the study group and 78% prisoners in the control group [table 2 ], and 11.33% prisoners in the study group and 22% prisoners in the control group were not willing to quit tobacco. also, 64.28% prisoners in the study group and 70.51% prisoners in the control group wanted to quit tobacco due to health reasons. prevalence and characteristics of tobacco users in the study population distribution of the study population according to tobacco usage before and after intervention in the study group prisoners the intervention of tobacco cessation showed a positive percentage change of + 16% in the study group, who stopped using tobacco completely after intervention [table 3 ]. regarding the effect on physical and psychosocial problems faced by the prisoners, a positive percentage change of + 5.81% was noticed in prisoners having lack of sense of well - being. the other factors like headache (2.93%) and irritability (2.42%) showed a negative percentage change after intervention. the main reasons given by the prisoners who did not want to quit tobacco usage before intervention in the study group were : for enjoyment by 23.52% prisoners, 14.70% were afraid of being unable to abstain, peer pressure by 8.82% prisoners, and other reasons like stress by 47.05% prisoners. after intervention, the reasons given were : for enjoyment by 28.20% prisoners, peer pressure by 12.82% prisoners, and other reasons like stress by 51.28% prisoners. majority (44.66%) of the prisoners had co level between 0 and 6 ppm, 40% prisoners had a level between 7 and 10 ppm, and 15.33% prisoners had a level between 10 and 20 ppm before intervention. the percentage increased to 70% prisoners with co level of 06 ppm, and a decrease of 17.34% was observed in prisoners with co level 710 ppm and 8% in those with co level 1020 ppm after intervention. distribution of study population according to tobacco usage after 6 months in the study group and control group tobacco use is a major risk factor for cancer of the oral cavity, periodontal disease, and tooth loss. based on 12 studies that have estimated oral cancer risk in smokers compared with non - tobacco users, the pooled risk estimate is 3.43 times higher in smokers. the community of prisoners differs from other social groups in terms of psychosocial factors, their level of education, alcohol and substance abuse, attitude toward health, and lifestyle. all these factors account for the higher prevalence of tobacco usage among prisoners, in comparison with the general population. a smoke - free environment may increase the willingness of smokers to consider quitting smoking and also reduces the risk of fires. however, the principal benefit of smokeless environment in prison is the reduction in second - hand smoke. this study is the first of its kind in which mi method was used for smoking cessation. mi is a particular way of helping clients recognize and do something about their current or potential problems. the strategies of mi are more persuasive than coercive, more supportive than argumentative, and the overall goal is to increase the client 's intrinsic motivation so that change arises from within rather than being imposed from others. psychological studies indicate that smokers who successfully quit smoking were more frequently controlled by their intrinsic rather than extrinsic motives (e.g. the will to receive a reward). smoking cessation strategies, using pharmacotherapies (e.g. buprion or nortriptyline), nicotine replacement therapies, and counseling intervention based on cognitive intervention are readily accessible in the community. nicotine replacement gum was not allowed in prison because it could be used to block locks or form the key impressions. using nicotine transdermal patch was not economically feasible. in the present study, prevalence of smoking was 92.60% among the convicted prisoners. according to a study conducted by the national institute of mental health and neuro sciences (nimhans), 67.3% of the prison population (undertrial prisoners and convicted prisoners) reported ever using tobacco in some form in their lives. this was more than double the prevalence of tobacco use in karnataka (29.6%, figure for 2001). a recent study conducted by narkauskaite. in lithuanian prisons revealed a smoking prevalence of 85.3% among the prisoners. it was estimated that around 6488% of the prisoners smoke (tielking, becker, and stver 2003 ; department of health and prison service 2007 ; narkauskaite. prevalence rates of smoking in prison are at least double or even triple compared to the general population. prisons are unique settings with high rates of both smoking prevalence and individuals who have smoking - related health problems. these individuals also are unlikely to access community - based smoking cessation treatment ; therefore ; prison can serve as a unique point of contact for conducting smoking cessation intervention for these individuals. in the present study, 90.66% subjects in the case group and 84.66% in the control group consider prison stress as the factor that increases the need for tobacco usage. similar findings (77%) were reported by sieminska. in 2006. according to the study conducted by nimhans, undertrial prisoners are significantly more likely to have smoked or chewed tobacco, compared to convicted prisoners. the reason could be the stress of judgment in undertrial prisoners. in the present study, negative percentage of change (15.67) was obtained in the study group prisoners who were using 31 cigarettes smoked per day. the physical and psychological problems faced while attempting to quit were reduced in the case group and increased in the control group after intervention. this might be because the case group subjects knew how to tackle with these problems. in the present study, only 19% in the study group stopped smoking during the third month and 16% at the end of the sixth month and only 0.66% stopped smoking in the control group. in a similar study conducted by harcouet, prisoners were given motivational counseling to reduce tobacco use, along with nicotine transdermal patch. in a study done by cropsy. this study was conducted in southeastern united states and showed a prevalence of 14% at the end of 6-month follow - up. at the end of the sixth month follow - up, 21.66% had reduced the use of tobacco and 45.33% ended up in relapse in the study group. a review by hughes and carpenter indicated that smokers who significantly reduce their smoking can maintain these reductions for long periods. the high rate of relapses during smoking cessation, mainly within 6 months, is common both in correctional and general populations. the study conducted by sieminska. showed 67% relapse in prisoners who attempted to quit. prison inmates are able to quit or reduce tobacco consumption while in prison, but any smoking cessation intervention in this setting needs to address prison - specific issues such as boredom, stress, court appearance, and isolation from family and friends. it appears that quitting while incarcerated has no adverse effects on either the physical or mental health of an individual inmate. age and a history of illicit drug use seemed to influence the success of inmates participating in our smoking cessation treatment. first, we enrolled only adult male prisoners and we do not know how these results would apply to female prisoners or to juvenile offenders. as prison is a unique environment of long - term confinement, there is high possibility of relapse findings of the present study suggest that the intervention has suggestive significance on tobacco usage. this study indicates that smoking cessation interventions among male prison populations are feasible, acceptable, and effective, compared with similar interventions delivered in the general population. relatively high rate of relapse in our study indicates that some policies should be adopted to improve smokers information on consequences of tobacco for health and motivational intervention should be added to prisoners. relatively high rate of relapse in our study indicates that some policies should be adopted to improve smokers information on consequences of tobacco for health and motivational intervention should be added to prisoners. | background : tobacco smoking is an integral part of prison life and an established part of the culture. little attention has been paid to prevention of smoking in prison. approximately 7080% of prisoners have been identified as current smokers. aim : to assess the effectiveness of smoking cessation intervention among male prisoners at central jail, bangalore city.aim:to assess the effectiveness of smoking cessation intervention among male prisoners at central jail, bangalore city.materials and methods : a randomized controlled trial was planned among male prisoners in central jail, bangalore city. there were 1600 convicted prisoners. a self - administered questionnaire was given to the prisoners to assess their smoking behavior by which prevalence of tobacco smoking was found. exactly 1352 tobacco users were studied. among them, there were 1252 smokers. based on inclusion criteria and informed consent given by the prisoners, a sample of 600 was chosen for the study by systematic random sampling. among the 600 prisoners, 300 were randomly selected for the study group and 300 for the control group.results:prevalence of tobacco smoking among the prisoners was 92.60%. in the present study, after smoking cessation intervention, 17% showed no change in smoking, 21.66% reduced smoking, 16% stopped smoking, and 45.33% relapsed (p < 0.0001) at the end of 6-month follow - up in the study group.conclusion:tobacco use was high among the prisoners. tobacco reduction is possible in the prison even if the living conditions are not favorable. relatively high rate of relapse in our study indicates that some policies should be adopted to improve smokers information on consequences of tobacco on health and motivational intervention should be added to prisoners. |
complete ureteral triplication, the presence of three individual ureters originating from the kidney and draining into three separate orifices, is a rare congenital anomaly. patients with such anatomy may be predisposed to recurrent infections, renal colic, and incontinence secondary to associated obstruction and reflux. when patients with complete ureteral triplication require surgical management, the surgeon must evaluate the degree of renal compromise attributable to the pathologic portions of the urinary tract, and correctly distinguish the diseased portions of the urinary tract from its healthy counterparts. it is for this reason that the role of perioperative imaging to guide operative treatment can not be overemphasized. indocyanine green (icg), a fluorescent dye that can be visualized under near - infrared fluorescence (nirf), has been utilized intraoperatively during robotic urologic procedures to highlight relevant anatomy and assess perfusion. icg may be visualized using the firefly system, which is integrated into the da vinci si robot (intuitive surgical, sunnyvale, ca, usa). herein, we describe a case in which both intraureteral and intravenous icg was used to facilitate a complex robotic partial nephroureterectomy in a patient with complete ureteral triplication. a 31-year - old female with a congenital right ureteral triplication presented with recurrent right - sided pyelonephritis and persistent right - sided flank pain refractory to medical management. delayed phase computed tomography (ct) scan with intravenous contrast clearly showed that each of the three right - sided ureters was associated with its own collecting system, and emptied separately into the bladder. the uppermost moiety showed sequelae of chronic obstruction : significant parenchymal thinning, delayed contrast excretion, and dilated ureter. the two inferior collecting systems and their associated ureters appeared to be emptying normally (fig. 1). mercaptuacetyltriglycine (mag3) study showed symmetrical renal function with no evidence of obstructive uropathy bilaterally. the uppermost moiety was not well delineated on the mag3 study. the patient elected for robotic right uppermost pole moiety nephroureterectomy. the patient consented to off - labeled use of icg after a complete discussion of its risks and benefits. we used cystoscopy and retrograde pyelography to show that the three separate right ureteral orifices were consistent with weigert - meyer law, and identify the ureter of interest (fig. 2). after preparing the ic - green (akorn inc., lake forest, il, usa) by mixing 25-mg sterile icg in 10-ml distilled water, we used a 6-fr ureteral catheter to inject a 5-ml solution of icg retrograde into the lumen of the diseased ureter. the patient was placed in right - side - up flank position with the table in full flexion. we used our standard robotic port placement strategy for robotic nephroureterectomy that was previously described at our center. after mobilizing the right colon, we turned on the firefly modality to locate the diseased ureter. under nirf, the diseased ureter fluoresced green while the other two healthy ureters did not fluoresce (fig. two weck hem - o - lok clips (teleflex, morrisville, nc, usa) were secured on the ureter at its junction with the bladder prior to transecting the ureter. the diseased ureter was then dissected proximally to its associated renal pelvis. during our dissection of the uppermost pole renal pelvis, we encountered a significant amount of fibrotic tissue, likely attributable to the patient 's history of recurrent pyelonephritis. by toggling the nirf on and off, we were able to clearly distinguish between the green fluorescing renal pelvis and ureter, from the nonfluorescing fibrotic tissue. after identifying the arterial and venous branches of the main renal artery and vein supplying the uppermost pole, we clipped the proximal and distal ends using two weck hem - o - lok clips and transected the vessels., there was a perfusion defect in the uppermost pole moiety of the kidney where we had removed its blood supply ; the remainder of the kidney was well - perfused and fluoresced green (fig. 4). utilizing nirf to delineate our margins, we excised the diseased collecting system and its blood supply en bloc. ureteral triplication is a rare congenital anomaly ; only approximately 100 cases have been described in the literature. smith classified ureteral triplication into four major variants : type 1, complete ureteral triplication (35%)-three ureters originating from the kidney and emptying into three orifices ; type 2, incomplete triplication (21%)-three ureters originating from the kidney, with two of these ureters joining to empty into two orifices ; type 3, trifid ureter (31%)-three separate ureters originating from the kidney, with all three ureters joining to empty into a single orifice ; type 4, double ureter, one bifurcated (9%)-two separate ureters originating from the kidney, with one ureter birfuricating into an " inverse y " to empty into three orifices. our case was a type 1, complete ureteral triplication with all three ureters associated with its own renal collecting system and emptying into three separate bladder orifices. surgical management of patients with ureteral triplication, which may involve simultaneous treatment of the upper and lower urinary tracts, is most often indicated when there is a risk of renal compromise as in cases of veiscoureteral reflux, obstruction, ureteral ectopy, and recurrent infections. given the variations in anatomy and increased predisposition for pathology, prudent use of perioperative imaging to guide surgical management of ureteral triplications is essential. patel. described the role of various preoperative imaging modalities on assessing anatomy and function in a patient with a type 2 ureteral triplication with ectopia. a dimercaptosuccinic acid renal scan showed decreased perfusion and uptake by the right upper pole, suggesting a duplicated anomaly and ectopic ureter. cystoscopy and retrograde pyelography demonstrated that there were two ureters originating from the right kidney that joined intramurally to form a single bladder orifice. intravenous urography and ct scan revealed the ectopic third ureter, which was dilated and associated with the upper pole. flechsig. described using dynamic magnetic resonance nephrography to concomitantly assess anatomic and functional characteristics in a 5-year - old girl with a type 1 left ureteral triplication. initial evaluation with a mag3 renal scan suggested poor renal excretion in the left lower pole, but the authors noted that precise localization of the impaired region was difficult due to limited resolution. however, a dynamic magnetic resonance nephrography allowed for a detailed region - of - interest evaluation that showed 45%, 46%, and 9% function in the upper, middle and lower pole, respectively. furthermore, the authors noted that the correlation between preoperative imaging and intraoperative findings assisted in performing laparoscopic partial nephroureterectomy. an intraoperative imaging modality that assists in correctly identifying the pathologic ureter and associated renal pelvis, and in precisely determining the margin between compromised versus healthy renal parenchyma would facilitate surgical management of patients with ureteral triplication. such an imaging modality would be particularly helpful during robotic surgery, as the surgeon relies solely on visual cues to guide dissection due to the lack of tactile feedback. despite this, a means to intraoperatively image relevant anatomy and evaluate function in real - time during robotic surgical management of ureteral triplications has yet to be described. icg has recently been increasingly utilized in urologic procedures as an intraoperative contrast agent to highlight relevant anatomy. it is particularly helpful for intraoperative use as it has a low toxicity, high signal - to - noise ratio, and penetrates tissue. first detailed the use of intravenous icg during robotic partial nephrectomy to differentiate normal renal parenchyma (bright green fluorescence) from tumors, cysts, and areas of fat necrosis (decreased / no fluorescence). bjurlin. noted that intravenous icg may also be used to assess tissue perfusion during surgical management of the upper urinary tract. poorly perfused fibrotic scar tissue appeared dark while well perfused renal pelvis and ureter appeared green. we have found the use of intraureteral icg to assist in robotic ureteral reconstructions, which was first described at our center, to be particularly helpful in localizing the ureter despite severe periureteral inflammation and in determining margins of ureteral strictures. in our case, we utilized both intraureteral and intravenous icg intraoperatively to highlight relevant anatomy during robotic right uppermost pole nephroureterectomy in a patient with type 1 ureteral triplication. intraureteral icg aided in accurately identifying the pathologic ureter and associated renal pelvis in real - time ; the system of interest fluoresced green while the other two systems did not fluoresce. this aided our ability to focus our dissection to the ureter of interest without risking devascularization to the healthy ureters. intravenous icg aided in assessing perfusion to the right kidney and delineating the margins of defective renal parenchyma in real - time ; the well - perfused middle and lower collecting systems fluoresced bright green, while the upper - most collecting system did not fluoresce as brightly. the use of intraureteral and intravenous icg to highlight relevant anatomy intraoperatively may be of particular benefit in patients with complex urinary tract anatomy, especially in cases necessitating concomitant operative management of upper and lower tracts. | a patient with a complete right ureteral triplication presented with recurrent pyelonephritis and flank pain that was refractory to medical management. evaluation showed that the atrophic upper - most renal moiety had been chronically obstructed and was associated with a dilated ureter. intraureteral and intravenous indocyanine green (icg) were used as real - time contrast agents intraoperatively to facilitate right robotic partial nephroureterectomy of the diseased system. intraureteral icg was used to accurately distinguish the pathologic ureter and associated renal pelvis from its normal counterparts. intravenous icg was used to assess perfusion in the right kidney and delineate the margins of diseased renal parenchyma. |
from december 2005 to september 2006, drug - naive type 2 diabetic patients, aged 1879 years, with a1c 7% and 10%, were recruited at 98 clinical centers in the u.s. inclusion criteria included fasting c - peptide > 1.0 ng / ml, bmi 40 kg / m, and renal status as follows : glomerular filtration rate > 60 ml / min per 1.73 m, serum creatinine 240 mg / dl at weeks 4 and 6, > 220 mg / dl at week 8, or > 200 mg / dl at week 10 were discontinued from the study and were eligible to receive additional antidiabetic agents. the study was conducted pursuant to the declaration of helsinki and was approved by institutional review boards / independent ethics committees at participating sites. the primary objective was to compare mean a1c change from baseline for each dapagliflozin group versus placebo after 12 weeks. secondary objectives were comparisons of dapagliflozin versus placebo for fpg change from baseline, dose - dependent trends in glycemic efficacy, proportion of patients achieving a1c 5% reductions with dapagliflozin (range 15.329.1%) than with placebo (7.7%) ; the proportion with metformin was 16.1%. mean percent changes in waist circumference were 1.6 to 3.5% (dapagliflozin), 1.2% (placebo), and 2.2% (metformin). a : percent change from baseline in weight over the 12-week treatment period and 4-week follow - up period (observed values). b : adjusted mean percent change from baseline in weight after 12 weeks of treatment (locf). displayed generally, adverse events were reported at similar frequencies across all groups (table 2). hypoglycemic events were reported in 610% of dapagliflozin - treated patients with no dose relationship, in 4% of placebo - treated patients, and in 9% of metformin - treated patients (table 2). events relating to each category were pooled (e.g., preferred terms urinary tract infection and cystitis were pooled as infections of the urinary tract were seen in 512% of dapagliflozin - treated patients with no clear dose relationship versus 6% of placebo - treated patients and 9% of metformin - treated patients. genital infections were seen in 27% of dapagliflozin - treated patients, 0% of placebo - treated patients, and 2% of metformin - treated patients. hypotensive events were seen in 02% of dapagliflozin - treated patients versus 2% of placebo - treated patients and 4% of metformin - treated patients. decreased blood pressure was observed in all dapagliflozin groups (table 2). mean changes from baseline in supine systolic blood pressure (sbp) at week 12 ranged from 2.6 to 6.4 mmhg with no clear dose relationship. changes in diastolic blood pressure (dbp) and heart rate were small and inconsistent across dapagliflozin groups. the diuretic effect of dapagliflozin was assessed by 24-h urine volume, hematocrit, and serum blood urea nitrogen (bun) and creatinine (table 2). small dose - related increases in 24-h urine volumes (range 107470 ml above baseline of 1.82.2 l) were demonstrated at week 12. increases in hematocrit were also dose - related (range 1.52.9%). there were small changes from baseline in serum bun and no change in serum creatinine at week 12 across dapagliflozin doses. mean percent increases at week 12 in the bun - to - creatinine ratio ranged from 10.4 to 18.3%, with no apparent dose relationship. changes in urine volume, hematocrit, and bun - to - creatinine ratio returned toward baseline during follow - up. there was no clinically meaningful change in estimated glomerular filtration rate (modification of diet in renal disease formula) (19) in any group. meq / l above the baseline mean (1.7 meq / l) in serum magnesium and a larger relative decrease of 1.0 mg / dl below the baseline mean (5.5 mg / dl) in serum uric acid were observed, returning toward baseline after discontinuation of dapagliflozin. serum phosphate increased in a dose - related manner for doses 5 mg (range 0.01 to + 0.24 mg / dl from baseline of 3.63.8 mg / dl), although these changes were not statistically different from placebo (0.08 mg / dl) (table 2). there were no clinically relevant mean changes from baseline in serum sodium, potassium, and calcium (table 2). with respect to bone metabolism, serum 1,25-dihydroxyvitamin d and 25-hydroxyvitamin d values were unchanged from baseline. mean changes in the 24-h urinary calcium - to - creatinine ratio were similar to those with placebo. small increases in mean parathyroid hormone concentrations (range 0.67.0 pg / ml above baseline of 31.135.0 pg / ml) were noted, which were generally greater than the 0.8 pg / ml increase for placebo. there was no clear treatment effect of dapagliflozin on fasting lipid parameters in this 12-week study. glucose reabsorption by the kidney is necessary from an evolutionary standpoint to retain calories but becomes detrimental in type 2 diabetes by contributing to perpetuation of hyperglycemia and caloric excess. paradoxically, the glucose resorptive capacity of the kidney may increase in type 2 diabetes (20). therefore, limiting renal glucose reabsorption through the inhibition of sglt2 represents a new approach to treating hyperglycemia in type 2 diabetic patients. this study provides evidence that inducing controlled glucosuria through selective sglt2 inhibition improves hyperglycemia consistently over 12 weeks of treatment in type 2 diabetic patients. dapagliflozin produced decreases in a1c, fpg, and ppg after 12 weeks, with reductions in fpg apparent by week 1. changes in fpg were dose - related ; however, there was little evidence of a dose response for either ppg or a1c. the impact of sglt2 inhibition was relatively greater on ppg than on fpg, with renal glucose excretion acting as a relief valve to blunt postprandial hyperglycemia. even the lowest dapagliflozin dose (2.5 mg) produced a near - maximal effect on ppg, consistent with reductions observed in a clinical ward study (16). in contrast, the effect on fpg, measured at the trough drug concentration, was dose - ordered and corresponded to expected residual trough pharmacodynamic activity (16). dapagliflozin exhibited a diuretic effect, with small dose - dependent increases in urine volume equivalent to 0.31.5 voids / day, small increases in bun, and small dose - dependent increases in hematocrit. the relevance of this diuresis in type 2 diabetic patients, who often require diuretics for controlling hypertension (21), warrants further investigation. although no effect on renal function was observed, longer - term studies and exploratory renal biomarker assessments are being undertaken. veterinary literature suggests that chronic administration of phlorizin in lactating cows induces lipolysis (22), and dapagliflozin in obese rats induces reduced adiposity (23). during treatment, weight loss was more pronounced during week 1 with dapagliflozin, particularly at higher doses. this observation, coupled with a rapid partial rebound in weight after discontinuation of higher doses, suggests that diuresis may contribute to some weight loss. overall, it appears likely that acute weight reduction during week 1 represents fluid loss, which may also result in lower sbp, whereas continued gradual weight loss represents decreased fat mass. longer - term clinical and body composition studies will help to establish the relative contribution of diuresis versus adiposity reduction to total weight loss. daily dapagliflozin was well tolerated with no major difference in adverse events across treatment groups. the hypoglycemia experience supports the potential for dapagliflozin to achieve meaningful glycemic efficacy with relatively low hypoglycemic risk. the number of reported urinary tract infections was similar among dapagliflozin, metformin, and placebo groups and is consistent with rates reported in type 2 diabetic patients (24). the incidence of genital infections was higher with dapagliflozin versus placebo, especially at higher doses, but without statistical significance for comparison. of note is the lower rate of genital infections reported for placebo group patients than previously reported for type 2 diabetic patients (25). dapagliflozin increased serum phosphate at higher doses, and all arms including placebo and metformin demonstrated increased serum parathyroid hormone. additional data are needed to understand the long - term effects of chronic glucosuria and dapagliflozin treatment on skeletal metabolism. this study demonstrated the clinical efficacy of inhibiting renal glucose reabsorption with dapagliflozin in type 2 diabetic patients and relative safety across numerous doses. our results suggest that dapagliflozin, as the first in a new class of sglt inhibitors, can improve glycemic and weight status of type 2 diabetic patients. although we evaluated monotherapy, the insulin - independent mechanism of dapagliflozin may complement other type 2 diabetes agents that act through insulin signaling pathways and thus enhance combination therapy. although human genetic case reports are reassuring, the chronic effects of pharmacologically induced glucosuria are unknown and require long - term assessment. on the basis of evidence to date, further clinical study of dapagliflozin is warranted to develop a more definitive benefit / risk profile for this novel therapeutic agent. | objectivedapagliflozin, a novel inhibitor of renal sodium - glucose cotransporter 2, allows an insulin - independent approach to improve type 2 diabetes hyperglycemia. in this multiple - dose study we evaluated the safety and efficacy of dapagliflozin in type 2 diabetic patients.research design and methodstype 2 diabetic patients were randomly assigned to one of five dapagliflozin doses, metformin xr, or placebo for 12 weeks. the primary objective was to compare mean change from baseline in a1c. other objectives included comparison of changes in fasting plasma glucose (fpg), weight, adverse events, and laboratory measurements.resultsafter 12 weeks, dapagliflozin induced moderate glucosuria (5285 g urinary glucose / day) and demonstrated significant glycemic improvements versus placebo (a1c 0.55 to 0.90% and fpg 16 to 31 mg / dl). weight loss change versus placebo was 1.3 to 2.0 kg. there was no change in renal function. serum uric acid decreased, serum magnesium increased, serum phosphate increased at higher doses, and dose - related 24-h urine volume and hematocrit increased, all of small magnitude. treatment - emergent adverse events were similar across all groups.conclusionsdapagliflozin improved hyperglycemia and facilitates weight loss in type 2 diabetic patients by inducing controlled glucosuria with urinary loss of 200300 kcal / day. dapagliflozin treatment demonstrated no persistent, clinically significant osmolarity, volume, or renal status changes. |
the ankle joints play an important role in gait. the structure of foot is adapted for locomotion and the alignment of the foot and ankle plays an important role in standing and gait1, and supporting weight during gait2. the proper functioning of the ankle joint is necessary for daily life, recreation and sports activities3, and it is important in ambulation and functional gait in terms of its mechanical properties4. the strength of the muscles around the ankle is related injuries from sports activities3, and this strength is essential for the prevention of falls5. the passive torque gained through the tension of muscular tissue and the stiffness of the tissues adjacent to joints like ligaments or tendons is required for the development of an appropriate strategy for the ankle joint. the central nervous system, which controls the spontaneous physical response to the external events, and stretches and contracts the muscles, generates the active torque. the active torque that is gained through the central nervous system is also required for the development of an appropriate strategy for the ankle joint6. postural control involves an adequate stepping strategy and the appropriate use of the muscles attached to the hips and ankles7. the primary functions of the ankle joints are the provision of balance adjustment in response to postural disturbance, absorption of shock during gait, and movement of the lower extremity8. the maintenance of balance is a complex prcess, and it is influenced by a range of several sensorimotor functions, including muscular strength, proprioception, and the visual and vestibular sensory systems9. the central nervous system uses ascending motor pathways that receive information from the feet to control the position of the body and coordinate posture in relation to the surrounding environment10. the ankle joint has the function of balance control, responding to the ground reaction force, it contracts muscles such as the gastrocnemius, the soleus and the tibialis anterior6. the plantar flexor and dorsiflexor play a central role in maintaining balance during single - limb support and bilateral - limb support, and the gastrocnemius and soleus play a significant role in postural correction11. muscular strength and muscle activation are required for balance, to keep the center of gravity within the base of support12. in one study, subjects performed short - term isometric and isotonic exercise, and isometric exercise increased muscular strength significantly at the low speed of 60/sec, while isotonic exercise did the same at the higher speeds of 120/sec and 180/sec13. another study analyzed how the function and muscular strength of the lower limbs are influenced by isotonic and isokinetic exercise in a closed kinetic chain type exercise for 6 weeks, and reported that muscular strength, muscle power and muscle endurance of the knee joint extensors and flexors did not show any statistically significant differences14. also, a study of a muscle strengthening program, including isometric and isotonic contractions and stretching exercises, performed by elderly women for a period of 12 weeks, reported that increased muscular strength improved their balance15. another study analyzed the isokinetic torque and the absolute peak torque of the invertor and evertor muscles of females with functional ankle instability who performed eccentric contraction and concentric contraction at the respective speeds of 60/sec and 120/sec. deficits in eccentric invertor strength contributed to symptoms of functional ankle instability, and it was noted that eccentric contraction decrease of the invertor had a significant impact on the results16. another study analyzed the equilibrium and emg (electromyography) activity of a sports player using a single - limb stance on four different unstable surfaces and reported that the emg activities of the peroneus longus, extensor digitorum longus, and tibialis anterior muscle were greater on a small wood board than on to hemi - cylindrical and large plastic wood boards17. the calf muscle - tendon unit of the ankle plays an important role in basic human movement activities18, such as balance control while standing19, and the enhancement of the effectiveness of walking20. in spite of the fact that the ankle joint is of such importance, most studies have performed exercises on the knee joint muscle rather than on the ankle joint and analyzed the variables of muscular function such as muscle strength and muscle power. therefore, this study examined the effects of isotonic and isokinetic exercise performed by the essential muscles of the ankle joint, which play an important role in balance and gait. the purpose was to investigate how the muscle activity and balance were affected by isotonic and isokinetic exercise. in addition, the effects of isotonic and isokinetic exercise on the tibialis anterior, which is responsible for dorsiflexion, and on the peroneus longus and gastrocnemius, which are responsible for ankle plantarflexion were investigated. twenty students (10 men, 10 women ; 18 right dominant, 2 left dominant) attending namseoul university in cheonan - si, republic of korea, volunteered for this study. they were clearly informed of the purpose and experimental procedures before providing their written consent. none of the subjects had impairments of the musculoskeletal or neurological systems, and none had a history of athletics or had received any training for muscle strengthening within the past 6 months21. two types of exercises were performed and 10 subjects each were randomly assigned to the isotonic and isokinetic exercise groups. a body composition analyzer (inbody720. biospace o. ltd, seoul, korea) was used to measure subjects physical characteristics. a functional rehab system (primus rs, bte tech., hanover, usa) was used to asses maximal voluntary isometric contraction and to perform isotonic and isokinetic exercise. a free emg system (free emg, bts inc., milan, italy) was used to measure muscle activity. a computerized balance platform (hur bt4, burlabs, tampere, finland) prior to the commencement of this experiment, the subjects were informed of the contents of the experiment and were allowed to participate in the experiment only after submitting their written consent. the subjects were placed in either the isotonic exercise group or the isokinetic exercise group with 10 individuals in each group. the subjects were ordered to kick a ball on the ground, and the side they used to kick the ball was considered to be their dominant side22. surface electrodes were then attached to the bilateral tibialis anterior, the peroneus longus and the medial gastrocnemius of each participant. the subjects were seated on a dynamometer with their hip joint and knee joints fixed in the mid - positon with a brace belt. the rotation axis of the equipment was then adjusted to match the line that links the medial malleolus, and the foot was fixed for the isokinetic exercise23. no movement was possible, and the exercise was performed on the non - dominant side24. balance was measured immediately after completion of the exercise protocol11. all exercises and measurements were performed with the subjects barefoot to prevent any potential confusion being caused by different shoe types or heel heights18. for surface electromyography, electrodes were attached to the muscle at maximum static contraction to prevent any technical difficulties from influencing the experiment25. the skin was abraded with sandpaper and shaved, then cleaned with an alcohol - soaked cotton swab, to reduce the electrical impedance of the skin to less than 5 k. active electrodes (carbon electrode, 3 m, usa) were placed 2 cm apart, in parallel with the muscle fibers, on the tibialis anterior, the peroneus longus and the medial gastrocnemius26. during the electromyography measurements, the electromyography signals were recorded at a sampling rate of 1 khz, bandpass - filtered between 20 and 500 hz. the root mean square value was calculated from the raw emg data27. the emg data of balance measures was processed after excluding the first and last 5 seconds. the subjects performed a series of 5 submaximal contractions as a warm - up to familiarize themselves with the dynamometer28. the highest peak torque of 3 maximum voluntary isometric contractions (mvics) was used in this study30. in the isotonic group, the istonic group performed dorsiflexion and plantar flexion 10 times for 3 sets and had a 1-minute rest between sets to prevent fatigue13. in the isokinetic group, the angular velocity of 60/sec that is generally used for muscle strengthening exercises for healthy adults and athletes was chosen from among the angular velocities of 60/sec, 180/sec and 300/sec, which are appropriate for isokinetics33. in the isotonic group, the ankle was placed in the neutral position34, and subjects performed dorsiflexion and plantarflexion in a non - painful range of motion 10 times for 3 sets, for a total of 30 times35, and had a 1-minute rest between sets to prevent fatigue24. the one - leg standing test with eyes open was performed on a computerized balance platform35. during the trials, the subjects had to stand barefoot on one leg for 30 seconds with their eyes open36, while the other leg was held off the surface with a knee flexion of 30 degrees along the anterior - posterior axis of the foot plate35, and with both arms in a relaxed state at the sides. for visual information, the subjects were asked to stand in as stable a position as possible, watching a point on a monitor that was 65 cm in front of them during experimental testing37. for visual information directions, a reverse countdown was started from 4 before beginning the measurements, and stop was said after the measurements had been taken. the measurements were taken twice, and the mean value of the two measurements was calculated. the dependent variables were tested for normality using the kolmogorov - smirnov test, and for homogeneity of variance using levene s test. manova (multivariate analysis of variance) was used to examine the significance of differences in balance and compare emg and the balance of the tibialis anterior, the peroneus longus and the medial gastrocnemius between the groups (isotonic and isokinetic exercise) and time (pre - exercise and post - exercise). this study was approved by the institutional review board of namseoul university (cheonan, korea, nsu-140528 - 1). twenty healthy adults took part in this study, and their physical characteristics are presented in table 1table 1.subject characteristicsgroupnage (years)height (cm)weight (kg)bmi (kg / m)msdmsdmsdmsdisotonic1020.31.7170.29.970.014.623.92.6isokinetic1020.21.8165.59.965.820.223.64.8msd : meansstandard deviation, bmi : body mass index. the results of the comparison of muscle electromyography between pre - test and post - test by group are shown in table 2table 2.comparison of muscle electromyography between pre- and post - test (mv)variablegrouppre - testpost - testmsdmsdnon - dominant tibialis anteriorisotonic0.350.300.330.14(support by non - dominant side)isokinetic1.471.521.180.83dominant tibialis anteriorisotonic0.160.130.420.64(support by non - dominant side)isokinetic0.620.590.710.52dominant gastrocnemiusisotonic0.570.591.372.14(support by dominant side)isokinetic2.351.622.422.25dominant peroneus longusisotonic0.620.581.010.83(supported by dominant side)isokinetic1.400.901.871.23significant difference, p<0.05. statistical analysis revealed that both the isotonic exercise and isokinetic exercise groups showed neither an interaction effect between group and time as, nor a main effect of time. a main effect of group was found in the non - dominant tibialis anterior, the dominant tibialis anterior, the dominant gastrocnemius and the dominant peroneus longus (p<0.05). the changes in balance between pre - test and post - test by group are shown in table 3table 3.comparison of balance between pre- and post - test (mm)variablegrouppre - testpost - testmsdmsdsway area(support by non - dominant side)isotonic435.0139.1615.1244.1isokinetic389.3215.6515.6277.7 significant difference, p<0.05. statistical analysis revealed that both the isotonic exercise and isokinetic exercise groups showed neither an interaction effect between group and time, nor a main effect of group. a main effect of time was statistically significant for the sway area when support was provided by the non - dominant side in both groups (p<0.05). msd : meansstandard deviation, bmi : body mass index significant difference, p<0.05 significant difference, p<0.05 loss of balance is caused by the center of foot pressure leaving the limits of stability of the base of support38. the sensory process of balance is interaction among the somatic senses, which include proprioception, visual sense, and stereotactic input from the vestibular system40. the lower limb activity sequence is an important factor for the neuromuscular function of the ankle joint41, and the sway is related to the postural variable, which provides important information about stability and postural control. fatigue increases the sway36 and analyzing the sway is essential for understanding of the postural control mechanisms and the treatment of patients with balance dysfunction42. in this study, healthy adults performed isotonic and isokinetic exercise for the ankle joint, and how the respective exercises influenced muscle activity and balance was investigated. when muscle activity was compared between pre - test and post - test by group, both the dominant tibialis anterior and the non - dominant tibialis anterior presented significant differences when support was provided by the non - dominant side, and the dominant gastrocnemius muscle and the dominant peroneus longus presented significant differences when support was provided by the dominant side. razaeimanech and farsani43 reported that performance of isotonic exercise for the lower limb for 6 weeks increased emg and improved the muscle strength of volleyball players. onambele.38 reported that in both elderly and young adults, emg of the tibialis anterior was higher than that of the gastrocnemius when their eyes were closed. they also reported that the tibialis anterior emg of the elderly was higher than that of the gastrocnemius, but the gastrocnemius emg of the young adults was higher than the tibialis anterior emg their eyes were open. in this study, the dominant tibialis anterior showed a significant increase when support was provided by the non - dominant side, and the dominant gastrocnemius muscle and dominant peroneus longus showed a significant increase when support was provided by the dominant side. thus, our present study s results are similar to those of previous studies. kennedy.44 studied neuromuscular fatigue caused by isometric contraction of the plantar flexor and dorsiflexor, and reported that central fatigue could be determined by comparing the level of voluntary activation of the dorsiflexor and plantar flexor between pre - fatigue and post - fatigue. however, the two muscle groups had different characteristics of fatigue mechanism and recovery, and because the mechanisms of the two muscle groups were not similar, they might not be influenced by the same method. they also reported that it took 7 minutes for the plantar flexor to recover from fatigue, while it took only 1 minute for the dorsiflexor. in addition, the possibility of compensation between muscles was higher in the plantar flexor than in the dorsiflexor, which is mainly composed of the tibialis anterior, contrary to that of the plantar flexor, which is composed of synergistic muscle. so we conjecture the reason why both sides (regardless of dominant or non - dominant side) of the tibialis anterior had a better recovery speed than the plantar flexor (as indicated by the significant difference in this study) is due to compensation among muscles. berger.45 reported that the emg of the exercised tibialis anterior decreased in their study of the bilateral effects of unilateral lower limb muscle fatigue on emg. they reported that the fatigued muscle activity was related to passive tension increase, and to resist increasing the stretch of the fatigued muscle, the decrease of the exercised tibialis anterior emg might restrict tension and increase the risk of injury to the fatigued triceps surae. thus, the reason why the non - dominant tibialis anterior showed a significant difference when support was provided by the non - dominant side, in this study, can be attributed to the fact that the muscle tension was influenced by the fatigue of the non - dominant tibialis anterior. in this study, the dominant gastrocnemius muscle and the dominant peroneus longus showed significant differences when support was provided by the dominant side. south and george46 studied the effect of fatigue of the peroneus muscle on proprioception and reported that fatigue of the peroneus muscle did not affect ankle position sense after inducing muscle fatigue using an isokinetic dynamometer. mcloda.47 concluded that muscles around the ankle other than the peroneal muscle are able to compensate for fatigue of the peroneal muscle as they are less fatigued than the peroneal muscle and can process the sensory information. they also reported that only 5 out of 12 muscles passing through the ankle, are restricted by the ankle joint. giulio.48 suggested that the tibialis anterior is not always the best muscle for preventing postural sway, and that the triceps surae is not always an agonist of ankle muscles. they also suggested that these roles are dynamic and can change according to the location of the center of gravity relative to the ankle joint. paillard.49 studied the impairment of postural control in the contralateral (non - exercised) lower limb after stimulation and voluntary contraction of the ipsilateral (exercised) lower limb. they observed a cross - over fatigue effect after the sensory stimulus, and suggested that voluntary contraction might impair postural control, since the sway area showed a significant difference after fatigue of the lower limbs had been induced by voluntary contraction and electrical stimulus. it is also known that the effect of one leg balance training can be transferred to the untrained contralateral side by the cross - education effect50. accordingly, we consider the dominant gastrocnemius and peroneus longus showed a significant difference in the current study due to the fact that fatigue induced by exercise being transferred to the dominant tibialis anterior by the cross - over fatigue effect, and the non - exercised muscle transmitting the sensory information, because the location of the center of gravity changed during the one leg stance. in this study, the sway area showed a significant difference between pre - test and post - test by group when support was provided by the non - dominant side. boyas.11 studied postural control impairment after muscle fatigue induced by isokinetic exercise of the ankle joint in healthy adults. they reported that the sway area during the one - leg stance with eyes open was greater when the plantar flexor and ankle dorsiflexor were exercised simultaneously than when the two muscles were exercised separately11. yaggie and mcgregor51 conducted a study in which they examined the balance maintenance and postural limits after inducing muscle fatigue by isokinetic exercise of the ankle joint at 60/sec in healthy and young adults. they reported that fatigue of the ankle plantar flexor and ankle dorsiflexor significantly affected the sway and the range of posture control. bisson.52 studied the acute effects of fatigue on the plantar flexor and reported that postural sway increased as fatigue increased, especially during the one - leg stance. boyas.36 studied postural changes related to postural control impairment due to fatigue of the plantar flexor during the one - leg stance and reported that the sway area was greater during the post - fatigue period than during the pre - fatigue period, showing that fatigue of the plantar flexor is directly connected to changes in the postural sway. the results of our present study show that fatigue of the lower limb, induced by isotonic or isokinetic exercise, significantly affected the mechanism of postural control and the sway area. hence, we consider the results of our present study are similar to those of previous studies in that the sway area showed a significant difference between pre- and post - fatigue. in conclusion, this study was conducted to examine how the balance of the lower limbs and the muscle activities of the tibialis anterior, the medial gastrocnemius and the peroneus longus are influenced by isotonic and isokinetic exercise of the ankle joint. the subjects of this study were healthy adults in their 20s, and the following results were obtained. (1) the changes in muscle activities between pre- and post - test in both the isotonic and isokinetic exercise groups did not show an the interaction effect between group and time, and a main effect of time was not shown by either of the groups. a main effect was shown by in the non - dominant tibialis anterior, the dominant tibialis anterior, the dominant gastrocnemius muscle and the dominant peroneus longus. (2) the changes in balance between pre - test and post - test in both exercise groups did not show an the interaction effect between group and time, and a main effect was not shown by either of the groups. a main effect of time was shown by both the isotonic and isokinetic exercise groups for the sway area when support was provided by the non - dominant side. the two exercise groups showed significant differences in muscle activities, and the group performing isokinetic exercise showed higher muscle activities. in terms of balance, there was no difference between the groups, but there was a significant difference between the pre - test and the post - test results. the results of this study may help in the selection of exercises for physical therapy, because they demonstrate that muscle activity and balance vary according to the type of exercise. | [purpose ] this study was performed to examine how the balance of lower limbs and the muscle activities of the tibialis anterior (ta), the medial gastrocnemius (gcm), and the peroneus longus (pl) are influenced by isotonic and isokinetic exercise of the ankle joint. [subjects ] the subjects of this study were healthy adults (n=20), and they were divided into two groups (isotonic=10, isokinetic=10). [methods ] isotonic group performed 3 sets of 10 contractions at 50% of mvic and isokinetic group performed 3 sets of 60/sec. muscle activity was measured by emg and balance was measured by one - leg standing test. [results ] for muscle activity, a main effect of group was found in the non - dominant ta, and the dominant ta, gcm and pl. for balance, a main effect of time was found in both groups for the sway area measured support was provided by the non - dominant side. [conclusion ] in terms of muscle activity, the two groups showed a significant difference, and the isokinetic group showed higher muscle activities. in terms of balance, there was a significant difference between the pre - test and the post - test. the results of this study may help in the selection of exercises for physical therapy, because they show that muscle activity and balance vary according to the type of exercise. |
a new trial conducted by the canadian critical care trials group investigated the impact of different diagnostic approaches on outcomes of patients suspected of having ventilator - associated pneumonia (vap). immediate administration of adequate antibiotic therapy is critical to improving survival in patients with vap. at the same time, appropriate antimicrobial stewardship includes not only limiting the use of inappropriate agents in patients with vap, but also improving our ability to diagnose and exclude infection in the intensive care unit (icu) setting in order to avoid administering antibiotics to patients without bacterial infection. this recent published randomized trial comparing the quantitative culture of bronchoalveolar lavage (bal) fluid and the culture of endotracheal aspirate in critically ill patients with suspected vap adds to the information presented by four previous trials [5 - 8 ]. the canadian critical care trials group found that the two diagnostic techniques were associated with similar clinical outcomes and similar overall use of antibiotics (table 1). several considerations should be taken into account, however, to appropriately evaluate the possible impact of diagnostic techniques on the individual (patient morbidity and mortality) and on the collective (emergence and dissemination of antibiotic - resistant strains) outcomes. outcomes and antibiotics in the canadian critical care trials group study no differences were statistically significant. first, as clearly underlined by kollef in his related editorial, the exclusion of patients previously colonized or infected with methicillin - resistant staphylococcus aureus or pseudomonas species and the exclusion of those patients having previously received the ' study drugs ' (that is, meropenem and/or ciprofloxacin) resulted in a low rate of studied patients with ' high - risk ' pathogens responsible for vap. a proportion of less than 12% of difficult - to - treat pathogens, such as p. aeruginosa, acinetobacter spp., stenotrophomonas maltophilia, and/or methicillin - resistant s. aureus, as compared with more than 30% in the french study, diminishes the usefulness of the results of this study in real life. second, 29% of patients managed using bal had new antibiotics initiated within 3 days before randomization, probably after the onset of the first signs in relation to vap, which is problematic when using quantitative culture techniques. in this case, a negative finding or a result below the usual threshold of 10colony - forming units / ml could indicate either that the patient has been successfully treated for pneumonia and the bacteria are eradicated, or that there was no lung infection to begin with. these authors did not give any information on how decisions regarding antibiotic treatment were taken in this group of patients. third, the authors report a relatively high rate (14%) of inappropriate initial empirical therapy in the bal group. as indicated above, the low frequency of high - risk, difficult - to - treat pathogens responsible for pneumonia can not explain such a disappointing result, when compared with the 0.5% rate of inappropriate initial therapy reported by fagon and coworkers in the invasive strategy group. the most probable explanation is that all patients included in this study were also randomized to receive a fixed combination therapy or monotherapy as initial treatment : meropenem plus ciprofloxacin or meropenem alone. several studies have clearly established that initial antimicrobial therapy in patients with vap should be customized to local epidemiology at the icu level. fourth, even on day 6 the rate of targeted therapy was only 74.2% in the bal arm, underlining the fact that, in many patients managed using this diagnostic technique, early de - escalation was not performed although clearly indicated. unfortunately, information on how decision algorithms were followed in the two study arms once cultures were available (as soon as day 2 or day 3) was not given. obviously, the potential benefit of using a diagnostic tool such as bal for safely restricting unnecessary antimicrobial therapy in such a setting can only be obtained when decisions regarding antibiotics are closely linked to bacteriological both direct examination and cultures results. in the current study, bal was not used for identifying patients with vap who needed antimicrobial therapy ; this decision was essentially left to the icu physicians in charge of the included patients on the basis of their clinical judgment, even when bal culture results were < 10colony - forming units / ml. interestingly, the proportion of ' confirmed pneumonia ' was 86% in the bal group and 83% in the endotracheal aspirate group. in contrast to previous recommendations concerning the use of quantitative bal, therefore, many patients with quantitative culture results below the cut - off point of 10colony - forming units / ml continued to receive antibiotics, even after day 3. this could entirely explain why there was a similar use of antibiotics in the two study arms. finally, a major benefit of a negative bal specimen may be to direct attention away from the lungs as the source of fever and, in the absence of antibiotic interference, to more readily diagnose other potential sites of infection. delaying diagnosis or definitive treatment of the true site of infection may lead to prolonged antibiotic therapy and to induction of additional dysfunction. in the current trial, we are left with uncertainties regarding the numbers of extrapulmonary infection in the two arms of the trial, as well as how long the recommended duration of therapy in patients with vap should be and the how patients were managed in case of a second episode. in summary, even if the results of the canadian study are consistent with those of the three spanish trials (table 2) in which antimicrobial treatment was also initiated in all suspected patients and rarely withheld in patients with negative cultures, our own bias is that additional studies will be needed before one can conclude that a strategy based on the systematic collection of distal pulmonary secretions prior to the introduction of new antibiotics and quantitative culture techniques is useless. in real life, the key issue is to be able to adhere to a de - escalation strategy, which is the only way to curb the unnecessary use of antibiotics in the icu. the predominant impact of pretest opinion and the absence of clear bacteriological - based decision algorithms in the current study may unfortunately encourage physicians to pursue antibiotics in most patients after 2 days, even once results of bacterial cultures are available. ruiz. reported the total duration of antibiotic treatment ; p = 0.48. the canadian critical care trials group reported antibiotic - free days ; p = 0.86. bal = bronchoalveolar lavage ; icu = intensive care unit ; vap = ventilator - associated pneumonia. | the results of a recently published canadian study suggest that bronchoalveolar lavage and endotracheal aspiration are associated with similar clinical outcomes and similar overall use of antibiotics in critically ill patients with suspected ventilator - associated pneumonia (vap). the study, however, does not provide convincing information on the best strategy to diagnose vap, to accurately choose initial treatment and to exclude vap in order to avoid administering antibiotics to patients without bacterial infection. in fact, this trial has several limitations or drawbacks : patients at risk for developing vap due to pseudomonas aeruginosa or methicillin - resistant staphylococcus aureus were excluded, far from the real - life scenario ; a significant number of patients were receiving recent antimicrobial therapy at the time of sampling, with, consequently, difficult - to - interpret culture results ; randomization of included patients for initial treatment meropenem plus ciprofloxacin or meropenem alone resulted in a high rate of inappropriate initial empirical therapy due to the absence of customization to local epidemiology ; and the initial decision to treat and the re - evaluation at day 3 were, in fact, based on clinical judgment and not on direct examination and quantitative culture results. in summary, because antimicrobial treatment was initiated in all suspected patients and was rarely withheld in patients with negative cultures, the study does not suggest an appropriate strategy for improving the use of antibiotics in intensive care unit patients. such a strategy has two requirements : immediate administration of adequate therapy in patients with true vap, and avoidance of administering antibiotics in patients without bacterial infection. |
nephropathy is widely encountered among the people of entire world irrespective of the age, racial, environmental, and geographical variability. the etiology behind this complication is broad ranging from substance - induced to various metabolic and physiological disturbances, paneling nephropathy amongst the 10 leading causes of death across the world. cisplatin (cis - diaminedichloroplatinum ii, cddp) is extensively used for the treatment of several cancers like testicular and lungs cancer. unfortunately, the gracious drug cisplatin is conjoined with a brutal side effect since it induces nephrotoxicity., locally known as kanchana, belonging to the family caesalpiniaceae, is a widely distributed plant throughout the subtropical and tropical regions of the world. it is used in abundance for medicinal and cattle feed purposes by the tribes of india and also being used in various indigenous systems of medicine like ayurveda and unani. different parts of this plant are used traditionally in various ailments owing to its folkloric claims such as liver tonic, antibacterial, to suppress the edema, dysentery, ulcers, eye disease, skin diseases, piles, and hemorrhoids. the stem of this plant is reported to contain many active phytochemicals including stigmasterol, flavone glycosides, lupeol, kaempferol-3-glucoside, -setosterol, etc. in this present context, the in vivo nephroprotective activity of the ethanolic extract of b. variegata (bv) whole stem was evaluated in albino male rats (wistar strain). the whole stem of bv was collected from young matured plant from bharatpur forest range, bhubaneswar, orissa, india, during the month of november and december and identified by dr., gopabandhu ayurvedic college, puri, orissa. the plant specimen (vide voucher no. : 081) was deposited in herbarium of university department of pharmaceutical sciences, bhubaneswar, orissa, india. after authentication fresh plant materials were collected in bulk, washed under running tap water to remove adherents, shade dried, and pulverized in a mechanical grinder to get coarse powder of bv whole stem. healthy albino male rats of wistar strain weighing between 150 and 200 g were selected for the investigation. the animals were kept under maintained laboratory conditions with adequate supply of drinking water ad libitum and pallet diet. the experimental protocol was approved by the institutional animal ethics committee and the conditions in the animal house approved by committee for supervision on experiments on animals (vide registration no. : 990/c/06/cpcsea). about 200 g of coarse powder of bv whole stem was taken in a soxhlet apparatus and extracted successively with petroleum ether, chloroform, and ethanol. the extraction for each solvent was carried out for 18 - -20 h. the ethanol extract was collected by evaporating the solvent by slow heat treatment. total 1.4 kg of pulverized stem was used to produce the required amount of test extract. calculated amount of dried ethanol extract was suspended in 0.5% w / v of sodium cmc in a normal saline solution to prepare the test doses (200 and 400 mg / kg / ml.). the dose limits were selected on the basis of previously performed oral acute toxicity studies in mice, in accordance with the oecd guidelines. group i (normal control) received oral dose of 0.5% sodium cmc (1 ml each) for 14 days. group ii (toxic control) received single dose of cisplatin (7 mg / kg of body weight ; i.p.) on day 1. group iii (standard group) received standard polyherbal drug cystone (5 ml / kg ; p.o.) bangalore, india) for 14 days with single dose of cisplatin (7 mg / kg of body weight ; i.p.) on day 1, group iv (eebv200) and group v (eebv400) received ethanolic extract 200 and 400 mg / kg b.w. once in a day for 14 days respectively along with the single dose of cisplatin (7 mg / kg of body weight ; i.p.) on day 1. urine was collected over 24 h on 14th day by keeping the test animals in individual metabolic cages. the volume of collected urine samples was measured followed by estimation of biochemical parameters, namely urine creatinine and urine albumin. blood samples were collected from the test animals under anesthesia (phenobarbiton sodium ; 40 mg / kg of body weight ; i.p) by cardiac puncture before sacrifice and serum parameters including creatinine, urea, albumin, and total protein were estimated. the biochemical estimations were done in a biochemical - semi - auto analyzer (ebra - chem-5-plus, v2. west germany) by standard procedures using commercial kits (ecolin : merck specialties, india). the kidneys were removed from the rats before sacrifice and organs were fixed using a formosal solution (10% v / v of formaldehyde in normal saline), embedded with paraffin wax followed by preparation of tissue sections using a microtome for histopathology study. statistical significance of data was assessed by analysis of variance (one - way anova) followed by a comparison between different groups using tukey - kramer multiple comparison test. the toxic control group was compared with the normal control group and all other treatment groups were compared with the toxic control group. data obtained in the experiment were expressed in terms of mean sem. statistical significance of data was assessed by analysis of variance (one - way anova) followed by a comparison between different groups using tukey - kramer multiple comparison test. the toxic control group was compared with the normal control group and all other treatment groups were compared with the toxic control group. data obtained in the experiment were expressed in terms of mean sem. statistical significance of data was assessed by analysis of variance (one - way anova) followed by a comparison between different groups using tukey - kramer multiple comparison test. the toxic control group was compared with the normal control group and all other treatment groups were compared with the toxic control group. the results as cited in table 1 includes change in body weight, kidney weight, urine volume along with the urine, and serum biochemistry data. cisplatin administration - induced renal injury was prominent as evidenced by significantly depressed renal functions, body weight, and urine volume as compared to the normal control group. parameters studied for the nephroprotective effect of the ethanol extract of bauhinia variegata the eebv400 group (ethanolic extract 400 mg / kg treated rat group) showed significant (p<0.001) elevation in body weight (7.16 1.10) with a significant (p<0.05) increase in urine volume output (11.95 0.79). however, the urine creatinine (1.81 0.32) and albumin (0.31 0.06) decreased significantly (p<0.01) as compared with the toxic control group. the serum creatinine (0.65 0.09) and urea (32.86 5.88) were found to be significantly (p<0.001) low when compared with the toxic control group. the histological features found from the tissue sections of different groups are mentioned in table 2 and the photomicrographs of tissue sections are presented in figure 1a -- d. the histopathology of tissue sections suggest that the toxic control group had encountered vast histological damages as evidenced by the glomerular and tubular congestion with abnormal bowman 's capsule, blood vessel congestion, epithelial cell desquamation, and presence of tubular cast with few inflamatory cells. the histological features of the eebv400 group showed minimal cellular damage in contrast to the toxic control group. histological features found from l.s of kidneys of different groups photomicrographas of l.s. of kidney of different groups (a) normal control group, (b) toxic control group, (c) standard group, (d) eebv400 group. the present study aimed to evaluate the protective effect of whole stem extract (ethanol) of bv linn. plant against cisplatin - induced nephropathy in rats. cisplatin - administered rats (toxic control group) had encountered acute kidney dysfunction as evidenced by elevation in serum urea and creatinine, decreased urine output and body weight with multiple histological damages. treatment with the ethanol extract of bv at the dose level of 400 mg / kg b.w. for 14 days (eebv400 group) significantly lowered the serum level of creatinine and urea, decreased urine creatinine and albumin with a significant weight gain, and increased urine output when compared with the toxic group. the histological damages in the bv extract - treated group were minimal in contrast to the toxic rats. the statistical significance of the nephroprotective activity of bv - treated group and the polyherbal drug cystone (standard group)-treated group (both the groups were compared against toxic control) were found almost equal as both groups gained same level of significance (p<0.001) against the toxic group in most of the parameters including serum urea and creatinine. the results of our study suggest that the ethanolic extract of bv possesses nephroprotective potential depending on the dose levels. extensive and multidimensional further research is needed to elucidate the exact mechanism of nephroprotective action of this plant extract. | the nephroprotective activity of the ethanolic extract of bauhinia variegata (linn.) whole stem against cisplatin - induced nephropathy was investigated by an in vivo method in rats. acute nephrotoxicity was induced by i.p. injection of cisplatin (7 mg / kg of body weight (b.w.)). administration of ethanol extract at dose levels of 400 and 200 mg / kg (b.w.) to cisplatin - intoxicated rats for 14 days attenuated the biochemical and histological signs of nephrotoxicity of cisplatin in a dose - dependent fashion. ethanol extract at 400 mg / kg decreased the serum level of creatinine (0.65 0.09 ; p<0.001) and urea (32.86 5.88 ; p<0.001) associated with a significant increase in body weight (7.16 1.10 ; p<0.001) and urine volume output (11.95 0.79 ; p<0.05) as compared to the toxic control group. the ethanol extract of b. variegata at 400 mg / kg (b.w.) exhibited significant and comparable nephroprotective potential to that of the standard polyherbal drug cystone. the statistically (one - way - anova followed by tukey - kramer multiple comparison) processed results suggested the protective action of b. variegate whole stem against cisplatin - induced nephropathy. |
the pemphigus diseases, which include some of the most severe bullous autoimmune skin reactions, are seen predominantly in middle - aged and elderly individuals. all nonendemic pemphigus diseases, including paraneoplastic pemphigus, have been reported to occur in adolescents and even very rarely in children younger than 10 years. pemphigus herpetiformis (ph) is considered a variant of pemphigus displaying clinical features similar to dermatitis herpetiformis and a diverse histopathologic pattern with intraepidermal and subcorneal microabscesses, eosinophilic spongiosis, or superficial bullae with usually scant acantholytic cells [1, 2 ]. the diagnosis is based on detection of ig g antikeratinocyte cell surface antibodies, both bound in vivo and in circulation. in the literature, few cases of pemphigus are reported in children. this rarity in childhood may be only apparent as a result of its difficult diagnosis. an 12-year - old boy was seen at our department in march, 2007, with erosive plaques, vesicles, bulls, and crusted lesions associated with severe itching persisting for six months. the boy 's family history and personal history were unremarkable, and his growth and development had proceeded normally. on examination, we observed vesicular, bullous lesions, some having clear contents and some being purulent with erosive arciform plaques and crusted lesions (figures 1(a) and 1(b)). they were seen especially over the back, buttocks, chest, abdomen legs, and arms. histologic examination of one of the lesions showed an intraepidermal bulla containing rare acantholytic epidermal cells, eosinophil and neurophil cells. the superticia1 and reticular dermis had an inflammatory infiltrate of eosinophile and neutrophile around some of the blood vessels (figures 2(a) and 2(b)). iga intercellular deposits on epidermis were not seen (figures 3(a) and 3(b)). a diagnosis of ph was made, and treatment with dapsone 2 mg / kg per day resulted in total clinical remission. however, two months later, new vesicles reappeared and treatment was begun with prednisone at a dose of 2 mg / kg daily. there was a very good response ; lesions regressed slowly, until they completely disappeared after 4 weeks of treatment. the daily dose of the prednisone was lowered gradually according to the clinical improvement, and, at the time of this report, one year after onset of the disease the skin involvement was being kept under control on a low maintenance dose of 10 mg of prednisone daily. autoimmune blistering diseases are extremely rare in children. a review from a paediatric dermatology referral center for a population of 4 million people identified only 23 cases of immunobullous disease in patients less than 18 years of age over a 16-year period. a recent review of pemphigus vulgaris (pv) in children found only 46 cases reported in the literature. pemphigus herpetiformis (ph) was first introduced by jablonska and colleagues as a variant of pemphigus. various terms have been used to describe this condition, such as acantholytic herpetiform dermatitis, pemphigus controlled by sulfapyridine, and mixed bullous disease. clinical manifestations consist of erythematous, urticarial plaques, and vesicles that present in herpetiform arrangement. histological findings of hp are variable and include eosinophilic spongiosis and subcorneal pustules with minimal or no apparent acantholysis [9, 10 ]. immunofluorescence findings show that most patients with ph have igg antibodies against keratinocyte cell surfaces. dapsone is the first choice of drug in the ph treatment given as monotherapy or combined with systemic corticosteroids. in most reported cases, the use of systemic corticosteroids was necessary to achieve clinical remission. in some cases, however, the use of immunosuppressive drugs such as azathioprine and cyclophosphamide becomes necessary to induce clinical remission or to help in reducing steroid dose so that its collateral effects are minimized. based on its clinical features, some researchers reported the disease as a distinct entity and consider it to be different from classic pemphigus because of its clinical peculiarity and benign course. however, others described it as a variant of pemphigus foliaceus (pf) or pv. we believe the boy presents a pemphigus herpetiformis variant because of the clinical features of dermatitis herpetiformis (vesicles, bulls, and itching) and the immunologic (igg and c3 deposit on intercellular epidermal) and histological features (intraepidermal bulls and acantholysis) of pemphigus with an eosinophilic spongiosis. various types of pemphigus including vulgaris, foliaceus, erythematosus, vegetans, and paraneoplastic pemphigus can be seen in childhood. the disease must not be forgotten as a possible cause of erosive mucocutaneous disease in children. it is important that antibody titers, management plans, and outcomes for this disease be reported so that greater knowledge of the effects of various treatment regimens can be obtained. | pemphigus herpetiformis (ph) is one of the less common forms of pemphigus. ph in children is unreported. we describe a case of a child who developed ph. observation. a 12-year - old boy was seen at our department with erosive plaques, vesicles, and crusted cutaneous lesions associated with severe itching persisting for six months. histologic examination showed an intraepidermal bulla containing rare acantholytic epidermal cells with eosinophilic spongiosis. direct immunofluorescence demonstrated intercellular ig g and c3 deposit. the serum titer of antibodies against intercellular epidermal was 1/200 ui / l. diagnosis of ph was made, and treatment with dapsone 2 mg / kg per day resulted in total clinical remission. however, two months later, new vesicles reappeared and treatment was begun with prednisone at a dose of 2 mg / kg daily. there was a very good response. discussion. childhood pemphigus herpetiformis is a rare disease, often initially misdiagnosed. it must not be forgotten that the disease is a possible cause of erosive mucocutaneous disease in children. |
in the cancer research field, vitamin d has emerged as the most prolific topic in the last decade with work connecting it with risk reduction in various epithelial cancers. aside from calcium homeostasis, vitamin d exerts a wide range of immunogenic and antiproliferative activities in the body. of particular interest to the oncologists is the reduced incidence of breast, colon, and prostate cancers with higher sun exposure, higher intake, or higher serum levels of vitamin d. vitamin d exerts its antiproliferative effect by binding to vitamin d receptor (vdr) found in various tissues and cells of the body. several human genes contain vitamin d response elements (specific dna sequences) that encode for proteins important in regulation of cell proliferation, differentiation, apoptosis, and angiogenesis. when the serum vitamin d levels are suboptimal these activities are impaired and as a result enhanced cellular growth, neoangiogensis, and cancer development takes place. the breast cells have vdrs in their nuclei and it is postulated that polymorphism of genes for these vdrs results in increased risk for breast cancer. vitamin d from both diet and sun exposure is metabolized in the liver to 25-hydroxy vitamin d (25(oh)2d) and then further hydroxylated by 1 alpha hydroxylase enzyme in kidneys and other tissues like breast cells to 1,25- dihydroxy vitamin d (1,25 (oh)2d), the most biologically active form and the natural ligand for vdr. serum concentration of 25(oh)2d are more sensitive to exogenous sources (dietary and supplemental intake) and endogenous production (through synthesis in the skin) of vitamin d and have a long half - life of 3 weeks and is the predominant form of vitamin d in plasma and the major storage form ; hence the circulating 25(oh)2d is the best indicator of vitamin d status of the body. serum 25(oh)2d concentrations as well as treatment with vitamin d supplementation are significant independent predictors of breast cancer risk. women with serum levels of 25(oh)2d more than 50 ng / ml had a 50% lower risk of breast cancer compared to those with serum values less than 30 ng / ml in various studies from the developing world. it has been documented that consumption of oral calcium and serum levels of vitamin d are associated with reduced risk of breast cancer in premenopausal women but the results were not statistically significantly in postmenopausal women. there are data showing that locally advanced breast cancer patients have more severe vitamin d deficiency than those with early stage disease. low serum levels of vitamin d are common at breast cancer diagnosis and are associated with a poorer prognosis in terms of overall survival and distant disease free survival particularly in postmenopausal females. in another group of breast cancer patients it was found that 94% women with serum levels of vitamin d less than 20 ng / ml were likely to develop metastases and 73% were likely to die of advance disease. vitamin d deficiency is associated with secondary hyperparathyroidism which results in increased bone resorption, release of calcium from bones, and may precipitate or exacerbate osteoprosis with consequent ill effects on bone mineral density (bmd). there has been significant documentation of osteopenia and osteoprosis in breast cancer patients primarily due to early menopause and vitamin d deficiency and later amplified by chemotherapy and endocrine therapy particularly the aromatase inhibitors. thus breast cancer patients must undergo a baseline metabolic bone evaluation with serum vitamin d levels and bone mineral densitometry. the aim of the study was to determine serum levels of 25-(oh)2d in pakistani breast cancer patients at the time of presentation, to assess its risk association with grade and stage of the tumor and to evaluate the bone density in breast cancer patients. the study was approved by the scientific research committee and institutional review board at shaukat khanum memorial cancer hospital and research centre, lahore. all newly diagnosed breast cancer patients who presented to the medical oncology department were recruited into the study as cases after informed consent over a period of 6 months from november 2010 till may 2011. age - matched healthy females who accompanied the nonbreast cancer patients to hospital were recruited as the control group. the socio - demographics were documented by direct questioning on to the proforma for the whole study population. postmenopausal status of females was defined as last menstrual bleeding at least 12 months before the date of interview or a history of bilateral oophorectomy. the histopathological diagnosis of breast cancer, grade, stage of the tumor, and hormone receptor status (estrogen receptor - er, progesterone receptor pr, and her2neu) was recorded from the pathology reports of breast cancer patients. serum 25-(oh)2d levels were studied by the elisa technique on the blood samples drawn of the study population at their initial presentation and the values were documented in ng / ml. vitamin d deficiency was considered at serum level less than 20 ng / ml, suboptimal vitamin d levels were considered between 20 and 39 ng / ml and optimal levels were more than 40 ng / ml. bone mineral density was calculated according to the who criteria from ct bone density scan of the breast cancer patients. the demographic variables and the descriptive measures in cases and controls were presented in frequency and percentages. relation of vitamin d deficiency with grades, stages, histology, and receptor status of tumor was determined by using the chi - square test. comparison of vitamin d levels among various histopathological parameters of tumor was done by using one - way anova. comparison of vitamin d levels between pre- and postmenopausal status was done by using a t - test. the association of vitamin d status with bmd in cases and the serum vitamin d levels among the cases and controls was calculated by using the chi - square. the demographic variables and the descriptive measures in cases and controls were presented in frequency and percentages. relation of vitamin d deficiency with grades, stages, histology, and receptor status of tumor was determined by using the chi - square test. comparison of vitamin d levels among various histopathological parameters of tumor was done by using one - way anova. comparison of vitamin d levels between pre- and postmenopausal status was done by using a t - test. the association of vitamin d status with bmd in cases and the serum vitamin d levels among the cases and controls was calculated by using the chi - square. the mean age of cases was 47.5th 9.8 years and for the control group was 46.2th + 2.6 years. there were 46.7% premenopausal females and 53.3 % postmenopausal females among the breast cancer population. age, marital status, menopausal, residential area, and parda observing status had almost similar distribution among cases and controls. a total of 70% of the cases were multiparous and the 50% of the healthy controls had more than three children. regarding the occupational history, 92% of the cases were house wives while 33% of the controls were office workers. the mean serum vitamin d level in the breast cancer patient was 9.3 ng / ml and in the control group was 14.9 ng / ml and the p value calculated was 30), revealing a small but sinister causal link of low vitamin d levels with high bmi in breast cancer patients. lower serum levels of vitamin d have been associated with obesity and lower physical activity in various epidemiological studies. serum levels of vitamin d did not correlate inversely with the advance stage and grade of breast cancer in our study. regarding the receptor types of breast cancer (luminal types a and b, triple negative or her2 neu over - expressed) all had similar deficient vitamin d levels and there was no difference of statistical importance. recently kim., from korea reported poorer outcomes of vitamin d deficient patients with luminal type breast cancer. also there are data suggesting that triple negative breast cancer patients have the highest percentage of vitamin d deficiency. studies with both pre- and postmenopausal female populations have shown high prevalence of vitamin d deficiency despite supplementation. premenopausal females had slightly more low mean serum levels of vitamin d compared to the postmenopausal females and the results yielded minimum significance. recruited 1295 postmenopausal females and showed that low levels of vitamin d correlated with increased risk of distant recurrence and poor overall survival. examination of bone mineral density in breast cancer patients revealed that osteopenia and osteoporosis was common in postmenopausal female, as expected. more than half of vitamin d deficient breast cancer females had low bmd (osteopenia or osteoporosis) but the data did not show statistical significance and hence bmd can not be considered as an indirect marker for evidence of vitamin d status for an individual. vitamin d concentration may be a risk and/or a valuable prognostic factor in breast cancer patients on the basis of various studies but data from large randomized trials are still sparse. the optimal circulating level of 25(oh)2d for reducing breast cancer risk or reducing the risk of recurrence of breast cancer has yet to be defined. it is also known that there are ethnic and racial variations in serum vitamin d levels. asians have low exposure to sunlight, there is high prevalence of various malabsorption syndromes, and use of vitamin d supplementation is rare. few epidemiological efforts have investigated the association between vitamin d concentration and breast cancer risk in asian women. to our knowledge ours is the first study from pakistan to assess the association between breast cancer and serum vitamin d levels. the relatively small size of the study population limited our ability to detect statistically significant trends of vitamin d deficiency with respect to histopathological characteristics of the breast cancer. vitamin d deficiency is rampant in pakistan and also serum 25(oh)2d levels are reflective of a recent and not life time vitamin d intake ; hence one single measurement of vitamin d levels in our study may not be reflective of long term exposure. the relevant time period during which 25(oh)2d levels may affect breast cancer occurrence or survival is currently unknown. regardless of whether vitamin d helps prevent cancer development or its recurrence, the high frequency of vitamin d deficiency in the pakistani population with its adverse impact on bone health and further intolerance to various systemic cancer treatments makes it important for the oncologists to recognize, treat, and prevent vitamin d deficiency. as more studies confirm similar results, dietary vitamin d and casual sunlight exposure will be among the modifiable risk factors for breast cancer. | aim : the aim was to determine serum vitamin d levels in breast cancer patients and to assess its risk association with grade and stage of the tumor.materials and methods : ninety breast cancer patients and equal number of age - matched healthy females were recruited into the study by consecutive sampling over a period of 6 months for this case control study. serum 25(oh)2d levels and ct bone mineral density was done.results:the mean age was 461.5 years. age, marital status, menopausal, residential area, parda observing status, and body mass index were similar in distribution among cases and controls. the mean serum vitamin d level in the breast cancer patients was 9.3 ng / ml and in the control group was 14.9 ng / ml (p value < 0.001). vitamin d deficiency was seen in 95.6% (86) breast cancer patients and in 77% (69) of the control group (p value < 0.001). among the breast cancer patients the tumor characteristics (histology, grade, stage, and receptor status) did not show any significant associations with serum levels of vitamin d. premenopausal breast cancer females had a mean serum vitamin d level of 10.5 ng / ml and postmenopausal females had a mean value of 13.5 ng / ml (p value 0.015). low bmd did not correlate significantly with vitamin d deficiency (p value 0.787).conclusion : invariably almost all patients with breast cancer were vitamin d deficient. tumor characteristics did not show any significant associations with serum levels of vitamin d. bone mineral density did not correlate significantly with vitamin d deficiency. |
over the last 2 decades, advances in surgical technology combined with a growing consumer demand have created a shift towards gynecologic minimally invasive surgery (mis). the safety and efficacy of mis have been thoroughly documented in the literature and include decreased blood loss, a shorter hospital stay, faster recovery time, fewer wound - related complications, and superior cosmetic results [1, 2 ]. the da vinci surgical system (intuitive surgical, sunnyvale, ca, usa) for use during gynecologic procedures has quickly become the minimally invasive alternative to conventional laparoscopy by providing surgeons with greater dexterity and improved visualization of the surgical field. this shift towards mis in gynecology has required that academic institutions train their residents and fellows in new technology while simultaneously teaching the fundamentals of open surgery. however, these expectations are becoming increasingly difficult in an environment of limited resident work hours and increased financial concerns where respecting patient safety and satisfaction is essential. as a result, a push has been made to discover the most effective way to train residents and fellows in both laparoscopic and robotic surgery. becoming competent in mis requires adequate preparation time to acquire this unique set of skills. the importance of preparation via mis simulation labs prior to entry into the operating theater is well documented. however, whether or not the skills gained from laparoscopy translate into improved robotic surgery skills for physicians in training is yet to be explored. previous studies by surgeons investigating their personal experiences in learning robotic - assisted gynecologic surgery partly attributed their short learning curves to their extensive experience with advanced laparoscopic procedures prior to using robotic assistance [58 ]. while these previous studies suggest that surgeons with more laparoscopic experience learn robotic surgery more quickly, it is not yet known if that data can be extrapolated to resident physicians. current data is limited on the impact of previous laparoscopic exposure on learning robotic surgery skills for physicians in training. laparoscopic suturing is arguably the hardest task while learning laparoscopic surgery because it requires a mastery of a complex set of maneuvers and skills. although the increased dexterity provided by the robot has decreased the difficulty of suturing, it still represents one the fundamental skills that must be learned by trainees in robotic surgery. the aim of the study was to evaluate if previous experience with laparoscopic surgery among gynecology residents could influence and perhaps shorten the learning curve for robotic suturing. this study was approved by the institutional review board at the university of texas medical branch, galveston tx, usa. a total of 13 residents were recruited for participation in this study : 4 third - year residents (pgy3) and 9 fourth - year residents (pgy4). none of the residents who participated in this study had any previous robotic surgery experience. third - year residents had an additional dry laboratory with hands - on training in suturing prior to their initial experience in the operating room. all but 2 of the pgy4s already had exposure to mis suturing during their previous experience in the operating room, so they received no additional dry - lab teaching. the 2 pgy4s with group 1 consisted of all pgy3s and the 2 pgy4s considered to be mis - naive residents. the more mis - experienced pgy4s, all of whom had at least 3 previous mis experiences, made up group 2. both groups of residents were evaluated using 2 different techniques : robotic suturing with intracorporeal knot tying and laparoscopic suturing using extracorporeal knot tying. in both the laparoscopic and robotic approaches, the vaginal cuff was closed using a 0 polyglactin (vicryl, ethicon) on a ct-1 needle in an interrupted suture closure. the suture time was defined as the elapsed time for loading the needle, placing 4 square knots, and cutting the suture. a t - test compared mean suturing times by surgery modality and stratified by groups of clinicians. the satterthwaite correction was applied when the test for heterogeneity of variances between groups was significant. the mean suturing times of the pgy3 and pgy4 residents are shown in table 1. table 1 demonstrates that pgy3s had a shorter suturing time than their pgy4 counterparts in both robotic and laparoscopic suturing. for the pgy3 group, no statistically significant difference (p = 0.5) was found between the mean suturing time in robotic suturing (235 337 seconds) versus laparoscopic suturing (158 457) (table 1, figure 1). the pgy4 group 's mean suture time was 337 235 seconds for robotic suturing and 457 158 seconds for laparoscopic suturing, a statistically significant difference (p = 0.02) (table 1, figure 1). for all residents, it took on average 382 159 seconds for laparoscopic suturing and 326 196 seconds for robotic suturing, which was not found to be statistically significant (p = 0.12) (figure 2). the mean suturing times between pgy3 and pgy4 for combined robotic and laparoscopic suturing, was also compared (table 2). the difference between the mean suture times in the pgy3 (310 95.8 seconds) versus pgy4 (393 210.6 seconds) groups was found to be statistically significant (p = 0.01) (table 2). the surgical experience of the residents was also taken into account (table 3). residents in group 1 (n = 6) had 2 or fewer previous laparoscopic surgery experiences, and those in group 2 (n = 7) had 3 or more cases (average = 4.4) prior to enrollment in the study. the residents in group 1 had a significantly lower mean suture time than those residents in group 2 for laparoscopic suturing (p = 0.009). the residents in group 2 had a lower mean suture time on the robot compared to group 1 ; however, this difference did not achieve significance (p = 0.5). in the literature, a variety of publications report about surgical education using robotic systems [911 ] and the learning curve for robotic - assisted surgery [1214 ]. however, our study is the first to examine the effect that gynecology residents ' previous laparoscopic experiences have on their initial experiences with robotically assisted suturing in the operating room. the results from our study demonstrate that previous laparoscopy experience improves the learning curve for robotic surgery. as shown in table 1, a statistically significant difference between the means of the laparoscopic suturing time and the robotic suturing time occurred in the pgy4 group, which had more overall laparoscopic experience. the less - experienced pgy3s did well laparoscopically but did not show the same type of time reduction for robotic suturing. however, our findings also indicated the importance of structured teaching in dry labs prior to beginning in the operating room. described the robot as an enabling technology because it may allow those with less - advanced laparoscopic skills to perform minimal access procedures they would otherwise do via an open approach. our results support the idea that physicians in training must first learn the basics in a well - established dry - lab environment prior to relying on robot technology. the primary limitation of our study is that of sample size, as both study groups were composed of a small number of residents, likely affecting the power of the statistical analysis. we stopped enrolling participants in the study when the gynecology mis department implemented the use of a braided suture for vaginal cuff closure. a second limitation of our study involves selection bias with regard to surgical case difficulty assigned to the different resident groups. trials with larger number of trainees and using braided sutures to reflect the trend in closure are needed before coming to a definitive conclusion. in conclusion, introducing the robotically assisted suturing technique to our residents resulted in a steep learning curve regardless of their previous experience in laparoscopy. however, our data suggest that residents with previous laparoscopic - suturing experience will gain more from a robotic - surgery experience than those residents with minimal or no previous laparoscopic - surgery experience. furthermore, initial introduction in a dry - lab setting decreases the learning curve for residency training. as suturing and knot tying are the bases of minimal access surgery, the need for specific training for all surgeons who aim to become competent with the techniques is paramount. in an era of increasing financial concerns and decreasing resident work hours, the need to effectively train residents in a time efficient manner, while keeping patients safe, is also a priority. | objective. to assess the impact of gynecology residents ' previous laparoscopic experience on the learning curve of robotic suturing techniques and the value of initial structured teaching in dry lab prior to surgery. methods. thirteen gynecology residents with no previous robotic surgery experience were divided into group 1, consisting of residents with 2 or fewer laparoscopic experiences, and group 2, consisting of residents with 3 or more laparoscopic experiences. group 1 had a dry - laboratory training in suturing prior to their initial experience in the operating room. results. for all residents, it took on average 382 159 seconds for laparoscopic suturing and 326 196 seconds for robotic suturing (p = 0.12). residents in group 1 had a lower mean suture time than residents in group 2 for laparoscopic suturing (p = 0.009). the residents in group 2, however, had a lower mean suture time on the robot compared to group 1 (p = 0.5). conclusion. residents with previous laparoscopic suturing experience may gain more from a robotic surgery experience than those with limited laparoscopic surgery experience. in addition, dry lab training is more efficient than hands - on training in the initial phase of teaching for both laparoscopic and robotic suturing skills. |
oral and oropharyngeal cancers, including cancers of the lip, alveolar bone, gingiva, buccal mucosa, floor of the mouth, oropharynx, salivary glands, maxillary sinus, and mobile tongue, are the sixth most common type of cancer in the world.1,2 oral cancer accounts for 3% of all cancers in the united states with over 40,000 new cases each year and over 12,000 deaths annually. all the cases of oral cancer, 90% are squamous cell carcinomas (sccs) ; moreover, 95% of these patients are older than 40 years of age, with an average age at diagnosis of 60 years. scc of the tongue is usually observed in males between 40 - 50 years of age.3,4,5 scc of the tongue has an incidence of 40 - 50% in oral and maxillofacial cancers ; moreover, scc of the tongue is associated with various complications such as difficulty of speech, swallowing, and mastication ; poor nutrition ; and a high risk of nerve damage after surgical resection or radiation therapy.2 to date, various risk factors for scc of the tongue have been identified, such as age, tobacco status, alcohol consumption, and human papilloma virus (hpv) infection.6,7,8,9 furthermore, nutritional deficiency and poor oral hygiene with misaligned dentition have been identified as causative factors.10,11,12 the relationship between dental prosthesis and scc of the tongue remains controversial, although it has been proposed that chronic irritation or direct trauma to the oral mucosa resulting from dental prostheses can increase the risk of scc of the tongue. one global epidemiological study, which focused on factors that contribute to scc of the tongue, showed that ill - fitted prosthesis is a primary cause of oral cancer in groups with certain geographical characteristics and socioeconomic statuses.13 therefore, the purpose of this retrospective study was to investigate the relationship between scc of the tongue and dental prosthesis, including positional aspects. a total of 439 patients with scc of the tongue were diagnosed and treated in the department of oral and maxillofacial surgery, seoul national university dental hospital. patients were treated over a 12.5-year period from january 1, 2001 to june 30, 2013 and were managed by four maxillofacial surgeons specializing in oral cancer, along with their reconstruction teams. among the 439 diagnosed patients, 211 satisfied the inclusion criteria for this study. these criteria included a pathologic diagnosis of malignancy in the tongue, the availability of clinical information regarding intraoral prosthesis, and panoramic radiograph data revealing the type and location of the prosthesis. these 211 patients were categorized according to sex, age, position and type of prosthesis, and primary location of the tongue malignancy. this study protocol was fully approved by the institutional review board of seoul national university dental hospital. most patients exhibited advanced stage iii or iv cancer and received a radical, combined - modality treatment regimen consisting of chemotherapy, surgery, and/or radiotherapy. patients ' clinical and demographic information, such as age, sex, tobacco history, previous cancer therapy, specific primary site in the tongue, histological diagnosis, and tumor staging data, including t (tumor size), n (nodal), and m (metastatic) staging, were collected. this history included the frequency and presence of prosthesis, such as fixed or partial dentures. the inclusion criteria for this study were as follows : (1) diagnosed with both clinical and pathological malignancy of the tongue ; (2) available clinical information regarding dentition and previous treatment ; (3) available panoramic radiographs for identifying the types and locations of the prostheses. exclusion criteria included : (1) diagnosed with clinical or pathological malignancy of the tongue ; and (2) insufficient clinical and panoramic information for identifying the type and location of prosthesis. patients were categorized according to the following variables : (1) sex(2) age (0 - 19 years, 20 - 29 years, 30 - 39 years, 40 - 49 years, 50 - 59 years, 60 - 69 years, and older than 70 years)(3) position of crown and/or bridge (fig. 1) group 0 : no crown or bridgegroup 1 : right molar area (from the right first premolar to the right third molar)group 2 : anterior teeth area (from the right canine to the left canine)group 3 : left molar area (from the left first premolar to the left third molar)group 4 : bilateral molar areagroup 5 : right molar and anterior teeth areasgroup 6 : anterior teeth and left molar areasgroup 7 : right molar, anterior teeth and left molar areas (4) position of dentures (maxilla, mandible, or both)(5) location of scc of the tongue [right border (type a), left border (type b), bilateral borders (type c), and other location (type d)14 ] (2) age (0 - 19 years, 20 - 29 years, 30 - 39 years, 40 - 49 years, 50 - 59 years, 60 - 69 years, and older than 70 years) (3) position of crown and/or bridge (fig. 1) group 0 : no crown or bridgegroup 1 : right molar area (from the right first premolar to the right third molar)group 2 : anterior teeth area (from the right canine to the left canine)group 3 : left molar area (from the left first premolar to the left third molar)group 4 : bilateral molar areagroup 5 : right molar and anterior teeth areasgroup 6 : anterior teeth and left molar areasgroup 7 : right molar, anterior teeth and left molar areas group 0 : no crown or bridge group 1 : right molar area (from the right first premolar to the right third molar) group 2 : anterior teeth area (from the right canine to the left canine) group 3 : left molar area (from the left first premolar to the left third molar) group 4 : bilateral molar area group 5 : right molar and anterior teeth areas group 6 : anterior teeth and left molar areas group 7 : right molar, anterior teeth and left molar areas (4) position of dentures (maxilla, mandible, or both) (5) location of scc of the tongue [right border (type a), left border (type b), bilateral borders (type c), and other location (type d)14 ] univariate analyses were performed using the pearson chi - square test, student 's t - test, and one - way analysis of variance in spss for windows, version 12. frequency analysis was conducted to determine the relationships of scc of the tongue with patient sex, age, location, type of prosthesis (crown and/or bridge), and prosthesis position. cross analysis was also conducted to analyze possible relationships between the location of the scc of the tongue and the presence of a crown and/or a bridge, the position of each prosthesis, and the presence of other removable dentures. among the 439 patients initially identified, 62 lacked sufficient clinical information for inclusion, and 166 patients were not diagnosed with a tongue lesion that was both clinically and pathologically malignant ; thus, these 228 patients were excluded. the data from the remaining 211 patients, including their panoramic radiographs, clinical chart records, and pathological diagnoses, were reviewed and compared. of the 211 patients included in the study, 80 (37.9%) were female and 131 (62.1%) were male. the mean patient age was 54.9 years ; the age bracket with the most patients was 50 - 59 years (60 patients, 28.4%), followed by 60 - 69 years (40 patients, 19.0%), 70 years and above (40 patients, 19.0%), 40 - 49 years (33 patients, 15.6%), 30 - 39 years (27 patients, 12.8%), and less than 20 years (11 patients, 5.2%). regarding the location of scc of the tongue, 104 patients (49.3%) had a lesion located on the right lateral border of the tongue, and 93 patients (44.1%) had a lesion on the left lateral border (table 2). the distribution of prostheses included 134 patients (63.5%) with at least one prosthesis and 77 patients (36.5%) without a prosthesis ; this difference was statistically significant (p<.05). fewer patients had a crown (91, 43.1%) than did not have a crown (120, 56.9%) ; similarly, fewer patients had a bridge (99, 46.9%) than did not have a bridge (112, 53.1%). the number of patients with both a crown and a bridge was 56 (41.8%) (table 3). regarding the position of the crown and/or the bridge, group 7 had the most patients (43, 20.4%) followed by group 4 (33, 15.6%), and group 1 (19, 9.0%) ; regarding the location of the crown, group 1 had the most patients (29, 13.7%) followed by group 4 (21, 10.0%) ; regarding the location of the bridge, each group showed similar frequencies except group 7 (31, 14.7%) (table 4). of the patients included in the study, 73 (34.6%) wore dentures ; 47 of those had both upper and lower dentures, a 22.3% overall frequency (table 5). cross analysis was performed to investigate possible relationships between the location of the lesion and the presence of a prosthesis (table 6), the location of the lesion and the position of the crown and/or bridge (table 7), the location of the lesion and the presence of a prosthesis (table 8), and the location of the lesion and the position of dentures (table 9). no significant relationship was observed between the presence of a prosthesis and the location of the lesion (p=.723) ; thus, it is still unclear why patients with a crown and/or bridge have more frequent scc of the tongue compared with patients without prostheses. moreover, no significant association was observed between the presence of a crown and/or bridge and the location of the lesion (p=.230), the presence of a crown alone and the location of the lesion, or the presence of a bridge alone and the location of the lesion (p=.674 and 0.066, respectively). regarding the position of a prosthesis, no significant relationship was observed between the position of a crown and location of the lesion (p=.071) or between the position of a bridge and location of the lesion (p=.716). similarly, the location of the lesion was not significantly associated with the presence of dentures (p=.409), and the position of dentures was not associated with location of the lesion (p=.073). oral scc is commonly observed in the tongue, lip, gingival tissue, palate, and floor of the mouth.15 scc of the buccal mucosa is common among asian populations due to cultural betel quid and tobacco chewing habits ; for instance, 40% of all oral cancers in sri lanka are found on the buccal mucosa.1,9 the tongue is the most common site of oral cancer among european and us populations and accounts for 40 - 50% of all oral cancers.1,15 many contributing factors of scc of the tongue have been identified, and scc of the tongue is believed to be a multifactorial condition.9,16 alcohol and tobacco have been hypothesized to exert their carcinogenic effects via a contact mechanism ; for example, tobacco smoking is more strongly associated with tongue cancer when patients have been heavily exposed to inhaled smoke. on the other hand, alcohol consumption exerts a stronger effect on structures belonging to the food channel and reservoir systems, such as the tongue. this explanation is consistent with studies in animal models that have investigated the effect of ethanol on the mucosal penetration of nitrosonornicotine in the oral mucosa. the detection of dna from hpv subtypes 6 and 16 in exfoliated oral cavity cells has been shown to be strongly associated with an elevated risk for tongue cancer ; moreover, dna - based studies of hpv isolates found in exfoliated oral tissue from case subjects showed that hpv types 16, 18, 31, 33, and 35 were the most common. however, a definitive association between tongue cancer risk and the detection of high - risk hpv types has not yet been proven.1,15,16 this study was conducted to identify contributing factors in tongue cancer. the patients in this study exhibited a similar clinical / demographic distribution, including sex, age, and primary tumor site of the tongue, as has been observed in other studies of oral cancer patients. of the patients in the study, 62.1% were male and 28.4% were female, the mean patient age was 54.9 years, and patients aged 50 - 59 years old were the most prevalent. no suitable studies were available against which to compare the presence and locations of intraoral prostheses. this study did have a few disadvantages ; for example, the duration of the intraoral prosthesis was not included, the study did not examine whether patients received prosthetic treatment by professionals, and the study did not examine marginal adaptation to the prosthesis. additionally, the raw materials of the dental prostheses were not examined in this study. to fully investigate the possible relationships between dental prosthesis and scc of the tongue, more extensive data need to be obtained from patients with scc of the tongue, and prospective studies focusing on the physical properties of these prostheses and their biochemical influences on the tongue should be performed. cross analysis did not reveal any significant association between location of the prosthesis and scc of the tongue. however, the number of patients with a crown and/or a bridge (134, 63.5%) was significantly different from the number of patients without prosthesis (77, 36.5%). these retrospective findings suggest that any prosthetic margin, crown surface, or bridge surface is capable of inflicting mechanical irritation on the tongue, and that these irritations may contribute to the development of scc of the tongue. people who wear dentures are known to have a higher prevalence of oral mucosal lesions compared with people who do not wear dentures, crowns, or bridges ; these oral mucosal lesions can develop into malignancies in the oral cavity. among people who wear dentures, those who wear complete dentures have been shown to have a higher incidence of lesions compared with people who wear partial dentures.17 oral cancer can also be observed in the contact area between the teeth and the prosthesis ; in particular, tongue malignancies of the lateral border have been observed opposite from the flange extension of the lower denture.18 another related study found that metallurgically - flawed gold crowns contribute to scc of the tongue.19 however, these findings are controversial. other studies have found no correlation between denture wearing and oral malignancies,20 denture type and oral mucosal lesions, or the use of dentures and scc of the tongue.21,22 lockhart.18 observed 28 intraoral malignancies in the contact area of either the teeth or prosthetic appliances ; moreover, all 10 patients with scc of the tongue were associated with a flange extension of a lower denture. kinnebrew.19 reviewed a case of a 25-year - old woman with scc of the tongue and concluded that the lesion was physically associated with a metallurgically - flawed gold crown that had been used for 15 years. however, albuquerque.22 concluded that dentures are not an etiologic factor for scc of the tongue, since no definitive association between scc of the tongue and denture use was demonstrated. furthermore, jainkittivong.17 reported that no association was found between pathologic conditions of the intraoral mucous membrane and denture type. physical trauma has been proposed to act as a contributing factor that could determine the location of oral cancer, which may explain a possible relationship between trauma and cancer location. inflammatory reactions in the oral cavity induced by either dental trauma or physical irritation have been considered to be important contributing factors for oral cancer. in support of this hypothesis, inflammatory changes in the thin atrophic mucous membrane have been observed.23 many other possible relationships have been proposed, but these relationships have not yet been confirmed. chronic trauma is frequently found in people who wear dentures, and other pathological conditions associated with the use of dentures such as candida - induced denture stomatitis, denture - related hyperplasia, angular cheilitis, and traumatic ulcers are known to be related to scc of the tongue. unfortunately, this retrospective study did not investigate the presence of premalignant lesions in the patients. however, ulcerations of the tongue caused by chronic trauma resulting from poorly fabricated dentures, fractured restorations, sharp edges on worn teeth, and ill - fitting crowns and/or bridges were confirmed in most cases. the tongue mucosa is thought to be more permeable to noxious substances and therefore more vulnerable to external carcinogens. moreover, the mucosal epithelium of the tongue becomes thinner with age, and the rate of collagen synthesis by connective tissue also decreases. regarding trauma, chronic irritations caused by ill - fitted dentures, fractured restorations, and other erosive factors can alter the tongue mucosa. together with other factors such as alcohol and tobacco, these factors may lead to the development of oral cancer. some studies have reported that scc in the anterior two - thirds of the tongue is often accompanied by local traumatic irritation. clearly, many people with years of chronic mucosal irritation from a dental or prosthetic source do not develop cancer. however, our study suggests that, of the people who do develop cancer, a high percentage of these cancers will arise in areas that are in direct contact with teeth and appliances. neither this study investigate the quality of the prostheses such as marginal discrepancy of crown, the duration of prostheses, or quality of occlusion, and no significant differences were observed between the position of the prosthesis and the location of the scc of the tongue. these results may only support the hypothesis that mechanical trauma is the cause of scc of the tongue. one of the possible precancerous conditions for scc is leukoplakia.24 earlier studies have indicated that oral galvanism is a contributing factor for oral leukoplakia, and that the removal of the metal prosthesis resulted in the resolution of the leukiplakia.25 oral galvanism can be generated by the presence of more than two adjacent metal prostheses, which can introduce current flow.26 the intraoral electrical phenomenon of galvanism could increase the proliferation of leukoplakia cells, induce apoptosis, and simulate some morphological features of scc. this galvanic current, which affects ornithine decarboxylase, is upregulated in many cancers, which is important because the nak - atpase acts as an ion transporter.25 electrical actions from prosthetic metal materials of crowns and/or bridges should be considered an important etiologic factor of scc of the tongue. although the currents generated are minute, they can form a type of weak battery. over time, this electrical battery can result in irritating injury to the tongue mucosa and the surrounding muscles. galvanic current is a type of miniature battery formed by electric circuits resulting from the presence of dissimilar metals in the mouth. prosthetic metals, such as gold, silver, copper, and mercury, show different electrical potentials in the aqueous saliva of the oral cavity. thus, if two dissimilar prostheses are placed in the mouth, a simple galvanic battery, called an electromotive series, will be formed.26 as the tongue moves, the positions of the two metals (one above and the other below, one on the left and the other on the right, or one in an anterior location and the other in a posterior location) constitute a new electrolyte, resulting in peculiar taste perceptions. if these metals are connected externally, electric current flows from one metal to another by means of an external conductor circuit. the mobile tongue mucosa is a good conductor, and saliva can be considered a solution composed of several electrolytes. therefore, prosthetic metal, when it is bathed in saliva and placed near a tongue conductor, forms a simple galvanic battery in the oral cavity. this electrical action of prosthetic metals has been proposed to be one of the main contributing factors to scc of the tongue. three different aspects - galvanometer measurement, local action, and polarization - have been proposed as mechanisms through which the electrical actions contribute to scc of the tongue.26 firstly, galvanometer measurements of metals in situ have been shown to have a relationship with some accompanying pathologic changes. an oral cavity with more than one metallic restoration can conduct currents ranging as high as 80 microamperes, with the exception of an oral cavity containing only fillings of 24-carat gold. the value of the current dropped within five seconds to a steady minimum, which was approximately 10% of the initial value, when the contactors were held in place for an appreciable length of time. both high and low values were obtained in oral cavities with pathologic lesions ; these values were not significantly different from those obtained in mouths with no untoward symptoms. subsurface porosity in the vicinity of the discoloration, accompanied by a thin, silvery surface layer of gold, was found in the metallurgic study. peaks of copper, gold, silver, and palladium were also examined in the interior and on the surface of a crown. the elements identified on the discolored surface of the crown were copper, silicon, chlorine, silver, and gold. however, much more silver and much less gold were identified on the discolored surface compared with the interior of the crown. the discolored surface of the crown was found to be high in silver solder (agcu) or to contain gold solder with a high silver content. the resultant corrosion products of silver, revealed by the chlorine and silver peaks, suggest that electrogalvanism had occurred. the second relevant finding is that metal alloys have local actions and induce pathological changes. when these crystals coexist on the surface of a restoration, potential differences will exist between the crystals and the saliva, thereby constituting a miniature galvanic cell. in this instance, the external circuit is the restoration itself, and the internal circuit is completed by the saliva and related soft tissues. any white mucosal lesions, such as leukoplakia, can be produced by this local action effect. similar to the manner in which nickel dermatitis can be caused by wrist watches and spectacle frames, the nickel found in white gold alloys has an electrolytic battery action when it is in contact with dissimilar metals and an acid or an alkaline fluid. when an electrolyte perspirates and acts upon the base, metal salts are formed. polarization is present in the oral cavity immediately after restorations are inserted and is manifested by a reduction in the normal current. this polarization phenomenon is also known to cause a metallic taste, which is noticeable immediately after the insertion of a restoration and disappears shortly afterward. the presence of continuous or intermittent depolarizing agents can permit current to flow, resulting in damage to the tongue mucosa. these lesions can be traced to electrical causes ; however, galvanometer readings bear no relation to the normal current flow in these cases. moreover, current does not normally flow between restorations ; this observation is consistent with the resistance of metals to wear. electrical energy is produced at the expense of one of the electrodes in an ordinary battery. if currents on the order of those of a normal battery were flowing between restorations, some of the restorations would be eroded over the course of several years.25,26 among the present patients with scc of the tongue, 63.5% more had a crown or a bridge compared with the number of patients without prosthesis. we investigated the cause of this difference but did not find any significant association between these prostheses and location of the lesion (p=.723). however, the precise relationship between dental prostheses and scc of the tongue remains controversial. therefore, we could not conclude that prostheses and galvanic phenomena play a major role in the etiology of scc of the tongue. also, the biochemical effects of oral prostheses and their contributions to scc of the tongue need to be addressed in future studies. finally, early diagnosis remains a key element for adequate therapy of oral scc, including scc of the tongue. clinicians should be aware that single ulcers, tumors, red plaques, or white plaques, particularly if any of these persist for more than two weeks, may be manifestations of malignancy. in these cases, a prompt biopsy of the suspicious lesion should be performed.27 moreover, the entire oral cavity, including the tongue, should be routinely examined in all patients with any prostheses. this clinical retrospective study included a statistical analysis of 211 patients with scc of the tongue and investigated whether scc of the tongue is associated with the positional aspects of dental prostheses. the data below strongly support the hypothesis that mechanical trauma and the galvanic phenomenon play a role in the etiology of scc of the tongue. male patients showed a higher incidence of scc of the tongue compared with female patients ; patients with scc of the tongue were most likely to be 50 - 59 years of age. no relationship between location of the dental prosthesis and the presence of scc of the tongue was observed. no significant associations between the position of a prosthesis and location of scc of the tongue were observed. more patients with scc of the tongue had a crown and/or a bridge than did not have a prosthesis. physical trauma from ill - fitted prostheses is expected to be a main causative factor of scc of the tongue. the galvanic phenomenon arising from dissimilar prosthetic metals is also expected to be another factor contributing to scc of the tongue ; this phenomenon is composed of pathologic changes, local actions, and the polarization effect. | purposesquamous cell carcinoma (scc) of the tongue has a relatively high incidence of all oral cancers. some studies have reported a relationship between intraoral dental prosthesis and scc of the tongue ; however, this relationship remains controversial. the purpose of this study was to investigate the relationship between scc of the tongue and the positional aspects of dental prosthesis using a retrospective analysis.materials and methodsa total of 439 patients with scc of the tongue were diagnosed and treated in the department of oral and maxillofacial surgery, seoul national university dental hospital. patients were treated over a 12.5-year period ranging from january 1, 2001 to june 30, 2013. statistical analysis was performed to examine potential differences between the groups.resultsthe number of patients with a crown and/or a bridge (134, 63.5%) was significantly different than the number of patients without a prosthesis (77, 36.5%). even after accounting for different types of prostheses such as crowns, bridges, and dentures, no significant differences were observed between the position of the prosthesis and the location of the scc of the tongue, with significance defined as a p - value less than.05 by the pearson - chi square test.conclusionpatients with crowns and/or bridges exhibited more frequent scc of the tongue compared with patients without these prosthesis. these data support the hypothesis that mechanical trauma and galvanic phenomena play a role in the etiology of scc of the tongue. |
the dynamic kinetic resolution of -halo -keto esters via an asymmetric cross - benzoin reaction is described. a chiral n - heterocyclic carbene catalyzes the umpolung addition of aldehydes to racemic -keto esters. the resulting fully substituted -halo glycolic ester products are obtained with high levels of enantio- and diastereocontrol. the high chemoselectivity observed is a result of greater electrophilicity of the -keto ester toward the breslow intermediate. the reaction products are shown to undergo highly diastereoselective substrate - controlled reduction to give highly functionalized stereotriads. |
|
, the hemoglobin mainly distributes in the red blood cell (kundu 2003 ; weber and fago 2004). structurally, each molecule of hemoglobin contains four globin chains and heme (kundu 2003 ; weber and fago 2004). the four globin chains (two alpha chains and two beta chains) are depicted as a tetramer looking like folded worms (kundu 2003 ; weber and fago 2004). in the case of structurally abnormal hemoglobins, the following mechanisms can be possible : single nucleotide base substitutions leading to amino acid replacement or chain termination variants ; nucleotide deletions or additions leading to deletion and frameshift variants ; and nonhomologous crossing over, leading to the production of fused globin chains (forget 1979). the molecular basis of the hemoglobinopathies, disorders characterized by absent or decreased synthesis of alpha- or beta - globin chains, is quite heterogeneous (forget 1979). luckily, the new development in molecular bioinformatics can be applied in nanoscale genomics and proteomics research. here, the author used a new gene ontology technology to predict the molecular function and biological process of four important hemoglobin disorders with single substitution. the database, unitprot (bairoch 2008), was used for data mining of the amino acid sequence for normal hemoglobin. the author performed a prediction of molecular function and biological process of normal and each abnormal hemoglobin using a novel gene ontology prediction tool namely gofigure by department of computer and information sciences, university of delaware, newark, usa (khan 2008). gofigure is a computational algorithm tool which is recently developed for gene ontology study (khan 2008). the tool accepts an input dna or protein sequence, and uses the basic local alignment search tool (blast) to identify homologous sequences in gene ontology annotated databases (khan 2008). the purpose is to use a blast search to identify homologs in public databases that have been annotated with gene ontology terms (khan 2008). these terms include : swissprot, flybase (drosophila), the saccharomyces genome database (sgd), mouse genome informatics (mgi), and wormbase (nematode) (khan 2008). the contents of the results include molecular function as well as biological process of the studied protein (khan 2008). the database, unitprot (bairoch 2008), was used for data mining of the amino acid sequence for normal hemoglobin. the author performed a prediction of molecular function and biological process of normal and each abnormal hemoglobin using a novel gene ontology prediction tool namely gofigure by department of computer and information sciences, university of delaware, newark, usa (khan 2008). gofigure is a computational algorithm tool which is recently developed for gene ontology study (khan 2008). the tool accepts an input dna or protein sequence, and uses the basic local alignment search tool (blast) to identify homologous sequences in gene ontology annotated databases (khan 2008). the purpose is to use a blast search to identify homologs in public databases that have been annotated with gene ontology terms (khan 2008). these terms include : swissprot, flybase (drosophila), the saccharomyces genome database (sgd), mouse genome informatics (mgi), and wormbase (nematode) (khan 2008). the contents of the results include molecular function as well as biological process of the studied protein (khan 2008). the four studied important abnormal hemoglobins with single substitution included hb s, hb e, hb c, and hb j - baltimore. summary of the mutation of each abnormal hemoglobin using gofigure server, the molecular function and biological process in normal and abnormal hemoglobin were predicted. the molecular function and biological processes of normal hemoglobin and abnormal hemoglobin are similar, as presented in figure 1. the summary of the comparison of the molecular function and biological processes between normal and abnormal hemoglobin is presented in table 2. the four studied important abnormal hemoglobins with single substitution included hb s, hb e, hb c, and hb j - baltimore. using gofigure server, the molecular function and biological process in normal and abnormal hemoglobin were predicted. the molecular function and biological processes of normal hemoglobin and abnormal hemoglobin are similar, as presented in figure 1. the summary of the comparison of the molecular function and biological processes between normal and abnormal hemoglobin is presented in table 2. hemoglobin research reached a renaissance in recent few years due to the discovery of globins or their genes in all living organisms and in all tissues of higher animals (kundu 2003 ; weber and fago 2004). new developments brought to a re - evaluation of our understanding of the structure and function of hemoglobins (kundu 2003 ; weber and fago 2004). until now, the functional aberration due to the mutation in hemoglobin was not well demonstrated, and there is a need for improvement of our understanding on those mutated proteins function. based on the recent advances in the genomics, current technologies can permit the examination of gene expression patterns of tens of thousands of genes (bairoch 2008). a challenge for the biologist to interprete such data is recognizing the function of many of the hits identified in a single experiment (khan 2008). while one can classically search the literature, a more rapid mean of developing some idea of potential function of a gene product is via the ontology terms that describe the gene (khan 2008). many genes ontology tools have been constructed and launched for public usage. in this study, the author used a gene ontology tool to predict the function of normal and four important hemoglobin disorders with underlying single amino acid substitution. compared with normal hemoglobin, all studied abnormal hemoglobins have the same function and biological process. this indicates that the overall function of oxygen transportation is not disturbed in the studied hemoglobin disorders. this result is similar to another report by wiwanitkit (2006) on four rare hemoglobinopathies, hb agrino (29leu-->pro), hb siam (15gly - > arg), hb amsterdam (32met-->ile) and hb evanston (14 try-- > arg). clinical findings of oxygen depletion in abnormal hemoglobins should be due to the other processes rather than genomics, proteomics and expression levels. | hemoglobin is an important protein found in the red cells of many animals. in humans, the hemoglobin is mainly distributed in the red blood cell. single amino acid substitution is the main pathogenesis of most hemoglobin disorders. here, the author used a new gene ontology technology to predict the molecular function and biological process of four important hemoglobin disorders with single substitution. the four studied important abnormal hemoglobins (hb) with single substitution included hb s, hb e, hb c, and hb j - baltimore. using the gofigure server, the molecular function and biological process in normal and abnormal hemoglobins was predicted. compared with normal hemoglobin, all studied abnormal hemoglobins had the same function and biological process. this indicated that the overall function of oxygen transportation is not disturbed in the studied hemoglobin disorders. clinical findings of oxygen depletion in abnormal hemoglobin should therefore be due to the other processes rather than genomics, proteomics, and expression levels. |
erythrovirus (formally parvovirus) b19 (b19) causes a wide range of diseases in humans. b19 is a currently the only accepted member of the erythrovirus genus and the only erythrovirus known to be pathogenic in humans. several workers reported cases of children with fulminant or acute hepatitis with acute b19 infection and suggested that b19 could be the cause of the hepatitis 1 - 3. their diagnosis was based on the detection of the serum b19 dna by pcr assay. however, the cause of fulminant hepatitis and biliary atresia in many patients is unexplained and this is a serious issue, especially in newborns and infants. more than 80% of these cases ultimately require liver transplantation or die of hepatic failure. b19 has been proposed as the cause of the fulminant hepatic failure in patients with or without aplastic anemia, on the basis of the presence of b19 dna in liver specimens. an immunologically - mediated mechanism of b19-induced hepatocyte destruction has been postulated, but not documented. thus, there could be an unsuspected cofactor that is associated with b19 infection and is deleterious to the liver. to address these important issues, we conducted a molecular characterization of b19 genomes isolated from patients with fulminant hepatitis and biliary atresia. patients : we compared 2 different groups consisted with 47 japanese patients (aged from newborns to 66 years old ; 24 males and 23 females). group a consisted of 28 patients with fulminant hepatitis (14 males and 14 females ; 9 infants less than one year old, 8 children 1 - 10 years old, 3 adolescents 11 - 20 years old and 8 adults over 21 years old). on the other hand, group b consisted of 19 patients with biliary atresia ; 7 males and 12 females ; 4 infants less than one year old, 10 children 1 - 10 years old, 4 adolescents 11 - 20 years old and one adult over 21 years old). all patients examined had no evidence of hepatitis virus infections and diagnosed as non - b, non - c hepatitis. most of the patients tested underwent living - donor liver transplantation at kyoto university hospital from 1994 to 2000. majority of patients had history of blood transfusion or blood product infusion in their course of treatment. the liver tissues and serum samples from recipients obtained at operation stored at -80c until used. informed consent for participation in this study was obtained from each individual. nucleic acid extraction, pcr and sequencing analysis : nucleic acids (dna / rna) were extracted from frozen liver tissues and serum using sepagene rv - r kit (sanko - junyaku, tokyo, japan). the resulting pellet was resuspended in 100 l rnase - free water, following the manufacturer 's instruction. b19 dna was screened by nested pcr using primers designed from vp1 region ; pv1 (sense, 5'-gct gtt aag gat gtt aca ga-3 ', nucleotide (nt) 3520 - 3521) and pv1r (antisense, 5'-gga tcc gta taa ggg att gt-3 ', nt 3882 - 3901) for the 1st pcr and pv2 (sense, 5'-cag gtt act gac agc act ac-3 ', nt 3541 - 3560) and pv2r (antisense, 5'-tgt tga ctg cag ccc tct aa-3 ', nt the sensitivity of this pcr assay allowed detection of as few as 10 copies of b19 genome. in addition, we sequenced the b19 isolates consisted of 4561 bp covering ns1, vp1 and vp2 regions obtained from liver tissues of patients. for this purpose, three overlapping pcr products (fragments a to c) were generated ; for fragment a (1944 bp), pv3 (sense, 5'-ttt ccc gcc tta tgc aaa tgg gca g-3 ', nt 393 - 417) and pv5r (antisense, 5'-agc tcc cac atg gca gct ac-3 ', nt 2533 - 2552) for the 1st pcr and pv4 (sense, 5'-tgt aac ggt taa aat ggg cgg agc g-3 ', nt 457 - 481) and pv6r (antisense, 5'- ccc ctt aca ccg tcc cac ac-3 ', nt 2382 - 2401) for the 2nd pcr ; for fragment b (1835 bp), pv5 (sense, 5'-gca gca gtg gtg gtg aaa gc-3 ', nt 2143 - 2162) and pv1r for the 1st pcr and pv6 (sense, 5'-ggc gcc tgg aac act gaa ac-3 ', nt 2208 - 2227) and pv2r for the 2nd pcr ; for fragment c (1297 bp), pv1 and pv3r (sense, 5'-tac agt ctg ggt ggt act ggt ggg c-3 ', nt 5010 - 5034) for the 1st pcr and pv2 and pv4r (antisense, 5'-ctg gtg ggc gtt tag tta cgc atc c-3 ', nt 4994 - 5018). the pcr was done with amplitaq gold (perkin elmer, norwalk, conn.) and blend - taq - plus dna polymerase (toyobo co., tokyo, japan). the amplicons were separated by 1% agarose gel electrophoresis and purified using the qiaquick gel extraction kit (qiagen inc., chatsworth, calif.). recovered amplicons were subjected to direct sequencing from both directions using the abi prismtm big dye terminator cycle sequencing ready reaction kit (perkin elmer). sequences of amplified dna were determined using a sequencer (abi model 373a ; applied biosystems, foster city, calif.). additionally, hepatitis a, b, c and e genomes were also determined by the nested pcr. detection of b19 mrna : the purified rna obtained from liver tissues as described above, was digested by rq i dnase (promega co., madison, wis.) to remove any dna. b19 cdna was synthesized from the pretreated rna by reaction with 100 units of moloney murine leukemia virus reverse transcriptase (promega co.) and pv1r antisense primer. phylogenetic analysis : characterization of b19 isolates was examined by the phylogenetic analysis as described previously 4. the data were analyzed using a standard statistical software package (stat view ; brain power inc., calabases, ca, u.s.a.). statistic analyses : statistic analyses were performed by the chi - square test. a difference with a p value of less than 0.05 b19 dna in liver tissues was detected in 10 of 28 (35.7%) patients with fulminant hepatitis and 7 of 19 (36.8%) patients with ba, respectively (statistically not significant). except for two infants with fulminant hepatitis who were hbv dna - positive, all cases tested were negative for hav rna, hcv rna and hev rna. importantly, among the hepatic b19 dna - positive 8 patients who had paired samples of liver tissues and serum, the serum b19 sequence was detectable in only one case (table 1). to determine the form of viral replication, the presence of b19 mrna in the liver tissue was examined. the results showed that b19 mrna was present in all cases with fulminant hepatitis to be hepatic b19-positive, but only 1 of 7 (14.3%) cases in biliary atresia tested. amplification of b19 dna did not take place in the absence of reverse transcriptase, indicating minimal dna contamination. in addition to the above - described examinations, to characterize the b19 genomes, we obtained 14 isolates of the b19 genome (10 isolates with nearly full - length sequences (4561 bp) that covered the complete genes of ns1, vp1 and vp2 and 4 isolates with only one short sequence in the ns1). all b19 isolates showed an overall identity of 98% at the nt level among each isolate with 4561 bp recovered in this study and to prototypes of b19 hv (f162273) and b19 au (m13178), and an identity of 87% to the b19 v9 isolate (ay083234). interestingly, phylogenetic analysis based on the ns1 gene revealed three different clusters : two (genotypes 1 and 3) for isolates from fulminant hepatitis and the other (genotype 2) for isolates from biliary atresia (fig., specific sites of the deduced amino acid sequence change were identified in the ns1 gene (fig. the amino acid at position 181 and 201 showed methionine and aspartic acid, respectively. furthermore, in 5 out of 6 isolates belonging to group 3, the amino acid at position 183 substituted to alanine. nucleotide sequence data reported are available in the ddbj / genbank / embl database under the accession numbers : ab126262 for b19-an23, ab126263 for b19-an28, ab126264 for b19-an30, ab126265 for b19-an34, ab126266 for b19-an40, ab126267 for b19-an41, ab126268 for b19-an56, ab126269 for b19-an66, ab126270 for b19-an85 and ab126271 for b19-an87. the etiology of hepatitis remains obscure in 3 - 10% of cases in europe 5 and the united states 6 and up to 30% of cases in asia 7, 8. particularly, the cause of fulminant hepatitis in children is unexplained in up to 50% of cases in our hospital. once well - known hepatotropic agents and metabolic, toxic, and immunological causes have been excluded, an infectious origin remains a possibility. on the other hand, the concept that biliary atresia consists of two major types based on the clinical aspects has been accepted 9, 10. one is the fetal, embryonic or prenatal type, and the other is the acquired or perinatal type. the perinatal type is believed to account for 70 - 80% of all biliary atresia. the first is called the ductal plate malformation theory, which assumes a congenital anomaly of the intrahepatic bile trees. according to the other theory, the progressive occlusion of the extrahepatic bile duct is probably triggered by viral infection and worsened by the subsequent immunopathological process. reovirus, rotavirus 11, 12, cytomegalovirus 13, 14 and others have been proposed as likely causative viruses, but no clear relation has been found. to the best of our knowledge, there are only a few reports 1, 15, 16 on the analysis of viral infections based on direct detection of viral genomes from liver tissues. b19 was discovered serendipitously in the sera of normal blood bank donors while screening for the hepatitis b virus. b19, a member of the family parvoviridae, subfamily parvovirinae, genus erythrovirus, is a small single stranded dna virus. this virus is responsible for various clinical manifestations including erythema infectiosum in children, polyarthropathy syndrome in adults, transient aplastic crisis inpatients with chronic anemia, persistent infections manifesting as chronic anemia in immunocompromised patients, transplant recipients, and hydrops fetalis and fetal death by intrauterine infection. aplastic anemia, which occurs in 33% of children and 5% of adult patients who undergo orthotopic liver transplantation (olt) for non - a, non - b, non - c fulminant hepatitis is pointed out as a complication with a high rate of mortality 17. the cause of aplastic anemia after olt may include factors such as posthepatitic aplasia, b19, and drugs. 1 reported the possibility of b19 as the cause of non - a - c fulminant hepatitis - associated aplastic anemia. actually, b19 may be an etiologic agent for hepatitis - associated aplastic anemia, because of the known tropism of b19 for erythroid precursors 18. 19 reported no evidence for hepatitis e or parvovirus b19 infection in patients with acute liver failure. to investigate the possibility that b19 infection of the liver might cause fulminant hepatitis and biliary atresia, we conducted a study to obtain direct evidence of a viral sequence in the liver from patients who underwent liver transplantation. in this retrospective study of 47 patients with liver diseases of unknown etiology, we postulated that the virus might even be detected in liver that is serologically negative for the virus. in fact, our results showed that only one case was sero - positive for the b19 sequence among 8 cases with intrahepatic - b19 dna. these results suggest that seronegativity for the b19 sequence does not exclude the existence of present infection of b19 within the liver. they showed b19 dna is frequently present in livers of anti - b19 seropositive adults suggesting persistence of b19 in the liver. our results also indicate that direct detection of viral genomes in tissues by a highly sensitive method is as important as serology. in the present study, we found a high prevalence of b19 infection not only in fulminant hepatitis patients, but also in biliary atresia patients. however, all fulminant hepatitis patients infected with b19 had the replicative form of b19 mrna. while, low rate of b19 mrna detection in biliary atresia patients who had intra - hepatic b19 dna suggested silent infection of b19. this suggests that b19 may be a cause of severe hepatic diseases although this remains to be investigated. b19 encodes three major viral proteins : vp1 and np2, the viral capsid proteins, and ns1, a nonstructural protein. both vp1 and vp2 are derived from overlapping reading frames and share substantial amino acid sequences. ns1 is known to be implicated in viral replication, the activation of viral gene transcription, and target cell cytotoxicity 21 - 25. among the sequence records deposited in the database, only 16 isolates of b19 have been sequenced in the full or nearly full - genome so far. here we reported that the nearly full - length sequence of b19 was isolated from 10 patients with liver diseases. it is known that b19 has a genome length of 5.4 kb with hairpin structures at each extremity ; in addition, a partial deletion and rearrangement of the sequence exist in the extremity regions. these findings indicate the difficulty in amplifying the sequences in these regions. in our study interestingly, phylogenetic analysis based on the ns1 gene revealed three different clusters : two for isolates from fulminant hepatitis and the other for isolates from biliary atresia cases. viral genotype has diagnostic and clinical implications, including use for assessing responses to anti - viral therapy and future vaccine development. the possibility that such variation is involved in the severity of associated liver disease, and that the difference between symptomatic and asymptomatic infection, may also occur as well as hbv and hcv. furthermore, among each genotype, specific sites of the deduced amino acid sequence change were identified in the ns1 gene. clarification of the relation between the genotypes or variants of b19 and its pathogenicity in hepatic failure including hepatitis and biliary atresia is awaited with great interest. to solve these important issues, a geographical study of the b19 genomes and various diseases including liver diseases in different countries is now planning and we shall report on further investigations in the future. in conclusion, we found a high prevalence of b19 infection in livers from patients with fulminant hepatitis and biliary atresia. interestingly, all fulminant hepatitis patients infected with b19 had the replicative form of b19 mrna. it is noteworthy that serum b19 dna was not detected in 7 of 8 cases that were hepatic b19 dna - positive. our results presented here suggested that b19 may serve as an etiologic agent for severe hepatitis. phylogram generated by neighbor - joining analysis of genetic distances in the ns1 region of erythrovirus b19. alignment of amino acid sequences (upper) and nucleic acid sequences (lower) in the ns1 region of erythrovirus b19. | background : fulminant hepatitis and biliary atresia are serious problems and their causes have not been explained well. we investigated whether or not erythrovirus b19 is a candidate etiologic agent in such liver disease patients who had undergone liver transplantation.methods : liver tissues from 47 patients consisted of 28 fulminant hepatitis and 19 biliary atresia were examined to detect b19 genes by pcr and further analyzed their genomic characterization.results : b19 dna was detected by nested pcr in 10 of 28 cases (35.7%) livers in the fulminant hepatitis group and 7 of 19 (36.8%) livers in the biliary atresia group, respectively (statistically not significant). importantly, among the 8 hepatic b19 dna - positive patients who had paired samples of liver and serum, the serum b19 genome was detectable in only one case. b19 mrna was identified in all of 10 fulminant hepatitis cases with hepatic b19 dna, but only 1 out of 7 (14.3%) cases in biliary atresia tested. furthermore, we obtained ten isolates having the b19 genome with nearly full - length sequences. interestingly, phylogenetic analysis based on the ns1 gene revealed three different clusters : two for isolates from fulminant hepatitis and the other for isolates from biliary atresia.conclusions : our results presented here suggested that b19 may be an etiologic agent of fulminant hepatitis. |
the collaborative cross (cc) is a mouse genetic reference population conceived 10 years ago as a community resource for systems genetics (threadgill and churchill 2012 ; threadgill. it is a panel of recombinant inbred (ri) lines generated by randomly mixing the genetic diversity of eight extant inbred mouse lines : a / j, c57bl/6j, 129s1/svimj, nod / shiltj, nzo / h1ltj, cast / eij, pwk / phj, and wsb / eij. the genetic architecture of the cc population has been recently reported (collaborative cross consortium 2012). the cc was started with mice from the jackson laboratory in three separate locations : the oak ridge national laboratory in tennessee (chesler. 2008), whose population was moved to the university of north carolina at chapel hill ; the international research livestock institute in kenya, whose population was moved to tel aviv university in israel (iraqi. 2008) ; and western australian institute for medical research / university of western australia / geniad, ltd in perth (morahan. all cc lines were independently bred in a similar funnel breeding scheme that combined the genetic variation present in the eight founders into cc lines over three generations followed by inbreeding to homozygosity (churchill. an increasing number of reports have established the value of the cc to provide insights in the genetic architecture of multiple traits, the identification of novel loci associated with them, and the characterization of functional novel alleles at known genes (aylor. 2011 ; bottomly. 2012 ; durrant. 2011 ; kelada. 2012 ; mathes. 2010 the cc population will be maintained and distributed by distribution centers that reflect the needs of the research community and of the institutions involved in the generation of the cross. currently, independent sets of cc lines are being generated at chapel hill in the us (cc - unc), tel aviv in israel (cc - tau), and perth in australia (cc - gnd) (collaborative cross consortium 2012). eventually, distribution centers will have stocks of each cc line from the three production centers. however, as of the writing of this report, only the chapel hill site has breeders from all three populations (cc - unc, cc - tau, and cc - gnd). there are about 130 cc lines (cc - tau), between the 13th and 29th generations of inbreeding that are under development at tel aviv university and maintained in a conventional mouse facility. there are about 180 lines under development up to the 26th generation of inbreeding in australia. these lines will be available for the research community as they reach the milestones for inbreeding discussed below. cc - tau and cc - gnd lines are under rederivation to specific - pathogen - free (spf) condition at unc. the rederived cc - tau and cc - unc lines will be shipped to tau for maintenance in their spf facility. this current state reflects the fact that unc is equipped and funded to use dense genotyping to accelerate inbreeding through marker - assisted inbreeding (mai) in order to expedite access to all cc lines (collaborative cross consortium 2012 ; welsh and mcmillan 2012). the following sections provide a brief overview of the unc systems genetics core (http://csbio.unc.edu/ccstatus/index.py), using a structure that each distribution center has agreed upon to standardize operating procedures to ensure long - term genetic integrity of the cc population. given that tau has recently become a european mouse mutant archive (emma) node and is therefore fully equipped to manage the colony, it is expected that it will be a key part in the distribution of cc in europe. cc - gnd strains have been shipped to unc and it is envisaged that geniad will manage distribution in australia and asia. distribution centers will provide cc mice on a cost recovery basis such that the price per mouse should be similar to those of suppliers of genetically defined stocks (e.g., the jackson laboratory). historically, ri lines have been deemed inbred after 20 generations of brother - sister mating, and lines within a given panel have been made available as they reached this milestone. the development of genotyping arrays for the mouse (collaborative cross consortium 2012 ; yang. 2009) has made it possible to substitute this arbitrary threshold with an objective genetic criteria, namely, the level of residual heterozygosity in a given line. after consultation with the external advisory board of the unc cc effort (collaborative cross consortium 2012), and after discussion with investigators leading the cc - tau and cc - gnd breeding programs, we decided that lines that reach specific levels of inbreeding should be made available to the public : lines are declared complete once they have reached 98 % homozygosity.lines are declared distributable once they have reached 90 % homozygosity. the first threshold is informed by the observation that many commonly used inbred lines have similar levels of residual heterozygosity (yang. the need for the second more relaxed criterion reflects our goal to accelerate access by all investigators to the cc resource. we also expect that even incipient inbred lines will be a powerful genetic resource. additionally, it addresses the fact that some lines become increasingly difficult to maintain with terminal inbreeding and thus may be lost. researchers need to carefully evaluate whether completed and/or distributable cc lines meet the needs of their experimental design. however, we wish to note that for decades geneticists have used resources with similar shortcomings and that in contrast with these historical resources, users of cc lines will know, with an unprecedented level of detail, which regions are not fixed and what alleles are segregating in these regions (see below for discussion on genotypes). measuring heterozygosity in cc lines requires dense genotyping of key mice in each line using a cost - effective genotyping platform coupled with sophisticated methods for haplotype reconstruction. currently we are using the mouse universal genotyping array (muga) (collaborative cross consortium 2012) developed at unc to guide mai in the cc population. the array has 7,500 informative snps and has been critical for projects using the cc and do populations (collaborative cross consortium 2012 ; svenson. a key step in determining whether a line has reached distributable or completed status is the identification of obligate ancestors in the line of all extant mice or subsets of extant mice with limited heterozygosity (fig. 1). 2008) to generate pedigree reports using the cranefoot program (http://www.finndiane.fi/software/cranefoot/) (mkinen. 2005). cranefoot displays allow easy viewing of each cc line at any generation, showing branches or arms in the pedigree (fig. in addition, a custom python script, mrca.py, determines obligate ancestors for any subset of the current generation of mice.fig. 1 a partial view of the pedigree of the or3252 cc line mice that are present multiple times (because they participate in multiple matings) are linked by blue curved lines. mice shown at the top of the pedigree with arrowheads are the obligate ancestors of this line used to determine whether it passes the threshold for distribution (most recent obligate ancestors). we use standard colors and a single - letter code to represent the contribution of the eight cc parental strains (collaborative cross consortium 2012) to the genome of the two most recent obligate ancestors. briefly, a / j, a, yellow ; c57bl/6j, b, gray ; 129s1/svimj, c, pink ; nod / shiltj, d, dark blue ; nzo / h1ltj, e, light blue ; cast / eij, f, green ; pwk / phj, g, red ; and wsb / eij, h, purple. the two autosomes and the corresponding complement of x chromosomes for each mouse are drawn to illustrate the regions that are fixed (all four autosomes or three x chromosomes have the same haplotype) or segregating (shown in boxes). c the genome of the or3252 line. the figure represents fixed regions as single lines and segregating regions as double lines of the appropriate colors a partial view of the pedigree of the or3252 cc line. mice that are present multiple times (because they participate in multiple matings) are linked by blue curved lines. mice shown at the top of the pedigree with arrowheads are the obligate ancestors of this line used to determine whether it passes the threshold for distribution (most recent obligate ancestors). we use standard colors and a single - letter code to represent the contribution of the eight cc parental strains (collaborative cross consortium 2012) to the genome of the two most recent obligate ancestors. briefly, a / j, a, yellow ; c57bl/6j, b, gray ; 129s1/svimj, c, pink ; nod / shiltj, d, dark blue ; nzo / h1ltj, e, light blue ; cast / eij, f, green ; pwk / phj, g, red ; and wsb / eij, h, purple. the two autosomes and the corresponding complement of x chromosomes for each mouse are drawn to illustrate the regions that are fixed (all four autosomes or three x chromosomes have the same haplotype) or segregating (shown in boxes). the figure represents fixed regions as single lines and segregating regions as double lines of the appropriate colors genotypes from muga are used for haplotype reconstruction as described previously (collaborative cross consortium 2012). the haplotype reconstructions of the obligate ancestors are jointly considered to determine the maximum heterozygosity of a distributable line (fig. calculating the joint heterozygosity involves two steps : establishing recombination breakpoints and determining segregating regions within and between the obligate ancestors (fig. 2). recombination breakpoints are estimated by the midpoint of any ambiguous region found by the haplotype reconstruction (these tend to be no more than 23 snps). ambiguous heterozygous regions within an ancestor begin and end at the closest heterozygous genotype call. when genotype calls are consistently inconsistent with the intensity - based founder assignment (collaborative cross consortium 2012), we assume that this is a feature of the line s haplotype and treat the region as fixed. we then compute the genomic length of all segregating regions divided by the full genomic length to determine the maximum residual heterozygosity within a line. if lines have reached the required thresholds, we generate a special haplotype file for the entire distributable line indicating regions fixed for a specific cc founder and regions that are still segregating and between which cc founders they are segregating (fig. 1c). all computations are based on mb distances of the ncbi m37 version of the mouse assembly.fig. the figure shows two chromosomes from line or3252 (shown in fig. 1) to illustrate the identification of segregating regions in distributable lines., with the top part of each subheading representing the contribution of the eight cc parental strains to the genome of the two most recent obligate ancestors and the midsection and lower sections representing the haplotypes of the line as provided in the cc website as figure or as text, respectively. a chromosome 12 illustrates two segregating regions in which one of the most recent ancestors is homozygous while the other is segregating. the figure also illustrates that in some cases the most recent obligate ancestors may appear to have slightly different boundaries between parental contributions. we suggest that investigators rely on the haplotype reconstruction provided in the text file rather than on visual inspection of most recent ancestors. we expect these discrepancies to be resolved in the near future with use of megamuga. b chromosome 1 illustrates a segregating region in which each of the most recent ancestors parents was homozygous for a different parental allele residual heterozygosity in distributable lines. the figure shows two chromosomes from line or3252 (shown in fig. 1) to illustrate the identification of segregating regions in distributable lines., with the top part of each subheading representing the contribution of the eight cc parental strains to the genome of the two most recent obligate ancestors and the midsection and lower sections representing the haplotypes of the line as provided in the cc website as figure or as text, respectively. a chromosome 12 illustrates two segregating regions in which one of the most recent ancestors is homozygous while the other is segregating. the figure also illustrates that in some cases the most recent obligate ancestors may appear to have slightly different boundaries between parental contributions. we suggest that investigators rely on the haplotype reconstruction provided in the text file rather than on visual inspection of most recent ancestors. we expect these discrepancies to be resolved in the near future with use of megamuga. b chromosome 1 illustrates a segregating region in which each of the most recent ancestors parents was homozygous for a different parental allele the haplotype assignments for each line can be visualized (as shown in fig. 1c) or downloaded as text files (as shown in figs. 2 and 3) from the unc systems genetics core web site (fig. the haplotype can also be visualized at http://www.csbio.unc.edu/ccstatus/index.py?run=ccv with the updated version of the cc viewer (fig. 4) (collaborative cross consortium 2012).fig. 3the unc systems genetics core web site. the available lines tab is highlighted on the left side of the web site as well as inserts of the pages associated with information on the number, genome, and characteristics of the available cc lines. links from the menu take you to the ordering page and the cc viewerfig. 4the cc viewer allows comparative analysis and visualization of multiple collinear genomes (wang. 2012b) and follows the conventions reported previously (collaborative cross consortium 2012 ; yang. 2011). on the left side is a screenshot of the cc viewer web site showing the fields for selection of the genomic region (chromosome, start and end), type, and identity of cc line(s) to be viewed. note that in addition to the distributable lines, active lines, and cc founders, the browser has data for the entire cc population reported previously (collaborative cross consortium 2012). on the right side of the figure is the output for four of the seven possible tracks for three distributable lines for chromosome 14. the phylogenetic tree for a specific region can be selected by clicking on the appropriate chromosome location in the haplotype count track. users can zoom in by selecting a region in any of the tracks or by using the zoom tab. clicking also allows centering the view on a given region and sorting according to the type of data shown in that track the unc systems genetics core web site. tab is highlighted on the left side of the web site as well as inserts of the pages associated with information on the number, genome, and characteristics of the available cc lines. links from the menu take you to the ordering page and the cc viewer the cc viewer allows comparative analysis and visualization of multiple collinear genomes (wang. 2012b) and follows the conventions reported previously (collaborative cross consortium 2012 ; yang. 2011). on the left side is a screenshot of the cc viewer web site showing the fields for selection of the genomic region (chromosome, start and end), type, and identity of cc line(s) to be viewed. note that in addition to the distributable lines, active lines, and cc founders, the browser has data for the entire cc population reported previously (collaborative cross consortium 2012). on the right side of the figure is the output for four of the seven possible tracks for three distributable lines for chromosome 14. the phylogenetic tree for a specific region can be selected by clicking on the appropriate chromosome location in the haplotype count track. users can zoom in by selecting a region in any of the tracks or by using the zoom tab. clicking also allows centering the view on a given region and sorting according to the type of data shown in that track because in most cases several generations separate the obligate ancestors from the mice accessible to researchers, residual heterozygosity is overestimated (i.e., distributed mice are more inbred than advertised). on the other hand, currently we do not track the mitochondrial genome or chromosome y due to the lack of informative snps in muga. furthermore, the snp density in muga has limits of detection that will miss very short haplotype segments, especially in the telomeric ends of chromosomes (collaborative cross consortium 2012). to address these limitations we will repeat the procedure with new obligate ancestors from each cc line as the breeding progresses. we also plan to repeat the procedure with a new genotyping array that contains ten times more snps that will be available in the summer of 2012 (megamuga). at unc, the distributable and completed cc lines have one of two health statuses, both of which are specific - pathogen - free (spf) or cleaner (threadgill. 2011). the spf facility is designated health status 2 and is negative for all of the following by serology : edim, tmev gdvii, mhv, mycoplasma pulmonis, mpv, mvm, parvo ns-1, pvm, and sendai. additionally, some are tested for car bacillus, ectromelia, lcmv, mad1, mad2, mcmv, polyoma, and reo3. the barrier facility is designated health status 1 and is negative for all of the above plus the following by serology : mnv, some are tested for pvm ; by culture nasal swab : pasteurella pneumotropica ; and by fecal pcr : helicobacter. the different health statuses of the lines in the unc systems genetics core reflect their different origins and our efforts to rederive distributable and completed cc lines through ivf and c - section to the clean barrier 1 facility. currently at tau, all cc mice are maintained in a conventional facility, while the rederived lines will be maintained in individual ventilation cages (ivc) and housed at the newly established spf facility. mice will be tested for routine microbiology monitoring according to federation of european laboratory animal science association (felasa) recommendations (nicklas. all distributed cc animals fall under a conditions of use (cou) that is virtually identical to the cou from the jackson laboratory. in essence, cc mice are available to any institution for internal use and can not be redistributed to any researchers from other institutions without prior permission. please note that all mice are provided as is and without liability to the provider. we are striving to make all genetic information on cc lines available through a dedicated web site : http://csbio.unc.edu/ccstatus/index.py. the menu bar offers a link to the cc resource (cc mice) and specific pages for information on available lines, ordering, and the cc viewer page lists cc lines that are in the distributable and completed categories, the percentage of the genome still known to be segregating, and the health status of the facility from which these mice are available. researchers can obtain further information on the genotypes, breeding performance, physical characteristics, and handling information for any of these lines by checking more info and using the corresponding tab. importantly, this information includes the number of days since the birth of the obligate ancestors used to estimate maximum residual heterozygosity. this can be used to estimate additional inbreeding that has occurred in any line at a given date. since three wild - derived strains were included in the original eight founder strains, some strains still exhibit jumpy characteristics and extra caution in handling should be observed. interested parties must register on the web site before ordering mice. by clicking on the ordering tab data collection forms appear asking for pertinent shipping and ordering information. a custom genome browser for the cc has been described previously (collaborative cross consortium 2012). this cc viewer has been updated to include the genome of all cc lines in the distribution center (fig. users can visualize the genome of selected cc lines based on location and use interactive tools to zoom in and out, center, and order these lines as desired. in addition to the haplotype reconstruction, the site provides the subspecific origin based on diagnostic snps (yang. 2011) and a tree reflecting the phylogeny of the founder strains at any given location. there are currently 42 cc lines listed in the unc system genetics core as distributable or complete, but we expect a rapid increase in number during 2012 (> 50 lines distributable) and 2013 (> 100 lines distributable). we also expect to provide an ever more accurate picture of their genomes through additional genotyping using the new megamuga array. we suggest that interested parties check on availability and further developments such as the ability to download complete genome sequences for each cc line generated by imputations of the snps and indels found in each cc founder strain at the corresponding location of the genome. pseudogenomes will be based on imputation of sequence variants found in the eight founders through whole - genome sequencing (keane. these pseudogenomes (provided in fasta format) will be especially useful for investigators using ngs methods such as rnaseq. tel aviv university, geniad, ltd in perth, and the jackson laboratory are actively seeking the establishment of additional distribution centers. finally, we are keenly aware of the value and the effort invested in the cc resource. to preserve the resource for the future, there are active efforts to archive embryos of completed and distributable cc lines at the unc mutant mouse regional resource center, the wellcome trust, and emma consortium. | the collaborative cross (cc) is a panel of recombinant inbred lines derived from eight genetically diverse laboratory inbred strains. recently, the genetic architecture of the cc population was reported based on the genotype of a single male per line, and other publications reported incompletely inbred cc mice that have been used to map a variety of traits. the three breeding sites, in the us, israel, and australia, are actively collaborating to accelerate the inbreeding process through marker - assisted inbreeding and to expedite community access of cc lines deemed to have reached defined thresholds of inbreeding. plans are now being developed to provide access to this novel genetic reference population through distribution centers. here we provide a description of the distribution efforts by the university of north carolina systems genetics core, tel aviv university, israel and the university of western australia. |
methane is a powerful greenhouse gas, and its atmospheric concentration has been steadily increasing over the past 300 years. there are two major ways in which methane is removed from the environment : aerobic oxidation by a specialized group of bacteria and anaerobic oxidation by a specialized group of archaea. the former is important for keeping methane concentrations balanced in freshwater sediments and soils, whereas the latter is the major process in anoxic marine environments. the biochemistry of aerobic methane oxidation is relatively well understood, following intensive research efforts with a number of model organisms, but the biochemistry of anaerobic methane oxidation is not yet fundamentally understood and no anaerobic methane - oxidizer has been isolated in pure culture so far. three recent studies using global approaches [1 - 3 ] have shed new light on both aerobic and anaerobic systems. here, we first review background information on the two metabolic systems involving methane and then discuss the insights revealed through the three recent studies [1 - 3 ], as well as a fourth that is useful for interpreting the new results on anaerobic methane oxidation. type i methanotrophs are -proteobacteria that have stacked membranes harboring methane monooxygenase (pmmo), the enzyme for primary methane oxidation, and that use the ribulose monophosphate (rump) cycle, which converts formaldehyde into multicarbon compounds, for building cell biomass. type ii methanotrophs belong to the -proteobacteria, have rings of pmmo - harboring membranes at the periphery of the cells, and use the serine cycle, an alternative pathway for converting formaldehyde into biomass ; these bacteria also often contain a soluble (s) mmo in addition to pmmo. the third type, type x methanotrophs, belong to the genus methylococcus (-proteobacteria) and combine features characteristic of the other two types : they have stacked membranes and the rump cycle, but they also have elements of the serine cycle and smmo. the type x methanotroph methylococcus capsulatus has been a favorite model for research because of its robust growth on methane and its relative ease of use as a genetic system [6 - 9 ]. two almost identical gene clusters have been identified encoding the subunits of pmmo, which are expressed simultaneously and are functionally redundant, and another gene cluster encodes the subunits of smmo. copper has been shown to play an essential role in expression of the pmmo operons, whereas the smmo operon appears to be expressed only in low - copper conditions. the catalytic mechanisms for both pmmo and smmo are understood on a sophisticated level, but until recently no whole - genome sequence has been available for m. capsulatus or for any other methanotroph. two recent studies have used a whole - genome - shotgun sequencing approach to complement the mounting dataset on the biochemistry and regulation of aerobic methane oxidation. in contrast, understanding of the process of anaerobic methane oxidation is in its infancy. microbes involved in this process have been identified recently as archaea related to methanosarcinales that fall phylogenetically into two distinct groups, anme - i and anme - ii ; these are normally found in association with sulfate - reducing bacteria. there is no clear concept of how methane oxidation is linked to sulfate reduction ; figure 1 shows a possible model. this co - metabolism has to be viewed in the light of the thermodynamic constraints, however ; the free energy (g) for anaerobic methane oxidation in situ is estimated at -20 to -40 kj / mol), the lowest value described that enables microbial growth. there is agreement on the hypothesis that reverse methanogenesis plays a key role in the methane oxidation process : most enzymes of methanogenesis are easily reversible, and part of the methanogenesis pathway operates in reverse for energy generation in methanosarcina species growing on such substrates as methanol or methylamine. but the last step of methanogenesis and presumably the first in anaerobic methane oxidation (step 1 in figure 1), catalyzed by methyl - coenzyme m reductase (mcr), presents a mechanistic challenge given the fact that methane is chemically unreactive. nevertheless, data have been obtained showing that methanotrophic archaea have homologs of the genes for all three subunits of mcr, suggesting that mcr or a similar enzyme may indeed be responsible for anaerobic methane oxidation. two recent studies describe efforts to establish the roles of mcr homologs and of other genes potentially involved in reverse methanogenesis by directly assessing environmental dna and protein pools. in a paper recently published in plos biology, ward. describe the complete genomic sequence of methylococcus capsulatus (bath). they annotate the genome in terms of the specific adaptations this organism has evolved in order to succeed at a lifestyle solely dependent on utilization of methane. the genome of m. capsulatus (3.3 megabases, mb) is much smaller than the genome of a model facultative methylotroph, methylobacterium extorquens am1 (7 mb), a bacterium with a much more versatile lifestyle, but is comparable in size to the genome of another obligate methylotroph, methylobacillus flagellatus (2.9 mb), suggesting that the degree of specialization in methylotrophy may correlate with genome size. the tricarboxylic acid (tca) cycle is the pathway that converts acetyl - coa to co2 and is the major source of reducing equivalents during growth on multicarbon compounds ; the long - held hypothesis that m. capsulatus lacks a complete tca cycle has not been proven true by genome sequencing, as putative genes for all the enzymes of the cycle were identified in the recent study. in addition, the organism seems to encode an array of enzymes that could metabolize sugars, so the inability of m. capsulatus to grow on sugars remains enigmatic. analysis of the genes encoding enzymes involved in the metabolism of single - carbon compounds in m. capsulatus (figure 2) has been greatly simplified by the addition of data available from pre - genomic analyses [7 - 9,21 ] and from the initial analysis of the genome of m. extorquens. as expected, all the genes encoding enzymes of the rump pathway have been identified. in accordance with previous observations, most of the genes for the serine cycle were also found, as were the genes for the calvin - benson - bassham (cbb) cycle, the pathway that reduces co2 and converts it into biomass (figure 2f). the potential to operate all three known pathways for the assimilation of single - carbon compounds that are found in various methylotrophs makes this organism unique, but further analysis involving knockout mutations is needed to understand the functions of each of the three pathways. the first glimpses into the expression patterns of pathways enabling methanotrophy are coming from a proteomic analysis of m. capsulatus by a group that has independently sequenced the m. capsulatus genome to 8x coverage. in this work, quantitative proteomic analysis was performed in order to compare the response of m. capsulatus to low - copper and high - copper conditions. identified a total of 682 differentially expressed proteins using a cleavable isotope - coded affinity tag (cicat) technique. the authors demonstrated that, as expected, pmmo is overexpressed in conditions of high copper whereas smmo is expressed at low copper levels. equally interesting data from this work concern the expression of proteins other than mmos, indicating that, indeed, all three assimilatory pathways are simultaneously expressed. the oxidative pathway linked to tetrahydromethanopterin (h4mpt) is one of the pathways by which formaldehyde can be oxidized to co2 (figure 2b) ; all the enzymes in this pathway were identified, pointing to the importance of this pathway, as suggested previously by enzyme - activity measurements. peptides for the oxidative branch of the rump cycle were also identified, suggesting that it is operational in m. capsulatus (figure 2a). some of the major serine - cycle enzymes were found to be overexpressed under high - copper conditions. it is unlikely, however, that their expression would be directly regulated by copper ; it is more likely that they are responding to the higher flux of formaldehyde that occurs during growth under high - copper conditions. it is important to note that the serine cycle can not operate as a major assimilatory pathway in m. capsulatus unless the two - carbon compound glyoxylate that is depleted during the cycle can be regenerated, but no genes have been identified in the genome that potentially encode either of the enzyme systems that can convert acetyl - coa into glyoxylate : the isocitrate lyase and the glyoxylate - regeneration cycle. given these considerations, what might the function of the serine cycle (and the interconnected tca cycle) be in m. capsulatus ? we suggest that a possible role for this pathway could be to handle the extra flux of formaldehyde that the organism may encounter under certain growth conditions (figure 2c). the excess of formate generated in the h4mpt - linked pathway (figure 2b) could also be redirected into the serine cycle after reduction to methylene - tetrahydrofolate (methylene - h4f ; figure 2d). acetyl - coa and other intermediates generated in this way could serve as building blocks for cell biomass. the role of the cbb cycle in m. capsulatus (figure 2f) is not clear at present. given that the fixation of co2 is a far less efficient mechanism of carbon sequestration than the rump or serine cycles, a significant amount of carbon shunted through the cbb cycle would be predicted to decrease growth yield. it is possible, however, that it serves to reduce the local concentration of co2 and/or to generate intermediates for biomass production. once again to provide support for the hypothesis that reverse methanogenesis is important in anaerobic methanotrophy, a consortium of researchers focused on identifying the enzyme potentially involved in the initial step of anaerobic methane oxidation ; this enzyme is hypothesized to be similar to the bacterial mcr (figure 1, step 1). a microbial mat in the black sea largely consisting of anme-1-type archaea was chosen as a source of this hypothetical enzyme., a conspicuous protein consisting of three subunits similar to the,, and subunits of mcr is abundantly present in this microbial mat (7% of the total extracted protein), suggesting that it has an important role in anaerobic methane oxidation. the protein contains a variant of f430, a cofactor used by the classical mcr, but the two cofactors differ in molecular weight as determined by mass spectrometry. the genes encoding this protein were sequenced as a part of an insert detected in an environmental dna library. alignment of amino - acid sequences translated from these genes with the respective sequences of methanogen mcr subunits showed that residues involved in active - site formation in the and subunits were conserved, but one of the important residues in the active site of the subunit was substituted. it is interesting to speculate that this modification of the active site and the use of a modified f430 cofactor could provide a mechanism for the biochemical activation of methane and could make the first step of reverse methanogenesis thermodynamically and kinetically possible. in a recent paper published in science, hallam. describe a large environmental sequencing effort which aimed to provide further evidence for the hypothesis of reverse methanogenesis. the group isolated dna from a 52o - meter - deep sediment of eel river basin in california, known for a high abundance of anme-1 and anme - ii archaea, and used it for both whole - genome shotgun analysis and fosmid ' walking ' (fosmids are large - insert plasmids). a total of 111.3 mb of non - redundant sequence was generated by shotgun sequencing and another 4.6 mb more were generated by fosmid - end sequencing. fosmids containing either 16s rrna genes belonging to anme - i or anme - ii archaea or homologs of the mcra gene were analyzed in detail, producing an additional 7.4 mb of sequence. the main conclusion from this work is that anme archaea contain most of the genes involved in methanogenesis, with one exception : mer, the gene encoding methylene - h4mpt reductase (step 3 of reverse methanogenesis ; see figure 1). on the basis of the apparent lack of mer, the authors propose a model in which parts of the methanogenesis pathway function in two opposite directions : a novel mcr - like enzyme oxidizes methane to methyl - com (step 1), and methyl - h4mpt : com methyl - transferase catalyzes a reverse reaction to produce methyl - h4mpt (step 2), while the rest of the enzymes reduce co2 to methylene - h4mpt (steps 4 to 7 in reverse) ; that is, contrary to previous models, methane is not oxidized to co2 by anme archaea. firstly, the model aggravates the thermodynamic constraints mentioned earlier, given that reduction of co2 to formyl - methanofuran (step 7) is an energy - consuming reaction (g = + 16 kj / mol). secondly, the fate of the methylene - h4mpt produced in steps 4 to 7 is proposed to involve either the assimilatory serine cycle or formaldehyde oxidation, but the high energy cost of such schemes would suggest they could operate only as minor pathways, not as major assimilatory or detoxification pathways. thirdly, there is no discussion by hallam. of how net co2 would be produced from methane. are an attempt to explain how methane metabolism might function in the absence of mer, they highlight the many aspects of this metabolic mode that are still unknown. two different explanations might be that either mer has simply not been detected because of incomplete sequence data, or that the function of mer is fulfilled by a novel enzyme (a non - homologous substitution), possibly involving a cofactor different from f420, so the reverse - methanogenesis pathway might in fact be complete (as in figure 1). an example of such a non - homologous substitution is seen in methylotrophic bacteria, in which a version of the ' reverse methanogenesis ' pathway has been found to operate where an nad(p)-linked methylene - h4mpt dehydrogenase acts in place of unrelated f420-linked or h2-forming enzymes. in conclusion, recent studies involving both organismal and environmental genomics shed new light on the biochemical details of the two processes important for methane balance on earth - aerobic and anaerobic methane oxidation - and suggest that these processes have more in common than just the substrate, methane, and the final oxidation product, co2. both processes involve common cofactors, such as h4mpt, common single - carbon intermediates bound to h4mpt, and common or similar enzymes for core reactions. although some enzymes involved in reactions that shift single - carbon compounds between different levels of oxidation are evolutionarily related in both processes, the primary methane oxidation enzymes, mmo and the newly identified mcr homolog, must have evolved independently and are fundamentally different. a proposed pathway for anaerobic oxidation of methane involving the homolog of methyl - com reductase and a novel methylene - tetrahydromethanopterin (h4mpt) reductase (mer), and its connection with the sulfate reduction pathway. solid arrows represent enzymes predicted from the sequences found by hallam. ; the dotted arrow represents the one enzyme that was not predicted, methylene h4mpt - reductase (mer). enzymes performing steps 1 - 7 : 1, methyl - com reductase - like protein (mcr) ; 2, methyl - h4mpt : coenzyme m (com) methyl - transferase (mtr) ; 3, methylene - h4mpt reductase (mer) ; 4, f420-dependent methylene - h4mpt dehydrogenase (mtd) ; 5, methenyl - h4mpt cyclohydrolase (mch) ; 6, formyl - mfr : h4mpt formyltransferase (ftr) ; 7, formyl - mfr dehydrogenase (fmd). (b) reverse methanogenesis is thought to be connected to sulfate reduction through an unknown intermediate (x) ; erepresents an electron. hallam. suggest that steps 1 and 2 in (a) function in the down direction and methyl - h4mpt is used for biomass generation (c), while steps 4 to 7 function in the up direction and the methylene - h4mpt produced is either converted to biomass through the serine cycle or is oxidized to co2. pathways in the aerobic methanotrophic bacterium methylococcus capsulatus involved in the metabolism of single - carbon compounds, as determined by genome sequencing and proteome analysis. formaldehyde produced from methane can be metabolized in the following alternative ways : (a) through the ribulose monophosphate (rump) cycle, which can either generate biomass (via the assimilatory (a) rump cycle) or co2 (via the dissimilatory (d) rump cycle) ; (b) by conversion to formate via intermediates containing tetrahydromethanopterin (h4mpt) ; (c) via methylene - tetrahydrofolate (methylene - h4f) to the serine cycle and from there into biomass. under certain conditions, there can be an excess of formaldehyde and formate ; the former can be used up through pathway (c) and the latter by reduction to methylene - h4f (d) and thus directed into the serine cycle. co2 produced in any of these reactions can be converted to biomass by either (e) the serine cycle or (f) the calvin - benson - bassham (cbb) cycle. | recent sequencing of the genome and proteomic analysis of a model aerobic methanotrophic bacterium, methylococcus capsulatus (bath) has revealed a highly versatile metabolic potential. in parallel, environmental genomics has provided glimpses into anaerobic methane oxidation by certain archaea, further supporting the hypothesis of reverse methanogenesis. |
all surviving members of the 2012 outbreak in jordan, their exposed contacts, and their household members who were identified serologically as either mers - cov positive or indeterminate were asked to consent to further participation. participants were asked to provide a follow - up serologic specimen so we could compare 34-month results with 13-month results. specimens were prepared by the jordan central public health laboratory (amman, jordan) and tested at the us centers for disease control and prevention (atlanta, ga, usa). antibody titers in serum samples were determined by an anti mers - cov nucleocapsid indirect elisa and by mers - cov (hu / jordan - n3/2012 strain) indirect ifa (1). the presence of neutralizing antibody titers was determined by microneutralization with live mers - cov (hu / jordan - n3/2012 strain) in a biosafety level 3 laboratory as described previously (1). neutralization titers were defined as the reciprocal of the highest serum dilution completely protecting the vero cell monolayer from cytopathic effect in at least 1 of 3 parallel wells. of the 15 surviving persons with > 1 positive serologic test result, 13 (87%) consented to follow - up testing. all 7 (100%) surviving persons with > 2 positive serologic test results 13 months after the mers - cov outbreak also consented. each of these 7 persons was considered to be a probable mers - cov case - patient according to world health organization criteria ; each had had a symptomatic acute respiratory infection during the outbreak period and documented, unprotected exposure to > 1 person with a case confirmed by reverse transcription pcr. for the 7 probable case - patients, elisa titers at 34 months ranged from 1 case - patient. each of these 6 persons had negative serologic test results at 34 months and continue to be considered negative overall. antibodies against mers - cov, including neutralizing antibodies, persisted in 6 (86%) of 7 persons 34 months after the 2012 mers - cov outbreak in jordan. the observed persistence of these antibodies contributes to the understanding of individual immune responses to mers - cov infection, of population - based immunity in regions where mers - cov outbreaks have occurred, and to efforts for developing effective vaccines and therapeutics to counter mers - cov infections. notwithstanding improvements in public health awareness and infection control practices in affected countries on the arabian peninsula and in the middle east, emergence of the virus (e.g., its introduction to south korea and the resultant epidemic of 2015) ongoing. mers - cov continues to pose grave risks to international healthcare and socioeconomic systems (6). it has been hypothesized that mild or asymptomatic mers - cov infections are potentially associated with lower levels of mers - cov neutralizing antibodies over time (7). all 7 case - patients reported here had respiratory symptoms, were relatively young, and had few underlying medical conditions (table). any association between our mers - cov antibody results and clinical severity is therefore difficult to assess. nonetheless, of the 5 persons for whom chest radiographs showed substantial changes within 3 days of symptom onset, each remained positive by microneutralization (> 20) 34 months after the outbreak. although some similarities in the short - term development of antibodies against mers - cov and sars - cov (e.g., seroconversion 23 weeks after illness onset) have been observed (8,9), longer term serum antibody kinetics of these infections have not yet been compared. after sars - cov infection, robust igg titers were observed through the second year but declined substantially during the third year after infection (10). our finding of generally reduced but persistent mers - cov antibody responses even at 34 months suggests the potential for longer lasting antibody - mediated protective immunity against reinfection. however, whether such long - lasting antibodies can prevent reinfection or affect clinical outcome has yet to be examined. diverse individual antibody test results allude to a potential role of genetic factors in explaining observed differences in immunologic responses to mers - cov exposure and infection. the times at which mers - cov antibodies were measured in our study were chosen because of logistics and field practicalities. although limited outbreaks of mers - cov have occurred in jordan since 2012, contact tracing efforts by investigators in jordan lead us to believe that these persons were not subsequently exposed. the observed elisa titers and neutralizing antibody titers support this supposition ; otherwise, we would expect increases resulting from a booster effect after secondary exposure and infection. to further assess the duration and resiliency of mers - cov antibodies in human populations, continued follow - up serologic evaluations of these persons | to determine how long antibodies against middle east respiratory syndrome coronavirus persist, we measured long - term antibody responses among persons serologically positive or indeterminate after a 2012 outbreak in jordan. antibodies, including neutralizing antibodies, were detectable in 6 (86%) of 7 persons for at least 34 months after the outbreak. |
the importance of trace elements and their role in biological cycling and homeostasis has become evident in recent years [1, 2 ], due to deficiency of essential trace elements or excess of potentially toxic elements accumulation in human body for a long time, which are responsible for various diseases. so the formulation of trace elements reference values is indispensable prerequisite for the evaluation of body health, as well as the cost - effectiveness analysis. plasma is one of the preferred biological fluids and widely used for biomonitoring purposes in the surveys involving trace elements. but it is often limited by a small - size sample, complicated matrix, simultaneous multielemental analysis, and a wide range of concentrations of analytes from trace (g / l) to ultratrace levels (ng / l - pg / l). fortunately, high resolution sector field inductively coupled plasma mass spectrometry (hr - icp - ms) is the most suitable equipment for the requirements because it exhibits an excellent sensitivity and precision, more superior ability to depress spectral interference, low volume sample consumption, and the rapid simultaneous analysis of multiple elements over a large range of concentrations. currently, the completed method of hr - icp - ms combined with a simple - size biological sample preparation was established and applied for the multielemental analysis in practice, which can speed up analysis and limit potential sources of contamination. so far, many studies involving trace elements across population have been carried out in different areas and countries, such as italy [7, 8 ], germany, uk, and canada. however, in china, there is no large - scale and multiregional survey including so many trace elements in population, especially in children, which is still scarce and fragmentary. however, children are usually faced with higher health risk of concern because of their special physiological susceptibility, behavior, lifestyles and dietary habits, and other related health factors. in addition, many intervention programs involving trace elements on children had been conducted in recent years, but a partial cost - effectiveness analysis had not been performed for lacking of the original baseline values of trace elements. therefore, the study to obtain the original baseline values of trace elements for children is really urgent and necessary, especially the different types of economic regions. chinese national nutrition and health survey project 2002 (cnnhs 2002), which is a nationally representative and cross - sectional study, provides a good opportunity to examine the status of plasma trace elements of children in 2002. the objectives of our study were to apply the optimized hr - icp - ms combined with simple small - size sample preparation to determine 16 selected plasma trace elements (fe, cu, zn, rb, b, al, se, sr, v, cr, mn, co, as, mo, cd, and pb) in children aged 312 years and to formulate the reference values of 16 selected trace elements, which could be considered as the original baseline levels for the representative children living in china economical developed rural areas in 2002. high - pure water (18.2) was obtained from tka gen - pure apparatus (gen - pure tka, niederelbert, germany). the electronic grade nitric acid (hno3) screw - capped polystyrene liners, 15 ml corning costar polypropylene centrifuge tubes were used for standard solution and sample preparation. all disposable pipette tips and plastic tubes were rigorously cleaned before use by immersion in 10% (v / v) hno3 and rising with high - pure water. l of fe, cu, zn, b, al, se, mn, mo, co, cr, cd, sr, rb, v, pb, and as were got from national center for standard materials (beijing, china). internal standard elements, consisted of 1000 mg / l of sc, ga, y, and tl, were purchased from national steel materials testing center (beijing, china). the certified reference materials used for internal quality control were clichek blood plasma control level 1 (recipe, munich, germany). the hr - icp - ms system was a thermo finnigan element ii model (bremen, germany) equipped with a 100 l / min concentric glass nebulizer, a water - cooled scott double - pass spray chamber, and torch with guard electrode device, nickel interface cones. the major icp - ms operational settings were listed : rf power, 12801300 w ; argon gas flow rates, 16.0 l / min (cool), 0.94 l / min (auxiliary), and 1.1101.200 l / min (sample) ; sample uptake rate, 0.1 - 0.2 ml / min. daily instrument optimization was performed by monitoring the sensitivity and stability for masses li, in, and u in tune solution. the resolution (300, 4000, and 10000) for elements was selected by the interference that happened in plasma samples. the percentage of oxides and doubly charged ion formation (bao / ba and ba / ba ratios) was maintained less than 0.003 and 0.03, respectively. the plasma samples were stored in deep frozen state at 80c and kept very well without repeated freeze - thaw cycles prior to analysis. the selected plasma samples were reconstituted at room temperature, and then were shaken slightly and centrifuged at 2000 g for 15 minutes. 0.1 ml supernatant of plasma was diluted with 0.5% (v / v) hno3 and internal standard solutions in 1 : 20 (v / v) up to 2 ml. 16 single - element standards (fe, cu, zn, rb, b, al, se, sr, v, cr, mn, co, as, mo cd, and pb) were mixed in the calibration solutions and diluted with 0.5% (v / v) hno3 and internal standard solutions and then prepared in correspondence to the concentrations of trace elements in human plasma. the certified reference materials used for internal quality control were diluted according to the instructions of products, further prepared with 0.5% hno3 (v / v). for those elements which are not certified in the commercially available samples, were performed by analyzing pooled plasma control samples fortified with appropriate aliquots (approximately 1-times baseline level) of trace elements. 16 selected trace elements were determined by hr - icp - ms using external standard quantification mode in class 1000 clean room. the determination of isotope was chosen according to the abundance, type of interference, and the amount of analytes expected in the matrix. for b, rb, sr, mo, cd, and pb, the analyses were in low resolution (lr, 300 m/m), for al, v, cr, mn, fe, co, cu, zn, the analyses were in medium resolution (mr, 4000 m/m), and, for as and se, the analyses were in high resolution (hr, 10000 m/m). internal standards (sc, ga, y, and tl) were used and the final concentrations were 10 g / l, which may provide adequate means for dealing with nonspectral interference and instrumental drifts. the plasma samples were chosen from the list of blood species established in cnnhs 2002 using a random stratified sampling procedure with proportional allocation by gender and age groups. a total of 440 plasma samples were selected from the children (boys : 232, girls : 208) with an age of 8.02 2.86 years living in china economical developed rural areas, which was equal to approximately 10% of the children blood species in that area. the distribution of 16 selected trace elements was not normal according to kolmogorov - smirnov 's tests. basic descriptive statistics were estimated from untransformed data including selected percentiles (p5, p50, and p95), geometric mean (gm), and the 95% confidence interval of the geometric mean (95% ci gm) of 16 selected trace elements for the children. additionally, gm, 95% ci gm of 16 selected trace elements was described by gender in conjunction with age groups. the differences caused by gender and age groups were identified with the mann - whitney u test. all statistical analyses were completed by using the statistical package spss base 20.0 (spss, chicago, il, usa). the working linear ranges were over at least 5 calibration points, which were divided into three ranges such as 0100 g / l, 010 g / l, and 0 - 1 g / l. the linear equations were established by over 20 replicates per calibration solution indicating linearity between signal and concentration, and correlation coefficient (r) ranged from 0.9957 (al) to 0.9999 (cu and fe). the limits of quantitation (loq) ranged from 0.02 g / l (rb) to 1.89 g / l (se), calculated as 10 sd above the value for 0.5% hno3 solution (n = 20) plus the dilution factor, which in this case was 20, considering the matrix effects and the excellent sensitivity of hr - icp - ms. table 2 shows the trueness (or recovery) and precision of method, which were performed by checking against values given by the certified reference materials and by testing against pooled plasma samples fortified with a known quantity of reference standards. the resulting trueness (or recovery) spanned from 89.82% (al) to 119.15% (se). the precision was expressed by calculating the relative standard deviance (rsd% = (standard deviance / mean) 100) for intraday and interday, respectively. the rsd% for intraday indicated the stability of the determination in one day (one sample preparation, 10 replicate measurements of the same sample) and the rsd% for interday indicated the stability of the determination in 10 consecutive days (10 different sample preparations, 10 measurements in different days). the precision ranged from 1.1% (zn) to 9.0% (se) in rsd% for intraday and 3.7% (fe) to 12.7% (al) in rsd% for interday. as shown above, the optimization method of hr - icp - ms combined with the simple sample preparation with 0.5% hno3 was applied for the determination of 16 selected trace elements from ng / l to g / l ranges. the 20-fold dilution causes the minor fluctuation at low concentrations for certain trace elements, but the method was reliable for the validation of methodological parameters and there was no coagulation or precipitation of plasma matrix components resulting in tubing or nebulizer blockages during 12 h of analysis. compared with the other analytical procedures [12, 14 ], the method using 0.5% hno3 directly diluted in 1 : 20 (v / v) is more rapid and free of contamination, especially in 0.1 ml volume of small - size sample consumption, low loq, and high sample throughput hr - icp - ms, which can provide more flexibility in multielemental analysis. 16 selected trace elements for 440 plasma samples are successfully determined and the detailed results are summarized in table 3. the concentrations of cd, pb, v, cr, and as are usually very low (ng / l), even below the loq. during the analysis, the concentration of al in plasma sample is still easily affected because of its ubiquitous, so that the rigorous control of exogenous contamination is of great importance. the further description of 16 selected plasma trace elements in children is presented by gender and age groups, which are listed in tables 4 and 5. comparing between 36 years children and 712 years children, most of the gm concentrations of trace elements are higher for 36 years children with some exceptions (rb, sr, cd, as, and se) in boys and with some exceptions (rb, sr) in girls. overall, the concentrations of cr (z = 2.622, p < 0.01), v (z = 2.728, p < 0.01), mn (z = 2.470, p < 0.05), and cu (z = 6.071, p < 0.001) for 36 years children are significantly higher than 712 years children. the concentrations of v (z = 2.478, p < 0.05) and cu (z = 3.900, p < 0.001) for 36 years children are significantly higher than 712 years children in boys, and the concentration of cu is significantly higher in girls (z = 4.479, p < 0.001). in a comparison between boys and girls, the concentrations of b (z = 2.908, p < 0.01), sr (z = 2.587, p < 0.01), and fe (z = 3.186, p < 0.001) for boys are significantly lower than girls, and the concentrations of b (z = 2.371, p < 0.05), sr (z = 2.072, p < 0.05), and fe (z = 2.844, p < 0.01) are obviously lower in 712 years boys. in general, in the children who lived in fixed areas for a long period of time without any major changes, the status of trace elements is representative and relatively stable. age and gender are the major factors that affect the body burden of trace elements. furthermore, the plasma trace elements usually associate with the age and gender after controlling for other potential confounders. age - related changes may be caused by behavior, dietary habits, and lifestyle. gender - related changes might rely on a specific source of exposure in one sex or due to the difference in physiological factors or metabolic pathways. comparing the status of trace elements in plasma of our study with other previous studies [14, 16, 17 ], most of trace elements such as b, rb, sr, mo, v, cr, mn, fe, co, cu, zn, and se are mostly similar to the wide ranges reported before and only the concentrations of cd, al, pb, and as are at relatively low levels, which may be correlated with the surroundings. in addition, the limitation of this study is a relatively small sampling, which should be expanded in future studies. in this study, we have established the method for the determination of 16 selected plasma trace elements in children. the method, combining hr - icp - ms with the simply diluted 0.5% hno3 by 1 : 20 (v / v), is an excellent method for rapid, free - contamination, small - size sample requirement and enough low loq for the most of plasma trace elements analysis. in addition, the concentrations of 16 selected plasma trace elements of the children from china economical developed rural areas were determined. the first representative data can be used as one of the basic components for the formulation of original baseline reference values of trace elements for the children aged 312 years in 2002, which can be helpful in the subsequent health evaluation and the cost - effectiveness analysis of health intervention programs. | a rapid, accurate, and high performance method of high resolution sector field inductively coupled plasma mass spectrometry (hr - icp - ms) combined with a small - size sample (0.1 ml) preparation was established. the method was validated and applied for the determination of 16 selected plasma trace elements (fe, cu, zn, rb, b, al, se, sr, v, cr, mn, co, as, mo, cd, and pb). the linear working ranges were over three intervals, 0 - 1 g / l, 010 g / l and 0100 g / l. correlation coefficients (r2) ranged from 0.9957 to 0.9999 and the limits of quantification (loq) ranged from 0.02 g / l (rb) to 1.89 g / l (se). the trueness (or recovery) spanned from 89.82% (al) to 119.15% (se) and precision expressed by the relative standard deviation (rsd %) for intra - day ranging from 1.1% (zn) to 9.0% (se), while ranged from 3.7% (fe) to 12.7% (al) for interday. a total of 440 plasma samples were collected from chinese national nutrition and health survey project 2002 (cnnhs 2002), which represented the status of plasma trace elements for the children aged 312 years from china economical developed rural areas. the concentrations of 16 trace elements were summarized and compared by age groups and gender, which can be used as one of the basic components for the formulation of the baseline reference values of trace elements for the children in 2002. |
leishmaniasis is a vector - borne parasitic disease caused by intramacrophage protozoa of the genus leishmania, transmitted generally by at least 30 species of sand flies (either phlebotomus or lutzomyia genera) and rarely by congenital and parenteral routes [1, 2 ]. depending on the species of leishmania involved, humans and a wide range of mammals can act as reservoirs. at least four major clinical forms of leishmaniasis are recognised : cutaneous leishmaniasis (cl), either diffused or localized, mucocutaneous leishmaniasis (ml), visceral leishmaniasis (vl), fatal if untreated, and the post - kala - azar dermal leishmaniasis (pkdl). the disease is endemic in the tropical and subtropical regions of 88 countries [4, 5 ]. population displacements and increasing cases of leishmania / hiv coinfection brought new dramatic concerns to the disease especially in developing countries. leishmaniasis - affected individuals are estimated to be about 12 - 13 million worldwide with an annual incidence of 11.5 million new cases of cl and 500,000 of vl [4, 7 ]. however, the real burden of leishmaniasis is greatly underestimated and a substantial number of cases remain unrecorded and misdiagnosed [8, 9 ]. in sudan leishmaniasis represents a serious health problem and outbreaks occur periodically causing a high number of victims [1012 ]. it is endemic in the central and northern part of the country, however, recently, cl due to l. donovani has been well documented. vl is the most serious form caused by l. donovani, transmitted by phlebotomus orientalis [15, 16 ], and endemic in the eastern part of the country [17, 18 ]. however, scattered cases have been reported from areas not known to be endemic in the southern, northern, and western parts of the country. both anthroponotic transmission and zoonotic transmission of vl were thought to occur [15, 1921 ]. at least 50% of vl patients develop pkdl and this percentage is higher than in any other vl endemic area [2224 ]. sudanese mucosal leishmaniasis (sml) is a rare and particular form of ml. unlike ml, sml starts usually as primary mucosal disease, without being preceded or accompanied by cutaneous lesions. few studies have been reported on the naturally mixed infection with different species or strains of leishmania in immunocompetent patients. however, natural infection with more than one leishmania, especially where foci of two species overlap, is more prevalent than previously reported. the general aim of this study was to genetically characterize leishmania isolates from patients with various clinical manifestations. here we report for the first time the detection of three cases of mixed l. donovani and l. major infections in three out of five patients diagnosed as cutaneous leishmaniasis patients with no apparent clinical symptoms of vl. one hundred and eight samples composed of blood (hb), bone marrow (hbm), lymph node aspirates (hln), extracted dna and cultured parasites were obtained from 69 patients clinically suspected of leishmaniasis. from 21 of these patients, hbm, hb, and hln aspirates were taken. seven hb samples and one hln were collected from eight symptomatic vl patients at mdecins sans frontires - suisse (msf - ch) leishmaniasis clinic inside the local tabarak allah hospital (gedarif, sudan). other 20 hb and 5 hmb were collected from south gedarif (table 1). clinical samples were spotted on whatman filter paper grade 3 ; each filter paper sample was stored in a separate polyethylene bag at room temperature till further analysis. five samples were taken from five patients presented at the institute of endemic disease, university of khartoum, sudan (iend), with typical cl ulcers. in addition, ten extracted genomic dna of seven vl, one cl, one ml, and one pkdl samples were kindly provided by the same institute (table 1). cl patients were not from vl known endemic areas and did not show clinical symptoms of vl. a single cutaneous aspirate from the edges of the ulcer was inoculated into bottles containing biphasic media (nnn) and incubated at 24c. cultures were examined microscopically for parasite growth and the successfully cultured isolates were mass - cultured in 200 ml of rpmi-1640 supplemented with 10% foetal calf serum (fcs) and 1% of penicillin / streptomycin solution. l. major mon 25, kindly provided by the leishmania reference centre, italy, was included in this study. dna of the cultured parasites was extracted using phenol / chloroform method as described elsewhere. filter papers with spotted biological material (lymph node or bone marrow aspirates or blood) were punched out with a paper puncher. to prevent dna contamination among samples, clean sheet of paper was sprayed with dna decontaminant using microsol 3 + solution and was punched several times as recommended by the world health organization (available at http://www.who.int/hiv/topics/drugresistance/dbs_protocol.pdf) for hiv patients. dna extraction was performed using qiaamp dna mini kit according to the manufacturer 's instructions. as an initial screening, a real - time pcr was conducted to investigate the presence of leishmania complexes (l. viannia, l. mexicana, l. donovani / infantum, and l. major) in all samples. the primers lid - f and lid - r which generate a 80 bp fragment of the gpi gene were used with the probe taqman mgb lid - probe 5-atcggcaggttct-3 labeled with the fluorescent reporter dye fam (6-carboxyfluorescein) at the 5end and with the fluorescent quencher dye tamra (tetra - methyl carboxyrhodamine). real - time pcr was performed in a final volume of 20 l containing 3 l of dna, 10 l of faststart taqman probe master (rox)1x (roche mannheim, germany), 0.4 m of both primers, and 0.3 m of the probe. the thermal cycling profile consisted of an initial activation at 95c for 10 min, followed by 45 cycles each consisting of denaturation at 95c for 15 sec and annealing / extension at 60c for 30 sec. negative (sterile water) and positive (dna extracted from l. infantum mon-1, mhom / tn/80/ipt1, izsve. real - time pcr was carried out on a 7900ht fast real - time pcr system (applied biosystems). to determine the genetic profile of leishmania spp., dna from each positive sample was subjected to the amplification of the cytochrome oxidase ii gene. pcr was performed as described previously for the targeted cytochrome oxidase ii for all leishmaniasis positive samples. the reaction was performed in a final volume of 50 l containing 5 l of dna, 5 l of pcr buffer 1x (applied biosystems, foster city, ca), 2 mm of mgcl2 (applied biosystems, foster city, ca), 0.4 m of each primer, 0.2 mm of dntps (applied biosystems, foster city, ca), and 2 u of amplitaq gold dna polymerase (applied biosystems, foster city, ca). amplifications were carried out in a geneamp pcr system 9700 thermal cycler (applied biosystems, foster city, ca) with the following thermal cycling profile : denaturation for 10 min at 95c, followed by 35 cycles each consisting of 30 sec at 94c, 30 sec at 50c, 45 sec at 72c, and a final extension step for 7 min at 72c. all the pcr products were analysed on 7% acrylamide gel, visualized by silver staining, and subsequently subjected to sequencing. pcr products were sequenced using the big dye terminator v3.1 cycle sequencing kit (applied biosystems, foster city, ca, usa). the products of the sequencing reactions were purified using performa dtr ultra 96-well kit (edge biosystems, gaithersburg, md, usa) and sequenced in a 16-capillary abi prism 3130xl genetic analyser (applied biosystem, foster city, ca, usa). sequence data were assembled and edited with seqscape software v2.5 (applied biosystems, foster city, ca, usa). its pcr was performed in the three cutaneous samples that showed overlapping nucleotides peaks in the coii sequences. the its region (1044 pb nucleotides) was amplified with the leishmania specific primers : litsr and a new designed primer itsrr (5-agagtgagggcgcggata-3) that amplify l. donovani complex and l. major. the reaction was performed in a final volume of 50 l containing 0.5 l of dna, 5 l of pcr buffer 1x (applied biosystems, foster city, ca), 2 mm of mgcl2 (applied biosystems, foster city, ca), 0.4 m of each primer, 0.2 mm of dntps (applied biosystems, foster city, ca), and 2.5 u of amplitaq gold dna polymerase (applied biosystems, foster city, ca). amplifications were carried out in a geneamp pcr system 9700 thermal cycler (applied biosystems, foster city, ca) with the following thermal cycling profile : denaturation for 10 min at 95c, followed by 35 cycles each consisting of 30 sec at 94c, 30 sec at 50c, 1 minute at 72c, and a final extension step for 10 min at 72c. all the pcr products were analyzed on 7% acrylamide gel and visualized as described above and cloned. the three pcr products of the coii and the three pcr products of the its genes were cloned separately into the pcr - ii vector using a dual - promoter topo ta cloning kit (invitrogen, carlsbad, ca) according to the manufacturer 's instructions. plasmids with the desired inserts were isolated from positive escherichia coli colonies by using a genelute plasmid miniprep kit (sigma - aldrich, st. sequencing data were assembled and edited with seqscape software v2.5 (applied biosystems) and analyzed. phylogenetic analysis was conducted by using the neighbor - joining method with 1000 bootstrap replicates implemented in mega 5 software. overall, the diagnosis of leishmaniasis was confirmed by real - time pcr in 40 patients out of 69 (58%). the real - time pcr confirmed 58 samples out of 110 (53%) as leishmania positive. the positivity in real - time pcr was higher for hln samples (66%) compared to hbm (57%) and hb (29%). samples associated with vl, pkdl, and ml clinical forms showed a unique sequence pattern with 100% genetic homology to l. donovani ay660023.1 reference sequences deposited in genbank (figure 1). one sequence associated with vl clinical symptoms (14 hbm) showed the highest identity (98.9%) to l. infantum mhom / tn/80/ipt1 reference sequence and a similarity of 98.1% to the abovementioned l. donovani genbank reference sequence (figure 1). the genetic homology between l. donovani genbank reference sequence and l. infantum sequence used in this study was 98.7%. two sequences associated with cl samples clustered within the l. donovani group (numbers 107 and 111) with 100% genetic identity (figure 1). the remaining three cl sequences showed overlapping nucleotides peaks (figure 2) and therefore were cloned. thirty - four, twenty - nine, and thirty - six sequences were obtained from cloning of cl samples numbers 108, 109, and 110, respectively. phylogenetic analysis of the abovementioned colonies (figure 3) revealed two different sequence patterns, corresponding to l. donovani with genetic similarity range between 99.5% and 100% to l. donovani ay660023 genbank reference sequence and to l. major with genetic similarity ranging between 98.9 and 99.3% to l. major ef633106 genbank reference sequence. twenty - three, fourteen, and eighteen sequences were obtained from cloning of samples numbers 108, 109, and 110, respectively. as for coii, the phylogenetic analysis of the its cloning sequences revealed two different sequence patterns, corresponding to l. donovani with 99.5100% genetic similarity to l. donovani aj634357 genbank reference sequence and 99.8100% to l. major fj753391 genbank reference sequence. the genetic similarity between l. major and l. donovani used in this study was 93.5% (figure 4). all the representative sequences showed in the trees (figures 1, 3, and 4) have been submitted to genbank (accession numbers from kf815198 to kf815226). the coii and the its genes gave concordant results and confirmed naturally mixed infection with l. major and l. donovani from a single cutaneous aspirate in the three cl patients who presented no evident vl clinical signs. l. major and l. donovani have different transmission vectors and distinct biologic properties and epidemiologic features and both have been reported as etiologic agents of cl in sudan. cl due to l. major lon1 is transmitted by p. papatasi in arid and semi - arid areas and circulates among rodents, while the vector that transmits cl due to l. donovani has not been established yet. coinfection with different leishmania has been reported in closely related leishmania species such as l. braziliensis, l. panamensis, l. major, and l. arabica. the only two reports on concomitant natural infection with l. donovani and l. major were reported in iraq where the two species were isolated from two different sites, the bone marrow and cutaneous lesions from kala - azar suffering patients and in kenya where isolation was achieved in a spleen aspirate culture from a clinically relapsed kala - azar suffering patient. the two species l. infantum and l. major have been isolated from the spleen of vl / hiv immune - compromised patient ; l. chagasi and l. amazonensis have been isolated from a diffuse cutaneous leishmaniasis case in bolivia. in our study, the three coinfected patients were presented with typical localized cutaneous ulcers, with neither evident signs of systemic illness nor immunosuppression or vl. all three patients originated from a vl non endemic area and had no history of travelling to a known vl endemic area. however, even if visceral involvement or immunosuppression was not clinically suspected, no other clinical or laboratory examinations were carried out. the occurrence and frequency of natural infection with more than one leishmania, especially where foci of two species overlap, it has been demonstrated that in co - cultivation of more than one leishmania the dominant species tend to inhibit the growth of the other ; consequently the degree of laboratory detection of such phenomenon remains unclear and likely underestimated [28, 42 ]. in our case, the two species were likely maintained because the cultured parasites were harvested and extracted during early growth at the third day of culture. however, after cloning, the number of colonies related to l. major genetic group was higher compared to that related to l. donovani genetic group. this may suggest the dominance of l. major species in the co - culture ; however, it is also possible that the pcr conditions were more efficient towards l. major genome. assumption of circulation of more than one strain / hybrid of l. major and l. donovani was demonstrated by the presence of more than one sequence pattern of l. major and l. donovani within the same patient (figures 3 and 4). recent studies have demonstrated the presence of genetically different populations of p. papatasi in sudan that could be able to transmit different strain / hybrids. moreover, high polymorphism has been attributed to l. major causing cutaneous diseases in iran. the hypothesis of the circulation of more than one strain / hybrid of l. donovani among sudanese patients is supported by our findings after analyzing clones of the its gene and the gp63 gene from different vl patients (data not shown). however, the intraspecific variations of leishmania species among sudanese patients and its correlation with clinical signs need to be explored. the other two cutaneous cases were identified to be caused by l. donovani only as reported by other the authors. p. papatasi is not a vector of visceral leishmaniasis and is refractory to the infection by l. infantum and l. donovani [45, 46 ]. however, an experimental evidence of p. papatasi infection with hybrid strains of l. infantum / l. based on these results, p. papatasi has been suggested as vector of this hybrid in nature. all the other cases of vl (except one case), ml, and pkdl were caused by l. donovani ; however, the limited numbers of ml and pkdl samples did not allow investigating whether other species of leishmania were involved. cytochrome oxidase ii gene confirmed its good capability to discriminate among different species of leishmania. previously, an extensive research on vl based on the isoenzyme classification led to the conclusion that l. donovani is the only cause of visceral leishmaniasis in sudan. in our study, cytochrome oxidase ii revealed one sample with sequences very similar to l. infantum genetic group and further studies are needed to confirm this finding. however, the correspondence between genetic and isoenzyme classifications is still debated and a revision of the taxonomy of the genus leishmania has been proposed by other researchers [49, 50 ]. to the best of our knowledge this is the first report of l. donovani and l. major co - infection in sudan. additionally no other cases of such co - infection from the same cutaneous sample were reported. this finding has important implications regarding the diagnosis, the choice of the most appropriate therapy, and the possibility of developing drug resistance. the limited number of cutaneous samples examined does not allow predicting the frequency of this coinfection. however, the presence of l. donovani in all the five cutaneous samples suggests caution in the followup of cl patients who could later develop vl symptoms. many aspects of leishmaniasis in sudan still need to be explored, such as the prevalence of different species and vectors and the competence of phlebotomus spp. in transmitting different leishmania species. | in sudan human leishmaniasis occurs in different clinical forms, that is, visceral (vl), cutaneous (cl), mucocutaneous (ml), and post - kala - azar dermal leishmaniasis (pkdl). clinical samples from 69 sudanese patients with different clinical manifestations were subjected to a pcr targeting the cytochrome oxidase ii (coii) gene for leishmania species identification. mixed infections were suspected due to multiple overlapping peaks presented in some sequences of the coii amplicons. cloning these amplicons and alignment of sequences from randomly selected clones confirmed the presence of two different leishmania species, l. donovani and l. major, in three out of five cl patients. findings were further confirmed by cloning the its gene. regarding other samples no significant genetic variations were found in patients with vl (62 patients), pkdl (one patient), or ml (one patient). the sequences clustered in a single homogeneous group within l. donovani genetic group, with the exception of one sequence clustering with l. infantum genetic group. findings of this study open discussion on the synergetic / antagonistic interaction between divergent leishmania species both in mammalian and vector hosts, their clinical implications with respect to parasite fitness and response to treatment, and the route of transmission with respect to vector distribution and or adaptation. |
it is a benign melanocytic lesion typically < 2 mm in thickness, with an annual malignant transformation rate of one in 8,845.1 polypoidal choroidal vasculopathy (pcv) is a recurrent and relapsing chorioretinopathy characterized by grape - like subretinal vascular lesions associated with retinal pigment epithelium detachments (peds).2 indocyanine green angiography (icga) is the gold standard for pcv diagnosis. however, patients with pcv are often mistaken for patients with exudative age - related macular degeneration and typical choroidal neovascularization (cnv). we previously reported the first case in the literature of a stable pcv associated with nevus, which was managed conservatively.3 in this article, we performed a retrospective chart review following the written informed patient consent of a 78-year - old caucasian female, who had active, symptomatic pcv secondary to nevus, and was successfully treated with photodynamic therapy (pdt). this case involved a 78-year - old caucasian female with a stable left - eye superotemporal extrafoveal pigmented nevus for 20 years. funduscopy showed a pigmented lesion measuring 4.83.2 mm in basal dimensions with overlying clumped soft drusen at the posterior pole along the 2 oclock meridian (figure 1a), corresponding to a nevus at 2.0 disc diameters from fovea. a discreet orange nodule adjacent to the pigmented lesion on the nasal aspect and associated subretinal fluid (srf) was noted. optical coherence tomography (heidelberg engineering, heidelberg, germany) showed typical features of a flat nevus with ped associated with an underlying discreet polyp - like lesion at the nasal edge of the nevus and extensive srf (figure 1d). fluorescein angiography (figure 1b and c) and icga (heidelberg engineering ; figure 2a and b) demonstrated early filling of a grape - like structure suggestive of pcv with leakage in the late phase. icga also revealed an associated small branching vascular network (bvn) in both early and late phases (figure 2a and b). a diagnosis of pcv adjacent to nevus was made. due to the peripheral location of pcv and the presence of srf threatening the fovea intravenous verteporfin (6 mg / m) was followed by standard - fluence pdt (50 j / cm) at the center of the pcv with a spot size of 1,600 m over 83 seconds from a 689 nm laser. following treatment, srf was absent ; however, a persisting small ped was observed (figure 1f). icga at 7 months showed an absence of leakage, with complete regression of the polypoidal lesion (figure 2c and d). no active polyp could be detected on icga at 2 years, with a stable best - corrected visual acuity of 6/6 (0.0 logmar). studies have found that patients with choroidal nevus may develop typical cnv.47 however, pcv secondary to nevus has rarely been reported. we previously reported a case of a quiescent pcv arising from a stable choroidal nevus where the polyp was located above the nevus, however, between the retinal pigment epithelium (rpe) and bruch s membrane. this location of the pcv corresponded to a study by uyama on icga findings of japanese patients with pcv, in which they proposed that pcv may be a peculiar form of cnv beneath the rpe and above the bruch s membrane.8 similar to this, histopathological examinations revealed that vessels found at the margins of type-1 choroidal neovascular membranes tend to be matured and dilated, and therefore, pcv seemed to originate from longstanding type-1 (occult) cnv above bruch s membrane and is not a primary choroidal vascular disorder.9 we postulated that a choroidal nevus may cause chronic inflammatory or degenerative changes overlying rpe, which may result in the growth of type-1 cnv and eventually lead to pcv lesions developing adjacent to the nevus.3 in our case, optical coherence tomography and angiography revealed a discrete orange grape - like lesion below the ped with early filling and leakage in the late phase associated with a bvn and an srf, which were more typical of active pcv. some may argue the current case to represent malignant transformation of choroidal nevus, since srf was observed with an orange structure, which could be interpreted as pigment associated with choroidal melanoma. however, the pigmented lesion was flat, and the orange structure was adjacent to, not overlying, the lesion. moreover, our case responded well to pdt being applied at the center of the pcv, not over the pigmented lesion, and has remained stable for 2 years post - pdt. these features support our case to be pcv secondary to choroidal nevus rather than malignant transformation. various treatments including thermal laser therapy have been used for pcv.1015 icga - guided pdt was found to be effective in treating cnv associated with choroidal nevus ; however, variable outcomes have been reported.11 pdt is also one of the most effective treatments for pcv, resulting in acuity improvement, leakage reduction, and complete pcv regression.13,14 however, a high recurrence rate and minimal bvn regression have been documented, with possible complications such as subretinal hemorrhage and rpe tears.16 the favorable outcome of pdt in our case was most likely due to the extrafoveal location of pcv and the patient s good initial acuity. in pcv patients, vascular endothelial growth factor (vegf) was found to be elevated in rpe and vascular endothelial cells.17 intravitreal anti - vegf injections for pcv have been shown to be effective in reducing srf and improving vision ; however, vascular abnormalities have often persisted.12,15 a previous study reported that combination treatment with pdt and anti - vegf resulted in better acuity outcomes and a lower risk of developing pdt complications than photodynamic monotherapy.15 lowering vegf levels after pdt may be the key to preventing pcv recurrence and cnv development. however, koh reported that in follow - up visits of up to 6 months, although pdt combined with ranibizumab was superior to ranibizumab monotherapy in achieving complete regression of pcv, no difference was found between the combination treatment and the photodynamic monotherapy.12 in this report, we present the case of a caucasian female who developed symptomatic, active pcv at the edge of a stable choroidal nevus, which was successfully treated with one session of pdt, resulting in improved symptoms and fluid resolution, and no further treatment was required. icga is invaluable for diagnosing pcv and differentiating pcv from typical cnv, as pcv is a highly variable disorder. further studies are required to understand the mechanisms of pathogenesis and evaluate optimal treatment options. | we report a case of a caucasian female who developed active polypoidal choroidal vasculopathy (pcv) at the edge of a stable choroidal nevus and was successfully treated with verteporfin photodynamic therapy. no active polyp was detectable on indocyanine green angiography 2 years after treatment, and good vision was maintained. indocyanine green angiography is a useful investigation to diagnose pcv and may be underutilized. unlike treatment of choroidal neovascularization secondary to choroidal nevus, management of pcv secondary to nevus may not require intravitreal anti - vascular endothelial growth factor therapy. photodynamic monotherapy may be an effective treatment of secondary pcv. |
a persistent sciatic artery (psa) is a rare vascular anomaly with an estimated incidence of 0.020.04% and with a high rate of complications such as aneurysm formation, thromboembolism, and ischemia, that may lead to amputation. we present a case of a female patient with complete symptomatic ambilateral psa and with unilateral aneurysm. the aneurysm was excised and the ptfe graft was interposed at the aneurismal sac (femoro - popliteal bypass could not be performed because of the high degree hypoplasia of the superficial femoral artery). the graft endured continuous compression and stretching during regular daily life of the patient. at check - up 18 years after the operation, the doppler ultrasound showed a patent graft and no new aneurismal dilatation of the sciatic artery. to our knowledge the follow - up of the presented case is the longest reported so far in the literature. the uneventful course of the patient confirms that classical aneurysmectomy still constitutes one of the treatment options of psa aneurysm. persistent sciatic artery (psa) is a rare, but potentially serious vascular anomaly. in mammals, during the early fetal period, it connects the internal iliac artery with the popliteal artery and is the major blood supply to the lower limb in embryonic development. at that time the trunk runs down along with the sciatic nerve on the dorsal surface of the hip joint and the extensor muscles of the thigh. during further fetal development (after 6 weeks), due to position changes of the pelvis, which deteriorates the blood supply, the sciatic artery undergoes gradual involution. in mammals remains of the sciatic artery take part in the formation of the inferior gluteal artery, deep femoral artery, fibular artery and the arteries of the foot. in the literature this vascular anomaly is called persistent sciatic artery, persistent axial artery, or are represented by a descending branch of the inferior gluteal artery, which at the level between the sciatic tuberositas and major trochanter forms a long and very thin branch called the committing artery of the sciatic nerve, which accompanies the sciatic nerve down to the popliteal fossa. in the most common (complete) form of this vascular anomaly, the internal iliac artery and its continuity psa leave the minor pelvis and enter the thigh through the lower portion of the greater sciatic foramen, below the pyriform muscle, running down to the posterior femoral compartment and accompanying sciatic nerve. psa has a variable relationship to the sciatic nerve, occasionally lying within the nerve sheath. at the level of the popliteal fossa it joins the popliteal artery. the profound femoral artery is usually preserved, whereas the superficial femoral artery in most cases (over 70%) is hypoplastic or even aplastic and ends in the form of several small branches at the distal part of the thigh [13,5,7,8,10,12,13 ]. the persistent sciatic artery usually runs a torturous course, is fragile and prone to vasculopathy, atherosclerosis, and aneurysm formation mostly due to hypertension and chronic trauma. the potentially serious complications of psa include ischemia due to occlusive thrombosis or thromboembolization, formation and rupture of an aneurysm. we present a rare case of the complete symptomatic bilateral psa with hypoplastic femoral arteries and unilateral aneurysm, treated successfully, with an 18-year period of uneventful follow - up. in 1991 a 63-year - old non - smoking female was referred to the outpatient vascular service of our clinic, with a 2-year history of pain of varying intensity, localized in the left buttock, radiating down along the left thigh with concomitant paresthesias of the left foot. she was initially treated at a neurological unit. during the second year she experienced 2 incidents of acute pain of the left toe accompanied by the symptoms of the blue toe syndrome. six months before, she began to feel discomfort, compression, and pulsatile mass in her left buttock. on admission, ankle / brachial index (ab / i) on the left was 0.67 and on the right 0.86. on physical examination, the initial angiograms (figures 1, 2) revealed ambilaterally enlarged internal iliac arteries which ran down the thighs in a posterior location and joined the popliteal arteries. on the left side the superficial femoral arteries were hypoplastic, and tapered gradually, ending as small branches at the distal thighs. it was diagnosed as psa (with aneurysm), type iia, according to pillet. the aneurysm was directly exposed and excised (figure 3). to restore the blood flow, a ptfe graft # 12 (12 cm long) was implanted in the place of the aneurismal sac in an end - to - end way (figure 4). the patient was discharged on the 12 postoperative day, free of symptoms and without any neurological deficit from the sciatic nerve. however, we managed to call her for check - up on june 2009, when she was 82. she did not report any complaints that could be directly attributed to the blood supply to the lower limbs. ab / i of her left foot was 0.76 and on the right ab / i was 0.81. the doppler ultrasound showed a patent graft and no new aneurismal dilatation of the sciatic arteries. it has been estimated that in approximately 0.020.04% of adults the sciatic artery persists as the main artery supplying the lower limb [1,4,5,8,10,1114 ]. one needs to be especially aware of the psa presence in the case of hip surgery, since there may be a certain overlap of symptoms, or during renal transplantation. division of the internal iliac artery for the graft renal artery anastomosis would result in limb ischemia if there was a psa. histological study of resected sciatic artery usually demonstrates severe atherosclerotic changes, with a deficit in the collagen components of the vessel wall. psa is a rare condition, but this artery is especially prone to develop atherosclerotic changes, embolism and aneurismal formation. whereas major arteries normally follow a course on the flexor aspects of articulations, the psa courses posteriorly in the buttock and thigh and is subjected to repeated mechanical trauma (eg, compression against the edge of a chair while sitting or overstretching when walking). this may result in aneurysmal formation (unilateral aneurysm is found in over 40% of cases of psa, while bilateral aneurysm in more than 12% of psa cases), typically under the gluteus maximus muscle at the level of the greater trochanter [1,4,5,912 ]. aneurysmal complications such as rupture, thrombosis or embolism constitute a high risk of acute ischemia and are still connected with a substantial rate of limb amputations, which is why early surgical intervention is increasingly regarded as preferable in case of the symptomatic psa with aneurysm. treatment of psa depends on the type of sciatic artery involved. in a case where the sciatic artery exists as the primary arterial supply to the extremity in the so - called complete form of psa (approximately two - thirds of reported cases) revascularization of the distal extremity multiple surgical techniques are available, including interposition grafting or femoro - popliteal bypass grafting in combination with ligation or resection of the aneurysm. recently, endovascular techniques such as percutaneous embolization with coils have been tried, providing symptomatic improvement, especially in cases of incomplete psa where revascularization is usually unnecessary [5,11,1719 ]. in cases requiring continuity of the sciatic artery, percutaneous endovascular management of the lesion with stent - graft represents a further treatment option [5,11,1719 ]. however, whether or not endovascular procedures represent a reasonable alternative to classic surgical techniques is yet to be determined, because a stent graft in this location bears the potential risk for graft dislodgment or compression over the hip joint, and its durability remains an open question. in our case, because of poor perfusion, and the fact that the left distal superficial femoral artery was hypoplastic and did not supply adequate flow to the lower extremity, we could not limit our procedure to the exclusion of the aneurismal sac, or perform a femoro - popliteal bypass. additionally, endovascular procedures were not available to us at that time. that is why we decided to interpose the graft at the place of the excised aneurismal sac. we were aware that, theoretically, placing the graft in the same position as the aneurysm was not the optimal solution, because such a graft could be exposed to incidents of repeated trauma or stretching, as it had been the sciatic artery. it could increase the risk of leaks at the anastomotic sites or cause graft thrombosis. however, in this case the graft endured continuous compression during regular daily life of the patient over a period of years. in an extensive english language review of this subject, van hooft noticed that presented follow - up was short (612 months) and poorly described in most articles, thus therapeutic strategies could not be deduced from the available literature. to our knowledge the follow - up of the presented case is the longest reported so far in the literature. | summarybackgrounda persistent sciatic artery (psa) is a rare vascular anomaly with an estimated incidence of 0.020.04% and with a high rate of complications such as aneurysm formation, thromboembolism, and ischemia, that may lead to amputation.case reportwe present a case of a female patient with complete symptomatic ambilateral psa and with unilateral aneurysm. the aneurysm was excised and the ptfe graft was interposed at the aneurismal sac (femoro - popliteal bypass could not be performed because of the high degree hypoplasia of the superficial femoral artery). the graft endured continuous compression and stretching during regular daily life of the patient. at check - up 18 years after the operation, the doppler ultrasound showed a patent graft and no new aneurismal dilatation of the sciatic artery.conclusionsto our knowledge the follow - up of the presented case is the longest reported so far in the literature. the uneventful course of the patient confirms that classical aneurysmectomy still constitutes one of the treatment options of psa aneurysm. |
archaea and bacteria were also believed to synthesize lysine through diaminopimelate until it was reported that the extremely thermophilic bacterium thermus thermophilus synthesizes lysine through -aminoadipate [58 ]. during lysine biosynthesis in the budding yeast saccharomyces cerevisiae, -aminoadipate is synthesized from 2-oxoglutarate and acetyl - coa by the enzymes lys20 or lys21 (homocitrate synthase [hcs ]), lys4 (homoaconitase), lys12 (homoisocitrate dehydrogenase), and -aminoadipate aminotransferase. lysine is synthesized from -aminoadipate by the enzymes lys2 (aminoadipate reductase), lys5 (phosphopantetheinyl transferase which posttranslationally modifies lys2), lys9 (saccharopine dehydrogenase, glutamate forming), and lys1 (saccharopine dehydrogenase, lysine forming) [1, 4 ]. it has been unclear why s. cerevisiae has 2 hcss (lys20 and lys21). for example, homocitrate is mainly synthesized through lys21 during growth on ethanol, while under fermentative metabolism, lys20 and lys21 play redundant roles. it was recently reported that lys20 of s. cerevisiae functions in nuclear activities involving chromatin regulation that are distinct from its previously established role in lysine synthesis. lys20 of s. cerevisiae is linked to the dna damage repair process via the histone acetyltransferase esa1 and the h2a.z histone variant. i selected 71 hcss (31 from saccharomycotina species, 30 from pezizomycotina species, 2 from taphrinomycotina species, and 8 from basidiomycota species) based on blastp results in the fungal genome database at ncbi (http://www.ncbi.nlm.nih.gov/projects/genome/guide/fungi/). multiple alignments were generated with clustal w. a maximum likelihood tree was reconstructed using mega version 5. the -distributed rate was considered, and the number of discrete gamma categories was 3. the nucleosome position profiles were compared between the promoter (1000 bases upstream of the translational start site) and coding regions (between the translational start and end site) of the hcs genes, according to a previously described method. similarity between the two nucleosome position profiles was estimated using the spearman 's rank correlation coefficient. the hcs phylogenetic tree (figure 1) indicates that the hcs gene has been duplicated multiple times in the course of ascomycete evolution. the 31 hcss of the saccharomycotina species (ascomycetous yeasts) are encoded in 17 organisms. in contrast, the 30 hcss of the pezizomycotina species (filamentous ascomycetes) are encoded in 28 organisms. thus, 14 of the 17 saccharomycotina species and 2 of the 28 pezizomycotina species have 2 hcss (figure 1). gene duplication is not found in lys1, lys2, lys5, lys9, and their homologues. in addition, no duplication was found in lys4, lys12, and their homologues (data not shown). therefore, among the fungal lysine biosynthesis - related genes, only the hcs gene has been duplicated. phylogenetic analysis of hcss in ascomycetous yeasts showed that the s. cerevisiae hcss (lys20 and lys21) are most closely related to each other (figure 1), suggesting that hcs gene duplication occurred during evolution of the genus saccharomyces. on the other hand, all saccharomycotina species except ashbya gossypii, vanderwaltozyma polyspora, and yarrowia lipolytica have duplicated hcs genes (figure 1). thus, hcs gene duplication may be related to genome duplication events in saccharomycotina [1618 ]. in addition to the phylogenetic analysis based on hcs amino acid sequences, i compared the nucleosome positioning of lys20 and lys21. interestingly, nucleosomes were mapped to the hcs gene promoters more often than to the coding regions (figure 2). nucleosome position profiles in the coding regions were highly correlated (spearman 's rank correlation coefficient = 0.833) between lys20 and lys21. on the other hand, those in the gene promoter regions were poorly correlated (spearman 's rank correlation coefficient = 0.396). this result suggests that these 2 hcs genes have different regulatory systems. on the other hand, lys20 expression is most similar to lys21 expression, and lys21 is most similar to lys20 expression, based on the spell version 2.0.2. in addition, recent comparative analyses of orthologous genes in evolutionarily close yeasts indicated that divergence of nucleosome positioning is not correlated with divergence of gene expression [20, 21 ]. although hcs (lys20 and lys21) is located in the nucleus of s. cerevisiae, hcs is located in the cytoplasm of penicillium chrysogenum [23, 24 ]. the phylogenetic tree (figure 1) showed that gene duplication is not found in basidiomycota and taphrinomycotina. in addition, gene duplication has occurred rarely in pezizomycotina, suggesting that a common ancestor of the dikarya lacked the nuclear function of chromatin regulation. considering that duplication of the hcs gene occurred in a limited number of ascomycetes, it may not be an essential event in the evolution of dikarya. i hypothesize that after divergence of the phyla ascomycota and basidiomycota, s. cerevisiae obtained hcs bifunctionality. | most ascomycetous yeasts have 2 homocitrate synthases (hcss). among the fungal lysine biosynthesis - related genes, only the hcs gene was duplicated in the course of evolution. it was recently reported that hcs of saccharomyces cerevisiae has an additional function in nuclear activities involving chromatin regulation related to dna damage repair, which is not related to lysine biosynthesis. thus, it is possible that the bifunctionality is associated with hcs gene duplication. phylogenetic analysis showed that duplication has occurred multiple times during evolution of the ascomycetous yeasts. it is likely that the hcs gene duplication in s. cerevisiae occurred in the course of saccharomyces evolution. although the nucleosome position profiles of the two s. cerevisiae hcs genes were similar in the coding regions, they were different in the promoter regions, suggesting that they are subject to different regulatory controls. s. cerevisiae has maintained hcs activity for lysine biosynthesis and has obtained bifunctionality. |
tuberculosis is an infectious disease that continues to be major public health problem in ethiopia due to the expansion of hiv epidemic. increased disease burden caused by tb and hiv coinfection makes it critically essential to accurately detect tb infection among persons with hiv. world health organization (who) has recommended screening of active tb in all hiv positive patients, with therapy provided to active tb infection or isoniazid preventive treatment for latent tb in order to reduce mortality. however, as of 2010, among 33.3 million individuals with hiv, only 2.3 million have been checked for tb infection, and of those without active tb 178,000 were offered preventive therapy. the lack of tb screening is due in large part to the limitations of currently used screening tests. different studies have shown that the existing clinical signs and symptoms of tb in hiv - infected individuals vary from those of people who are hiv negative ; for example, many hiv - infected and culture positive tb patients are not showing cough, which is one of the common tb symptom screening questions for tb control programs. ethiopian guidelines suggest that tb screening should be performed in hiv patients by combining tb symptom screening with that of sputum afb smear microscopy and chest x - ray. however, only limited numbers of hiv - infected individuals are checked for tb due to limited resources and lack of well formulated national implementation guideline. studies have suggested that smear afb microscopy and chest x - ray, which are the common accessible tb screening tools, may not be sufficient screening tools for hiv coinfected active tb patients. there are also increased smear negative and extra pulmonary tb among hiv positive individuals, which can make the diagnosis of tb challenging. sputum culture can increase tb positivity among hiv - infected individuals, but does not always exist in resource limited areas. the new who 2011 tools for screening of tb and ipt in hiv positive people were approved and employed in cambodia and south africa by the year 2010. the tools recommend symptom screening of current cough, fever, weight loss, and night sweats and giving ipt when these symptoms are not present. the negative predictive value of this screening tool in those hiv positive people with 5% tb prevalent setting is 97.7%. the progress of active tb in hiv - infected individuals depends on immune status, high - risk condition, and way of life and contact history with active tb patients [810 ]. therefore, this study aimed to assess the performance of 2011 who tb symptom screening algorithm for diagnosing pulmonary tb and to identify potential risk factors for the existence of pulmonary tb in hiv positive individuals at the referral hospital of gondar, ethiopia. institutional based cross - sectional study was conducted from february 2012 to november 2012 at the university of gondar teaching and referral hospital among hiv / aids adults attending art clinic. the referral hospital provides care as well as treatment service for tb and hiv patients. currently there are about 9,470 hiv patients on follow - up in art department of gondar ; out of these 6,000 patients are starting art and 3,470 patients are on pre - art. the sample size was determined by single population proportion method, assuming asymptomatic tb disease prevalence of 16% in newly diagnosed hiv patients based on shah., 2009, in addis ababa, ethiopia, and considering 95% level of confidence, 5% margin of error, and 10% contingency. hiv - infected patients aged 18 years visiting the university of gondar hospital, art clinic, and who have any of tb symptoms including cough, fever, weight loss, and night sweats at the time of study were included. sociodemographic and clinical data including tb symptoms were collected for each study patient using a structured questionnaire to evaluate the performance of who 2011 tb screening algorithm for diagnosing tb among hiv patients. in addition, known risk factors for tb including housing, alcohol drinking habit, previous contact with individuals with active tb, and employment history were also collected by a medical doctor and nurses via interview during the patient visit. hiv positive individuals aged 18 years who agreed to participate and provided consent and who were on pre art or are currently receiving art and those who had previous tb history were included. those hiv positive individuals with severe disease unable to give sputum specimen were excluded from the study. the spot - morning - spot sputum specimens were collected from eligible hiv patients to this study by the laboratory technologists at the university of gondar hospital art laboratory. a direct afb smear microscopy was performed using the conventional ziehl - neelsen staining technique at the school of biomedical & laboratory sciences research laboratory (biomedical complex laboratory), and then, the three sputum samples were pooled together in 20 ml sterile screw capped universal test tubes and kept in deep freeze at 20c and all sputum samples were transported in ice box (at + 4c) to the core research laboratory at armauer hansen research institute (ahri), addis ababa, for sputum culture. about 1 ml of the sediment was inoculated into the conventional lj medium with 0.6% sodium pyruvate and glycerol for primary isolation. after inoculation, lj slants were held for 8 weeks at 37c and were examined visually for bacterial development and growth every day in the first week and then twice per week thereafter for the total of 8 weeks for the presence of mycobacterial colonies. it means patients who are afb positive with two initial sputum smear microscopy examinations. the sensitivity and specificity, as well as predictive value of symptoms, were calculated using presence of individual symptoms and sputum culture as a gold standard method. analyses of univariate and multivariate logistic regression were also performed to identify potential risk factors associated with the development of pulmonary tb and p value 2 weeks. this result is consistent with another study conducted by cain., which showed that extra sensitive screening approach for diagnosis of ptb by combination of symptoms as admission for ptb [4, 7, 13, 14 ] without identifying the cough period would possibly end up in the ptb path. an approach that joins tb symptom screening with afb smear microscopy and chest x - ray (e.g., recent guidelines in ethiopia) would distinguish 62% tb patients, with 82% specificity and elevated negative predictive value. due to lack of widespread accessibility of tb culture, the 3-step approach may present a suitable stability as the tool misses the smallest number of tb patients, while reducing overidentification and therapy. in the current study, a study conducted in south africa showed, the gene xpert mtb / rif assay increased tb case detection rate by 45%. therefore, we need a diagnostic method with a minimum turnaround time than culture. in the present study, the results showed that the occurrence of tb disease and the frequency of the isolates of mycobacterium isolated from tb cases were not affected either by age, sex, residence area, occupation, marital status, educational status, alcohol intake, previous contact with tuberculosis patients, and tb symptoms in ptb (p > 0.05). our result is consistent with other observations made by alemie and gebreselassie, 2014, and iwnetu., 2009. this might be due to the fact that all patients in this study who had history of tb infection did complete the full dose of antituberculosis treatment and were declared as cured cases. secondly, we were incapable of comparing the performance of the new with that of the old who algorithm. thirdly, patients without symptoms were not included so we could not compare the performance of the guideline with those patients with tb symptom. in addition, we applied solid culture as standard for identification of pulmonary tb. culturing various samples on liquid media would have identified further tb confirmed patients [18, 19 ] and affected the sensitivity and specificity of diagnostic method in hiv - infected tb suspected individuals. the diagnostic accuracy of the new who tb symptom screening tool to diagnose pulmonary tb is good and minimizes tb morbidity and mortality among hiv patients. the guideline has also implications for conclusions to start tuberculosis preventive treatment for patients in the art service. | the new who 2011 guidelines on tb screening among hiv - infected individuals recommend screening using four tb symptoms (current cough, fever, weight loss, and night sweats). this study aimed to assess the performance of who 2011 tb symptom screening algorithm for diagnosing pulmonary tb in hiv patients and identify possible risk factors for tb. institutional based cross - sectional study was conducted from february 2012 to november 2012. a total of 250 hiv - infected patients aged 18 years visiting the university of gondar hospital, art clinic, were enrolled. information about who tb clinical symptoms and other known risk factors for tb was collected using structured questionnaire. spot - morning - spot sputum samples were collected and direct afb microscopy, sputum culture, and rd9 molecular typing were performed. statistical data analysis was performed using spss version 20.0 software. of 250 study participants, fever was reported in 169 (67.6%), whereas cough and night sweats were reported in 167 (66.8%) and 152 (60.8%), respectively. a total of 11 (4.4%) tb cases were identified. of these, 82% (9/11) tb patients reported cough, so that the negative predictive value was 98%. in addition, 66% (158/239) tb negative patients reported cough, so that positive predictive value of cough was 5%. according to the new whotb symptom screening algorithm, out of 250 hiv - infected persons, 83% (5/6) have been investigated by tb symptom screening and afb smear microscopy. therefore, the 2011 who tb symptom screening tool for the diagnosis of pulmonary tb is likely to reduce the diagnostic delay and lower tb morbidity and mortality rate particularly in hiv prevalent settings. |
reproductive tract infections (rti) including sexually transmitted infections (sti) present a huge disease burden and adversely impact the reproductive health. their consequences are far more devastating and widespread among women as compared to men [1, 2 ]. vaginitis is one of the commonest rti and is characterized by vaginal discharge, vulvar itching / irritation, and malodor. it encompasses three main etiologies, namely, bacterial vaginosis (bv), vaginal candidiasis (vvc), and trichomoniasis (tv), which generally account for 90% of all etiologies. it is associated with an increased vaginal ph and replacement of vaginal lactobacilli with gardnerella vaginalis and anaerobic gram - negative rods. bv is of special public health concern because of the high burden of reproductive and pregnancy related morbidity. likewise high coinfection rates with other sti raise the possibility that bv increases susceptibility to sti [4, 5 ]. though majority of the infections are caused by candida albicans the incidence of non - albicans candida (nac) infection is being increasingly reported nowadays probably due to low dosage azole maintenance regimens and the use of over - the - counter antimycotics. trichomoniasis is associated with infertility, enhanced predisposition to neoplastic transformation in cervical tissues, and an increased risk of transmission of other sti, including human immunodeficiency virus (hiv), by as much as twofold [7, 8 ]. the symptoms of vaginitis are nonspecific and diagnosis without laboratory confirmation can lead to inappropriate medication. furthermore the etiological profile varies from country to country and from one region to another within the same country. the present study was undertaken for the clinicoetiological characterization of infectious vaginitis amongst women of reproductive age group attending the outpatient clinic of obstetrics and gynaecology department from navi mumbai, india. the cross - sectional observational study was conducted in the department of microbiology in association with the department of obstetrics and gynaecology at mgm medical college and hospital, which is a referral and teaching hospital located in kamothe, navi mumbai, india. a part of the study was carried out at national aids research institute, pune. the study was approved by the institutional ethics committee of the mgm medical college and hospital. a total of 380 women attending the clinic from july to december 2014 were screened for symptoms of vaginitis. the inclusion criteria were women with age between 18 and 45 years and presenting with symptoms of vaginal discharge, vulval itching / irritation, and/or vaginal malodor. women who were pregnant, who were menstruating, who had received antibiotics in the past 4 weeks, and who were not consenting to participate were excluded. informed written consent was taken from patient before enrolment in the study, followed by detailed physical examination of the patient. per speculum examination was performed and the vaginal mucosa was inspected for presence of erythema, lesions, and discharge. the first swab was used to determine the vaginal ph by touching a ph strip and comparing the change against a reference reader ; it was then smeared on to a glass slide for gram staining and finally 2 drops of 10% potassium hydroxide were added to it to determine amine / fishy odour (whiff test). the gram stained slides were evaluated using nugent 's scoring system for detection of bv. a nugent score of 03 was interpreted as negative for bv and a score of 46 as intermediate while a score of 710 was interpreted as consistent with bv. the second swab was placed in screw - cap plastic tubes containing 0.5 ml of 0.9% saline to carry out wet mount microscopy for detection of tv. swab was vigorously rotated in the saline and pressed against the side of the tube to express as much fluid as possible. one drop of the expressed fluid was placed on glass slide with a cover slip and examined at magnification of 200x within 15 minutes of collection of the sample. the positive result was defined as the presence of one or more trichomonads with characteristic morphology and jerky motility. the third swab was used for candida culture on sabouraud 's dextrose agar followed by incubation at 37c for 48 h. candida isolates were identified by the standard mycological techniques like cultural characteristics, gram stain, germ tube formation, and the biochemical profile on api 20 c aux (biomerieux, france). all cases with candida as the etiological agent also had a clinical diagnosis of vvc. the graphpad statistical software (graphpad software inc., the chi - square or fisher 's exact test as applicable was applied for analysis of categorical variables. a p value of < 0.05 was considered to be statistically significant. the prevalence of infectious etiologies, namely, bacterial vaginosis, candidiasis, and trichomoniasis, was determined in these women. the median age of the women was 28 years (interquartile range, 2432) ; all of them were married and cohabiting with their husbands, while 13 (11.8%) were nulliparous, 48 (43.6%) primiparous, and the rest multiparous. the presenting symptoms were vaginal discharge 106 (96.4%), vulval itching / irritation 19 (17.3%), malodor 5 (4.5%), pain in abdomen 3 (2.7%), and dysuria 1 (0.9%). the attributes of vaginal discharge are presented in table 1. in the 110 women presenting with vaginitis, 54 (49.1%) infectious etiologies the etiological agent was identified in 47 (42.7%) women, of which 7 (14.9%) had mixed etiologies. the commonest etiology was candidiasis detected in 33/110 (30%) cases. c. albicans was isolated in 18/33 (54.5%) cases, while 15/33 (45.5%) cases had nac isolates. the nac isolated were c. glabrata (n = 10), c. tropicalis (n = 3), and c. krusei (n = 2). bv (nugent 's score 710) was observed in 19/110 (17.3%) cases. mixed etiologies detected were bv + vvc in six cases and bv + tv in one case. we observed a trend of decrease in the prevalence of infectious vaginitis with age as presented in table 2 ; however the difference was not statistically significant between the different age groups (p = 0.187). a statistically significant association between candidal infection and the presence of curdy - white discharge (0.001) and vulval itching / irritation (p = 0.007) was noted. vaginitis is the commonest rti in sexually active women and is associated with a significant risk of morbidity. the management of vaginitis remains largely empirical, though establishing correct diagnosis is the most important factor for successful treatment. variable prevalence rates of infectious vaginitis, attributable to the varied etiologies studied, the detection techniques applied, patient groups involved, and the geographical locales have been reported by studies conducted globally as well as in india [1318 ]. in the present study majority of symptomatic women had unidentified etiology. this finding requires due consideration and indicates the need for looking into other etiological agents including but not limited to gonorrhea, chlamydiasis, viruses that may lead to vaginitis. vvc was the commonest infectious etiology identified in the present study and its prevalence was consistent with earlier reports [13, 1924 ]. the distribution of candida spp. identified in women with vvc varies widely depending on the locations as well as the populations studied. recently a number of studies have shown an increasing trend of nac infection [20, 21, 26 ]. though c. albicans remained the predominant species isolated in the present study, nac were isolated in considerable proportions. furthermore in accordance with previous literature c. glabrata was the commonest nac isolated [21, 27 ]. with higher resistance levels of most nac species to the commonly prescribed azole - based treatments, the consequences for women affected by these strains might be incapacitating. hence microbiological investigation up to species level should be made mandatory for candida infections to ensure appropriate management. various studies show prevalence of bv ranges from 2.5% to 48% [14, 17, 2833 ]. these variations may be because of differences in study population, economic status, educational background, and method used for detection of bacterial vaginosis. the prevalence of bv in the present study is in accordance with other reports from india [4, 29 ]. it is noteworthy that the mixed microbial etiologies observed were all seen in association with bv. it may thus be speculated that women with bv tend to lose natural protection against genital tract infections leading to acquisition of coinfections like t. vaginalis and candida. relatively lower prevalence of trichomoniasis (1.8%) was observed in our study, a finding consistent with researchers ' earlier reports [14, 15, 18, 29, 30, 34 ]. on the contrary, a number of studies have reported a higher prevalence of tv than ours [3538 ]. these differences could be due to variation in personal hygiene practice, environment, and socioeconomic and cultural factors of the study participants. moreover, the detection of tv by conventional wet mount method in the present study might have reduced the actual prevalence. to conclude, we present here the clinical and etiological characterization of infectious vaginitis amongst women of reproductive age group from navi mumbai, india. we observed the etiological predominance of candida infection, with considerable prevalence of nac, indicating the need for microbiological investigation up to species level in cases of candida infections, to ensure appropriate management. | vaginitis is one of the commonest reproductive tract infections in sexually active women. in the present study clinicoetiological characterization of infectious vaginitis amongst 380 women of reproductive age group (1845 years) was done. bacterial vaginosis (bv) was detected by nugent 's scoring, candida infection by culture, and trichomoniasis (tv) by wet mount. one hundred and ten (28.9%) women presented with symptoms of vaginitis. the presenting symptoms were vaginal discharge 106 (96.4%), vulval itching / irritation 19 (17.3%), malodor 5 (4.5%), pain in abdomen 3 (2.7%), and dysuria 1 (0.9%). the commonest etiology detected was candida in 33 (30%) cases, of which 18 (54.5%) were c. albicans and 15 (45.5%) non - albicans candida (nac) infections. the nac isolates were c. glabrata (n = 10), c. tropicalis (n = 3), and c. krusei (n = 2). bv and tv were observed in 19 (17.3%) and 2 (1.8%) cases, respectively. a statistically significant association between candida infection and presence of curdy - white discharge (p = 0.001) and vulval itching / irritation (p = 0.007) was noted. to conclude, we observed the etiological predominance of candida infection, with considerable prevalence of nac, indicating the need for microbiological investigation up to species level in cases of candida infections, to ensure appropriate management. |
prostate cancer is one of the most prevalent cancers in male and the second most common cause of death due to cancer among males in the united states of america. men with a family history of prostate cancer are at increased risk of this disease. about 86% of men who have prostate cancer recent studies indicate that mortality from the malignancy itself is lesser than 40% in prostate cancer. androgen deprivation therapy (adt) is the basis of treatment for early and advanced stages of prostate cancer. in addition, it has some side - effects due to testosterone deprivation, including vasomotor symptoms, osteoporosis, anemia, gynecomastia and sexual dysfunction. affecting the metabolism and endocrine function of patients with prostate cancer, adt can lead to metabolic syndrome. since much prostate cancer mortality is due to causes other than malignancy and also increased use of adt for its boost to survival rates, we decided to evaluate the effect of adt on cardiovascular risk factors in prostate cancer. this cross - sectional study was performed in 2011 in seyyed - al - shohada hospital, isfahan, iran. all patients with documented metastatic prostate cancer without documented cardiovascular diseases, referred for adt to seyyed - al - shohada hospital, were included in the study. this project was approved by the ethical committee of isfahan university of medical sciences (project number 290145). after obtaining written consent from the patients, 5 cc of fasting venous blood sample was taken from each to evaluate the serum levels of total cholesterol (tc), triglyceride (tg) and fasting blood sugar (fbs) at the beginning and after 5 months of therapy. data was analyzed using the statistical package for the social sciences statistics version 20 software. spss 20 (spss inc., chicago, il, usa) paired t - tests were used for analysis. a total of 35 male patients suffering from metastatic prostate cancer (stage 4) were enrolled in this study. the mean standard deviation (sd) of patient age was 71.77 6.84 with a range of 60 - 88 years old. at the beginning of the study, the mean sd serum levels of fbs, tg and tc were 96.74 14.04 mg / dl (normal range : 65 - 100), 130.82 41.57 mg / dl (normal range 0.05). it is possible that in men who ages over 80 years old, the level of androgen are low enough that adt does nt have any effect on level of androgen. some studies indicate that there is a significant correlation between serum testosterone levels and insulin sensitivity and that insulin injection for males suffering from hypogonadism, could maintain insulin sensitivity and decrease insulin sensitivity over the time lead to impairing glucose tolerance. adt in prostate cancer may lead to an increase in tg and tc levels. in patients with a high risk of cardiovascular disease patient 's lipid profile we suggest performing another study with a larger sample size, to compare adverse effects of adt based on patients age ranges. limitations of the study are (1) although the patients were educated for diet and exercise, the uncertainly is still present. (2) the small changes in lipid profile may be due to small sample size. | background : androgen deprivation is the basis of treatment for advanced stages of prostate cancer. cardiovascular disease may be a risk factor for mortality in prostate cancer. therefore, we decided to evaluate the effect of androgen deprivation therapy (adt) on the cardiovascular risk factors in patients with prostate cancer.materials and methods : in a cross - sectional study on 2011, 35 patients suffering from metastatic prostate cancer as candidates for adt were enrolled. serum levels of fasting blood sugar (fbs), triglyceride (tg) and total cholesterol (tc) were measured at the beginning and after the 5th month of adt.results:the mean level of tg increased significantly from 130.82 41.57 mg / dl to 150.05 48.29 mg / dl (p < 0.012). furthermore, serum level of tc increased from 197.62 40.71 mg / dl to 212.54 38.25 mg / dl, which is statistically significant (p < 0.001). a non - significant increase in the serum level of fbs from 96.74 14.04 mg / dl to 99.17 15.23 mg / dl was also seen (p = 0.27).conclusion : adt in prostate cancer may lead to an increase in tg and tc levels. in patients with a high risk of cardiovascular disease patient 's lipid profile should be considered during adt. |
the primary objective of melanoma treatment is to specifically eradicate the tumor while minimizing damage to normal tissue. in order to accomplish this goal one group of proteins that exhibit selective expression in cancer includes a group of proteins whose expression is otherwise normally limited to germ cells. most of the research into germ cell proteins in cancer has focused on expression differences and immunogenic potential for vaccines. however there is an increasing effort to decipher the potential role germ cell proteins may play in oncogenesis. the first germ cell - specific antigen discovered was the melanoma antigen 1 (mage - a1) in a patient with prolonged survival despite bulky lymph node disease. ongoing research revealed a family of mage antigens expressed in many tumor types, and while also expressed in the testis, the antigens did not appear to be expressed in most normal adult tissues. with the expansion of known germ cell proteins in cancer, the term cancer testis antigen (cta) was coined to refer to those proteins that are expressed primarily in the testis or placenta and cancers but not generally in normal adult tissues. nevertheless, cta expression can be found in normal tissues, such as pancreas, brain, and liver. the term cta has been limited to proteins not expressed in more than two nongerm cell normal tissues. however, clearly germ cell proteins may still play a role in cancer even if they are found to be more widely expressed. there are now over 100 families and 255 cancer testis entries compiled in a database by ludwig institute for cancer research. the common ctas researched in cancer include mage, gage, and ssx families as well as ny - eso1 and prame. the large number of these proteins and their expression in cancer suggests a potential link between germ cell pathways and tumor development. however, it is now clear that melanoma tumors are heterogeneous, and subpopulations of cells with stem - cell - like features are present. germ cells may be considered the ultimate stem cells as they have the capacity to give rise to entirely new individuals. some of the germ pathways are thought to have evolved from dna repair pathways and serve to specifically create genetic diversity in offspring. in single cell eukaryotes, such as yeast, these pathways are activated by stress [8, 9 ]. it is possible that germ cell pathways play a critical role in allowing tumors to become more genomically diverse, survive under different metabolic conditions, and extend overall growth potential. despite recent advances in melanoma care, germ cell proteins may hold the key to new therapeutic approaches to prevent the development and evolution of cancer. this review will focus on differences in germ cell protein expression, role in diagnosis, prognosis, and therapy. further we will discuss critical germ cell pathways and consider the potential roles these pathways may play in malignant transformation. among malignancies, melanoma is one of the tumors with the highest frequency of cta expression. also included in this group are bladder, lung, ovarian, and hepatocellular cancers. the lowest levels of expression appear to be in lymphomas, renal and colon cancers. a correlation between the gene families expressed and cancer type has been identified. melanoma has been noted to express higher levels of magea, magec3, spanx, and ldh than other cta gene families. the finding that germ cell proteins are not only differentially regulated across tumor types but they are also differentially regulated within tumor subpopulations suggests a potentially complex role in tumorigenesis. this is critically important in melanoma as the diagnosis of early melanoma versus an unusual benign mole can be quite challenging. a study by farmer. on the discordance of the histopathologic diagnosis of melanoma found that 38% of the samples reviewed had two or more discordant diagnoses. recurrent nevi, combined nevi, acral nevi, deep penetrating nevi and spitz nevi, may be overdiagnosed as melanoma while certain types of melanoma such as the nevoid, desmoplastic, spitzoid, and regressed lesions may be underdiagnosed. misdiagnosis of certain lesions may lead to unnecessary invasive treatment or a malignant melanoma going untreated. in order to improve patient care it is vital to develop more accurate diagnostic tests that will aid dermatopathologists and clinicians in the difficult task of classifying ambiguous lesions. the high expression of ctas in melanoma and lack of expression in normal skin make the presence of these germ cell proteins a potential diagnostic tool (table 1). primary melanomas and benign nevi have been analyzed based on immune detection of three antigens : mage - a1 (ma454), mage - a4 (57b), and ny - eso-1 (es121). when the cta panel was expanded to include three additional antigens including mage - c1 (ct7 - 33), mage - a3 (m3h67), and gage (gage), 77% of the melanomas tested positive for at least one cta. therefore the sensitivity of potential diagnostic tests may be improved by increasing the number of antigens included in the array. other studies have found 100% specificity for mage-3 in melanoma nodal metastasis under optimal pcr conditions, but determined the need for a broader panel of ctas to increase the sensitivity. one study that used only anti - mage antibody 57b found the diagnostic potential of ctas was limited by their presence in several types of benign skin lesions. this reinforces the need for a wider range of antibodies in order to improve diagnostic abilities. the expression pattern revealed a statistically significant difference between normal skin, benign nevi, and melanoma. the prevalence of spanx in 80.9% of the melanomas tested makes it a useful target for diagnostic assays. normal skin did not show any presence of the antigen but benign nevi did display an intermediate level. this intermediate level was significantly lower than the expression in melanoma and significantly more than the expression in normal skin. additionally, mage-3 was found to be expressed in melanoma but not in normal melanocytes while prame was determined to be an adequate marker for differentiating between melanoma cells and benign nevi [15, 18 ]. the expression of prame in 88% of primary cutaneous melanomas and lack of expression in normal nevi make it particularly useful [19, 20 ]. thus germ cell protein / cta expression patterns may serve as a diagnostic tool to differentiate between malignant and benign skin lesions. in addition to potential diagnostic value, germ cell proteins may also have prognostic value. after melanoma diagnosis, clinicians determine the relative prognosis in order to anticipate further care needs. currently, prognostic information from the primary tumor is largely based on histopathologic criteria including breslow 's depth, ulceration, and mitotic rate. nevertheless, some lesions that should have had a good prognosis by these variables ultimately prove to be lethal and some with a poor prognosis never recur. a summary of ctas and their associated prognostic features is presented (table 2). according to the study by svobodov. expression of ctas has independent prognostic value for relapse - free survival (rfs), with cta negative tumors (mage - a1, mage - a4, and nyeso-1) having an rfs of 72 months compared to 45 months for positive tumors. this study found the cta prognostic value comparable to the current clinic - pathologic classifications. a study by mikhaylova. revealed a correlation between the level of expression of ctas and the differentiation of tumors with the more poorly differentiated cells exhibiting higher cta levels. further a study found a specific prognostic relationship between the stage of melanoma and antigen present. according to vourc'h - jourdain., mage - a3 was significantly related to an increase in disease - free survival when expressed in stage iii melanoma patients. there is also potential prognostic value in determining the type of cta present in the tumor. according to velazquez. primary tumors that are ny - eso1 positive are thicker than those tumors negative for this antigen, while barrow. found ny - eso1 expression in tumors of 1.1 to 4 mm significantly higher than in thin tumors less than 1 mm. also, the ny - eso1 antigen is often more associated with metastatic disease [24, 26 ]. combined thickness and metastasis properties result in a poor prognosis for ny - eso1 positive tumors. when comparing the presence of ny - eso1 to other antigens, like ctp11, there is a positive correlation between ny - eso and more advanced disease while the ctp11 antigen is found in less advanced melanoma. certain antigens have been found exclusively in melanoma metastases and not in primary tumors, such as xage. another antigen, mage - a3, was expressed at least partially in 90% of metastases, making it a positive indicator for metastatic melanoma. a study by curioni - fontecedro. found the expression of mage - c1 and mage - c2 in primary melanoma lesions to also be a significant predictor of lymph node metastasis. barrow. also found several correlations between mage - a1 and a4 and current prognostic criteria. mage - a1 showed a significant increase in expression in ulcerated and thicker tumors ; although mage - a4 displayed a similar trend, its correlation was not statistically significant. thus cta expression may have prognostic value in helping predict the potential aggressiveness of a tumor, potentially allowing clinicians to tailor therapeutic approaches accordingly. even without a complete understanding of germ cell protein function in cancers, there have been advances in germ cell protein - based therapies. however, until more is known about their function, the related therapies primarily revolve around germ cell protein immunogenicity. tumor cells are generally considered antigenic but not immunogenic, allowing them to evade host immune defenses. the limited expression of cancer testis antigens has made them a major target for immune - based therapies because their restricted expression should cause minimal side effects. melanoma - specific vaccines have been created to specific germ cell proteins in an effort to drive the patient 's own immune system to attack the cancer cells. there are several current trials to test the efficacy of such vaccines in melanoma patients, particularly mage and ny - eso1 antigens. in addition to vaccines it may be possible to make the tumors more antigenic by driving the expression of ctas. there is evidence that regulation of cancer testis genes, like mage, is dependent on dna methylation and histone acetylation. this makes epigenetic regulation by way of histone deacetylase inhibitors and dna demethylating agents a possible treatment [32, 33 ]. kit tyrosine kinase may also be important in the epigenetic control of mage and potentially a target for future therapies. found t cell receptor modification of transduced t cells, specific for ny - eso1, to be an effective therapeutic approach for melanoma. finally, combination therapies utilizing several of these approaches may have synergistic effect in treating melanoma. in addition to the role of ctas as direct immunologic targets, germ cell protein expression may also provide clinicians with insight into tumor sensitivity to certain drugs. cell lines which express at least one of three mage genes are more susceptible to tumor necrosis factor - mediated cytotoxicity, while the over - expression of genes like mage - a2 and mage - a6 may signal resistance to chemotherapy. the differences in susceptibility show the potential for tailoring therapies based on the expression of germ cell genes. thus germ cell proteins may not only serve as direct immunologic targets but they may also direct the pharmacologic therapeutic approach. while the presence of germ cell proteins in malignancies is well documented, the reason why they are expressed is not clearly understood. theories on germ cell protein expression can be grouped into three general categories including (1) accidental / nonfunctional, (2) accidental / functional, and (3) programmed / functional. the first is an accidental activation of germ cell - specific genes, which do not provide any functional benefit to the cancer cell. this scenario would include processes like widespread epigenetic regulation that would turn on germ cell genes secondarily but without any benefit to the cell. the most noted epigenetic controls in germ cell gene expression appear to be dna methylation and posttranslational histone modifications. the second category consists of the accidental activation of germ cell genes, but with a functional benefit to the cancer cells. by providing a benefit, the cancer cells that express these germ cell genes may survive and thrive better than those without expression thereby expanding the population expressing beneficial germ cell proteins. lastly, the expression of germ cell proteins may be programmed into the cell to be activated under certain conditions. primitive organisms, like yeast, tend to increase genetic recombination when they experience such stresses in order to create new phenotypes that may be better suited for the environment. a recent study by forche. found a correlation between loss of heterozygosity in candida albicans and the degree of stress the yeast was exposed to, suggesting increased levels of gene rearrangement. due to overgrowth of the local blood supply, a fraction of cancer cells would be expected to be hypoxic and possibly nutrient deprived. an evolutionary conserved programmed response to the lack of oxygen and nutrients could lead to the activation of germ cell proteins, causing genetic recombination, and genomic instability, similar to that seen in yeast, allowing cancer cells to adapt to the environment and become more suited to thrive in adverse conditions. in all three scenarios it would be expected that there would be differential expression of germ cell proteins in the tumor mass as the cancer progresses. specifically, heterogeneity has been noted in the expression of ssx, gage, and ny - eso1 [4547 ]. evidence has been found for an increase in germ cell protein expression with tumor progression. a study by barrow. found that both antigens mage - a1 and mage - a4 showed an increase in expression throughout disease progression. these patterns suggest that germ cell protein expression may be playing a role in tumor progression. the increase in expression may suggest that the germ cell phenotype confers adaptations more suitable for survival in adverse conditions. if these germ cell - specific genes are being turned on as a programmed response to stressors, their function is likely significant to survival of the cancer cells. therefore, disrupting this function may decrease the ability of cancer cells to adapt and thrive under stressful conditions. there is increasing evidence that germ cell pathways can be activated and may play a role in tumorigenesis. hypoxic stress has been noted to induce germ cell protein expression in rat kidney fibroblasts, suggesting that hypoxia by itself may be enough to turn on some of these pathways. the germ cell regulatory protein, plu1 (jarid1b), has been shown to mark a subpopulation of melanoma tumor cells required for continuous tumor growth. further an association between germ cell gene expression and brain tumors in drosophila has recently been identified. knock - down experiments of several of the germ cell proteins revealed that they played a critical role in tumor growth. together these studies support the idea that germ cell pathways may be activated due to stress or other means and that these proteins then functionally benefit the malignant state. although it is possible that germ cell protein expression is accidental / nonfunctional in cancer, given the association of upregulation with hypoxia and role in brain tumor development, it is more likely that expression of these pathways is programmed and functional in tumor development. although there have been significant advances in unraveling the pathways involved in tumorigenesis, the role of germ cell proteins in this process remains to be fully understood. a recent review by fratta. covered many of the known molecular functions of ctas ; these findings will also be briefly addressed below in the apoptosis / transcriptional regulation sections. in addition to these findings, germ cell proteins affect other major potential pathways such as metabolism, meiosis, and telomere extension that are likely to play critical roles (table 3). genes of the class i mage family have been found to be associated with the p53 corepressor, kap1. the suppression of mage by sirna and small compounds has been shown to inhibit tumor growth and induce apoptosis [51, 52 ]. the prame gene was found to repress retinoic acid signaling, a common proliferation inhibitor and apoptosis inducer. by interfering with retinoic acid receptors, thus expression of these germ cell proteins may help the cancer cells escape programmed cell death. mage - a1 was found to inhibit transcription by interacting with the transcriptional regulator, skip, and recruiting histone deactlyase 1 (hdac1). skip interacts with the notch pathway, which controls cell differentiation during embryonic and adult life. germ cells express a unique set of metabolic enzymes that allow them to utilize certain substrates more effectively. spermatocytes are able to utilize lactate, pyruvate, and glucose while spermatids are only able to use lactate. there are a number of glutamate transporters that are preferentially expressed in certain stages of spermatogenesis and allow for increased or decreased utilization of glucose. there are also specific glycolytic enzymes expressed only in spermatogenic cells, including hexokinase (hk), phosphoglycerate kinase-2 (pgk2), and glyceraldehyde 3-phosphate dehydrogenase (gapd). increased glucose utilization is an important finding in many cancers and has been referred to as the warburg effect. in this phenomenon cancer cells utilize the glycolytic pathway in a fermentative manner, resulting in an increase in lactate. even in the presence of oxygen the cancer cells rely more on fermentation than respiration. expression of germ cell enzymes may contribute to the preferential use of glucose by cancer cells. the ability of germ cells to utilize lactate is also dependent on a testis - specific enzyme, lactate dehydrogenase c or ldhc. expression of this enzyme is found in numerous human cancers, allowing the cancers to use lactate as a substrate for atp production. this is beneficial to the cancer cells in light of the warburg effect, which results in a buildup of lactate within the cell. melanoma cell lines, when compared to normal melanocytes, rely more heavily on the warburg effect. while under hypoxic conditions melanocytes and melanoma cells both showed signs of glucose fermentation to lactate, only the melanoma cells were able to use the tricarboxylic acid cycle to produce fatty acids from glutamine and lactate from glucose. the increase of lactate production and utilization in melanoma cells is accompanied by an upregulation of the germ cell protein ldhc. the change in metabolism seen in cancer cells is potentially attributable to this expression of alternate enzymes that are normally expressed in germ cells. tumors may exploit these proteins to adapt to changes in substrate concentrations that accompany abnormal cell proliferation. this display of metabolic adaptations supports the theory of programmed expression of germ cell proteins because by expanding a cell 's useable substrates for energy, the cell has an increased chance of survival. cell division requires cell cycling through mitosis ; however germ cells also have the capacity to undergo meiosis. unlike mitosis, in the first meiotic division the sister chromatids remain attached to each other and recombination occurs across homologous chromosome arms. the expression of meiosis proteins in cells attempting to undergo mitosis (termed meiomitosis) could cause genomic instability. these include spo11 a protein that creates double - strand dna breaks : scp1, a protein involved in the pairing of homologous chromosomes and hormad1 which may play a regulatory role in meiotic synapses. there are also several other meiotic proteins that may be present in melanomas such as rec8, a meiosis - specific cohesion (figure 1). during meiosis i rec8 is maintained at the centromeres and functions to bind the sister chromatids together. in meiosis the expression of rec8 during mitotic division could lead to failure of nuclear division, abnormal chromosomal segregation, and aneuploidy. thus meiosis proteins have been noted to be expressed in melanoma and could function to cause genomic instability. since telomeres shorten with every round of division, cancer cells must express a mechanism to maintain telomere length in order to protect the genetic material. in order to accomplish this task, over 90% of cancers reactivate telomerase, suggesting it is vital to their survival. one of the highest rates of normal telomerase expression is in the testis just prior to meiosis i in the primary spermatocyte. the levels of telomerase expression have been noted to be significantly higher in melanoma than in acquired and dysplastic nevi. in contrast to cell lines that fail to continue to grow in culture, immortal melanoma lines maintain telomerase expression. it is possible that the pathways leading to the activation of telomerase also activate other germ cell proteins. in summary, the expression of germ cell proteins in melanoma has the capacity to prevent cell death, alter transcription, improve energy options, promote evolution of the genome, and provide infinite growth potential. the expression of these proteins in melanoma has been demonstrated to have both diagnostic and prognostic value. it is likely that they contribute to altered metabolism, immortalization, and genomic instability in melanoma and other cancers. the high level of expression of germ cell proteins within melanoma makes the disease an ideal model for dissecting their role in tumorgenesis. these efforts may provide insights into microenvironmental processes that differentially regulate the germ cell genes which in turn likely serve to drive tumorgenesis. ultimately research unraveling these pathways may allow for the development of new therapeutic targets to control or potentially eradicate tumor cell growth. | germ cell protein expression in melanoma has been shown to correlate with malignancy, severity of disease and to serve as an immunologic target for therapy. however, very little is known about the role that germ cell proteins play in cancer development. unique germ cell pathways include those involved in immortalization, genetic evolution, and energy metabolism. there is an ever increasing recognition that within tumors there is a subpopulation of cells with stem - cell - like characteristics that play a role in driving tumorgenesis. stem cell and germ cell biology is intertwined. given the enormous potential and known expression of germ cell proteins in melanoma, it is possible that they represent a largely untapped resource that may play a fundamental role in tumor development and progression. the purpose of this paper is to provide an update on the current value of germ cell protein expression in melanoma diagnosis, prognosis, and therapy, as well as to review critical germ cell pathways and discuss the potential roles these pathways may play in malignant transformation. |
allergic rhinitis (ar) is an allergic inflammation of the nasal airways and is characterized by sneezing, nasal congestion, nasal itching, and rhinorrhea, in any combination. ar affects approximately 60 million people in the united states and the prevalence is about 1030% in adults and nearly 40% in children [13 ]. a nationwide questionnaire survey conducted with 42,886 koreans using the international study of asthma and allergies in childhood (issac) questionnaire demonstrated that 12-month prevalence of 612 and 1215 years of age with ar were 28.8% and 29.1%, respectively. patients with ar present an inflammatory ige - mediated response characterized by allergen type 2 helper t cells (th2) immunological pattern with mast cells and eosinophil activation and release of inflammatory mediators in response to exposure to allergens [57 ]. allergens are any substance, most often eaten or inhaled, that can be found in a variety of sources, such as dust, pollen, and pet dander. such allergens (antigens) induce eosinophil recruitment into the tissues and increase serum ige level. in allergic reactions, mast cells become active when an allergen binds to ige [9, 10 ]. in addition, not only mast cells but also b cells play an important role in the pathogenesis of ar [1116 ]. allergen type 2 helper t cells (th2) play a major role in the regulation of ige antibody producing b cells, mast cells, and eosinophils. moreover, th2 secrete il-4, il-5, il-6, and il-13 and regulate b cell and eosinophil mediated responses, whereas th1 produce interferon. th1/th2 cells are associated with a series of immune and inflammatory diseases, such as bacterial and viral infectious diseases [17, 18 ]. t cell - mediated immunity is an important process of elaborating antigen (ag) specific t lymphocytes to remove viral, bacterial, or parasitic infections or malignant cells. cytotoxic cd8 + t cells are t lymphocytes that kill infected, damaged, or malignant cells bearing the ag, while cd4 + t helper cells generate cytokines that can be directly poisonous to the target cells or can stimulate other t cell effector functions and b cell antibody production and mobilize powerful inflammatory mechanisms. the traditional herbal medicine hyeonggaeyeongyo - tang (hyt) is known to treat otitis media, sinusitis, tonsillitis, and a variety of otolaryngology symptoms. in japan and china, hyt is known as keigai - rengyo - to or jing jie lian qiao tang, respectively. antihistamines and steroids are the most common medications used to treat ar. however, long - term use of drug generates resistance or side effects requiring the development of new drug. therefore, in this study, we investigated the effects of hyt on allergic responses in ovalbumin- (ova-) induced ar mice model. we also measured the levels of cytokines in the serum and the level of leukocytes in the blood. hyt used in this study contains thirteen different herbal medicines : schizonepeta spica 0.47 g, forsythiae fructus 0.47 g, saposhnikoviae radix 0.93 g, angelicae gigantis radix 0.93 g, cnidii rhizoma 0.93 g, paeoniae radix alba 0.93 g, bupleuri radix 0.47 g, aurantii fructus 0.93 g, scutellariae radix 0.93 g, gardeniae fructus 0.93 g, angelicae dahuricae radix 0.93 g, platycodi radix 0.93 g, and glycyrrhizae radix 0.67 g. they were prepared by hanpoong pharm (jeonju, republic of korea) following good manufacturing practice (gmp) procedure. dried hyt powder was stored in aliquots at 80c until further analysis (table 1). male balb / c mice (aged 6 weeks) were purchased from orient (seoul, republic of korea). the average body weight of mice was 26 g. the animals were housed in air - conditioned room with a 12 h light / dark cycle. all procedures performed on the mice were approved by the animal care center of kyung hee university (approval number khuasp (se)-12 - 048). the mice were divided into five groups (n = 8) : group 1, normal saline ; group 2, ova + saline ; group 3, ova + hyt (101 mg / kg) ; group 4, ova + hyt (202 mg / kg) ; group 5, ova + hyt (404 mg / kg). the experimental procedures for allergic sensitization and challenge are summarized in figure 1. for establishment of ova - induced ar model, male 6-week - old balb / c mice were intraperitoneally sensitized with 25 ug of ova twice a week for three weeks. after three weeks, ova group of mice were sensitized by intranasal instillation of 500 ug ova in 30 ul pbs twice a week for one week. finally, mice were fed with hyt together with ova sensitization for 2 weeks. at the end of experiment, each section was stained with hematoxylin and eosin (h & e) for inflammatory cells or with toluidine blue (t.b) for mast cells counts and examined under light microscopy (olympus). mast cells and inflammatory cells were counted in 10 parts of high power fields (hpf) at 40x, 400x, and 1000x magnification. expression of cd4 + lymphocytes was detected by immunohistochemical analysis using the anti - cd4 + antibody. after a microwave treatment, the sections were treated with 3% hydrogen peroxide in pbs for 15 min to inhibit endogenous peroxidase activity of blood cells. following hydrogen peroxide treatment, the nasal sections were incubated with 5% bovine serum albumin (bsa) in pbs, contained a blocking reagent for 1 hour at room temperature. nasal sections were incubated with mouse monoclonal cd4 + antibody overnight at 4c and subsequently incubated with secondary biotinylated anti - rabbit igg for 1 h at room temperature. sections were treated with avidin - biotin hrp complex (vectastain abc kit, vector labs, ca, usa) for 30 min at 4c and stained with diaminobenzidine tetrachloride (dab) as the substrate. the slides were mounted with an aqueous mounting solution (dako, glostrup, denmark) and cover - slipped. all the sections were analyzed using an olympus microscope and images were captured using a digital video camera. blood serum was analyzed by the bio - plex multiplex cytokine assay (bio - rad laboratories, hercules, ca, usa) according to the manufacturer 's instructions. the assay was read using a luminex 100 (austin, tx) and analyzed using a bio - plex manager software. the mean concentration of cytokines (il-4, il-13, and lif) in supernatants from ova - stimulated cells over the unstimulated cells (background) was then calculated. the blood was placed in vacutainer tm tubes containing edta (bd science, usa). anticoagulated blood was processed to determine hematological parameters (wbc, lymphocytes, monocytes, eosinophils, basophils, and neutrophils) in a hemavet 950 hematology analyzer (drew scientific, inc., oxford) in accordance with manufacturer ' recommendation. suspension of spleen from normal mice under aseptic condition was prepared by homogenization in rpmi-1640 medium (containing 10% fbs, 1% ab, and 0.05 mm mercaptoethanol). the contaminating red blood cells were removed by using red blood cell lysis buffer (sigma, usa). mice splenocytes (1 10 cells / well) were plated in 96-well culture plates and incubated for 24 h. cells were treated with con a (2 ug / ml) or hyt (10, 50, 100, 200, 500, 1000, and 2000 ug / ml). after 24 h incubation, 10 ul of wst solution was added to each well of the plate, and the plates were incubated in the dark at 37c for another 2 h. optical density was measured at 450 nm using an elisa plate reader (versamax, molecular devices, ca, usa). all experiment results were expressed as the means standard deviations (sd) or means sem (n = 8) of at least three separate tests. student 's t - test was used for single variable comparisons, and a p value < 0.05 was considered statistically significant. we found that normal group shows higher weight as compared to other groups (figure 2(a)). we also found that hyt did not show any toxicity maintaining body weight (figure 2(a)). to determine whether hyt reduces infiltration of inflammatory cells into nasal cavity, we performed h & e staining on the nasal mucosa samples. the respective numbers of inflammatory cells in the nasal mucosa in ar mice model were shown to be higher than those in the normal mice. hyt decreased such infiltration of inflammatory cells into nasal cavity (figure 3(a)). the respective numbers of mast cells in the nasal mucosa in ar mice were shown to be higher than those in the normal mice. hyt decreased such infiltration of mast cells into nasal cavity (figure 4(a)). lowest hyt concentration showed the highest effect to reduce infiltration of mast cells into nasal mucosa. cd4 + t helper cells generate cytokines that can stimulate other t cell effector functions and b cell antibody production. we also performed immunocytochemistry to measure intracellular level of cd4 + (total t cells). we found that ova increased numbers of cd4 + (total t cells), while hyt decreased them in nasal cavity (figure 5). therefore, we measured the levels of cytokines in the blood samples by multiplex cytokine assay. we found that ova increases the levels of il-4, il-13, and lif, while hyt inhibits such increase (figures 6(a), 6(b), and 6(c)). it is known that allergic diseases activate eosinophils, neutrophils, monocytes, basophils, and lymphocytes [24, 25 ]. to investigate whether hyt suppresses inflammatory phenomenon, we measured leukocytes levels in cardiovascular blood samples using hemavet 950 hematology analyzer. we found that ova increases the levels of eosinophils, neutrophils, monocytes, basophils, lymphocytes, and wbc, while hyt inhibits such increase (figures 7(a), 7(b), 7(c), 7(d), 7(e), and 7(f)). finally, we measured the effect of hyt on splenocyte viability. for that purpose, we treated splenocyte with various concentrations of hyt (10, 50, 100, 200, 500, 1000, and 2000 ug / ml) and measured cell viability using wst assay. we found that con a increases cell viability, while hyt decreases it (figure 8). in this study, we investigated the effect of hyt on allergic responses in ova - induced ar mice. increase of infiltration of inflammatory and mast cells into nasal cavity is known in ova - induced ar mice. immunohistochemical study showed thathyt reduced increased number of cd4 + t cells induced by ova. moreover, induction of various cytokines due to ar was also suppressed by hyt in mice. the activation of eosinophils releases a variety of chemicals to cause inflammation and tissue injuries [26, 27 ]. a variety of cytokines are released by inflammatory stimulation [2830 ]. we found that ova increases the levels of il-4, il-13, and lif, while hyt inhibits such increase. we also found that ova increases the levels of eosinophils, neutrophils, monocytes, basophils, lymphocytes, and wbc, while hyt inhibits such increase. ar, also known as hay fever, affects approximately 20 percent of people of all ages. the risk of developing ar is much higher in people with asthma or eczema and in people who have a family history of asthma or rhinitis. ar can begin at any age, although most people first develop symptoms in childhood or young adulthood. the symptoms are often at their worst in children and in people in their 30s and 40s. the severity of symptoms tends to vary throughout life ; many people experience periods when they have no symptoms at all. the treatment of ar includes reducing exposure to allergens and other triggers, in combination with medication therapy. nasal glucocorticoids (steroids) delivered by a nasal spray are the first - line treatment for the symptoms of ar. hyt is known to treat otitis media, sinusitis, tonsillitis, and a variety of otolaryngology symptoms. in this study our present study clearly demonstrates that hyt suppresses the progression of ar induced by ova. | allergic rhinitis (ar) is an allergic inflammation of the nasal airways. the prevalence of ar is increasing worldwide. we investigated whether hyeonggaeyeongyo - tang (hyt) is effective to suppress the progression of ar induced by ovalbumin (ova). male balb / c mice were used for this study. allergic rhinitis was induced by ova. treatment with hyt was assessed to study the effect of hyt on allergic rhinitis in mice. histological analysis, immunohistochemistry, multiplex cytokine assay, blood analysis, and cell viability assay were performed to verify inhibitory effect of hyt on allergic rhinitis. hyt did not show any toxicity maintaining body weight. food intake was steady without variation in mice. hyt reduced infiltration of inflammatory cells and mast cells into nasal cavity. hyt reduced the levels of cytokines and leukocytes in the blood. hyt decreased the splenocyte cell viability. antihistamines and steroids are the most common medications used to treat allergic rhinitis. however, long - term use of drug generates resistance or side effects requiring the development of new drug. our present study clearly demonstrates that hyt suppresses the progression of allergic rhinitis induced by ova. this suggests that hyt might be a useful drug for the treatment of allergic rhinitis. |
the prevalence of epilepsy in the general population is estimated to be 40 - 70/1000 people ; people with intellectual disability (i d) have a higher rate of epilepsy diagnoses than the general population. i d was known as mental retardation earlier, and we have used these terms interchangeably in manuscript. the prevalence of epilepsy increases with the severity of i d, which approaches 7% in people with mild to moderate i d, 67% in people with severe i d, and 50 - 82% in people with profound i d. dodson reported that children with epilepsy have an intelligence quotient (iq) score that is 10 points lower than their healthy, age - matched peers. epilepsy can affect a person 's education, career, general health, mental health, and marriage, among other things. for example, epilepsy affects their health - related quality of life outcomes and their ability to function normally. children with both epilepsy and intellectual disabilities experience psychiatric disorders in 50 - 59% of cases. epilepsy does not only affect the individual but it also negatively affects an individual 's family members. in a review article, bowley and kerr reported that having i d along with epilepsy affects a person 's physical morbidity, which in turn leads to increased mortality and a greater burden on their family. the risk of death is five times higher in children with epilepsy compared with children without epilepsy. this risk is even higher in children with i d that also have neurological disorders. because epilepsy is a known neurological disorder, it follows that children who have low iq scores and epilepsy have an even higher death risk. given these correlations, a strong relationship seems to exist between epilepsy and low iq, but very few studies have investigated the nature of this relationship. if the relationship was linear within the i d population, one would potentially be able to predict how a person 's iq score relates to their likelihood of having or developing epilepsy. this organization is located in one of the poorest districts of india, in barwani. a total of 53% of the population in this district lives below the poverty line. the district has both a tribal population (68%) and a nontribal population (32%). the tribal population is more disadvantaged in terms of health, education, and employment compared with the nontribal population. agt implemented a community - based rehabilitation project in 63 villages of the barwani block with financial help from action aid india. a total of 63 villages were surveyed door - to - door, and a total of 262 children were identified as having intellectual disabilities. consent to test for epilepsy and receive comprehensive rehabilitation services as part of the cbr project was obtained from the children 's parents or grandparents. initially, two tests a developmental screening test (dst) and an indian adaptation of the vineland social maturity scale (vsms) were used to determine each child 's iq score. the average of development quotient obtained from dst and social quotient from vsms makes iq. on place of other standardized test malins intelligence scale for indian children, an adaptation of wechsler intelligence scale, dst and vsms were preferred because these tests can be administered with less time even in nonclinical settings on nonschool going children. children were also administered other standardized intelligence tests as needed. all children diagnosed with i d were referred to psychiatrists and physicians to assess and treat, respectively, any medical condition, including epilepsy. while taking case history, professional asked for any symptoms of epilepsy, if there was any symptom than that case was referred for medical evaluation for epilepsy. then, the child underwent an electroencephalogram (eeg) administered by the psychiatrist or a general physician. eeg was administered for 20 - 30 min to record brain activities in awaken condition. the eeg results were used to support a positive diagnosis but were not used to rule out epilepsy. clinicians heavily relied on the clinical history that was obtained from parents or any other eye witness family member. if available, reports from previous consultations were reviewed and taken into account before making each diagnosis. statistical software for social science (spss version 21) was used for statistical analysis. spearman 's correlation used to examine association of epilepsy with iqs and age of the study participants. x statistics is used for gender, severity of i d, socioeconomic status, cerebral palsy, down syndrome, behavior problems, coexisting disorders, and family history of mental illness, mental retardation, and epilepsy. this organization is located in one of the poorest districts of india, in barwani. a total of 53% of the population in this district lives below the poverty line. the district has both a tribal population (68%) and a nontribal population (32%). the tribal population is more disadvantaged in terms of health, education, and employment compared with the nontribal population. agt implemented a community - based rehabilitation project in 63 villages of the barwani block with financial help from action aid india. a total of 63 villages were surveyed door - to - door, and a total of 262 children were identified as having intellectual disabilities. consent to test for epilepsy and receive comprehensive rehabilitation services as part of the cbr project was obtained from the children 's parents or grandparents. mental retardation professionals (including the author) assessed children using standard diagnostic tests. initially, two tests a developmental screening test (dst) and an indian adaptation of the vineland social maturity scale (vsms) were used to determine each child 's iq score. the average of development quotient obtained from dst and social quotient from vsms makes iq. on place of other standardized test malins intelligence scale for indian children, an adaptation of wechsler intelligence scale, dst and vsms were preferred because these tests can be administered with less time even in nonclinical settings on nonschool going children. all children diagnosed with i d were referred to psychiatrists and physicians to assess and treat, respectively, any medical condition, including epilepsy. while taking case history, professional asked for any symptoms of epilepsy, if there was any symptom than that case was referred for medical evaluation for epilepsy. then, the child underwent an electroencephalogram (eeg) administered by the psychiatrist or a general physician. eeg was administered for 20 - 30 min to record brain activities in awaken condition. the eeg results were used to support a positive diagnosis but were not used to rule out epilepsy. clinicians heavily relied on the clinical history that was obtained from parents or any other eye witness family member. if available, reports from previous consultations were reviewed and taken into account before making each diagnosis. statistical software for social science (spss version 21) was used for statistical analysis. spearman 's correlation used to examine association of epilepsy with iqs and age of the study participants. x statistics is used for gender, severity of i d, socioeconomic status, cerebral palsy, down syndrome, behavior problems, coexisting disorders, and family history of mental illness, mental retardation, and epilepsy. we found epilepsy highly associated with male gender than female (x 7.399, p = 0.005) and not equally distributed with severity of i d (x 57.21, p = 0.001). epilepsy found strongly associated with the children those have family history of mental illness (x 7.389, p = 0.007), mental retardation (x 9.42, p = 0.003), and epilepsy (x 53.2, p = 0.001). except psychiatric disorders and down 's syndrome, epilepsy is found more among i d children those had cerebral palsy (x 33.97, p = 0.001), and behavior disorders (x 5.709, p = 0.01). the occurrence of epilepsy is not different in population groups (x 0.742, p = 0.427), and among socioeconomic groups (x 2.608, p = 0.456) [table 1 ]. the association of epilepsy with different variables : gender, socioeconomic status, population type, severity of intellectual disability, family history of mental illness, mental retardation, epilepsy, and coexisting disorder. values are given for x statistics and their corresponding p values table 2 it expresses spearman 's rho statistics of epilepsy with the ordinal variables age and intelligence quotient. epilepsy had inverse correlation with age (p = 0.683), and intelligence quotient (p = 0.605) among children with intellectual disability. graphs demonstrate that prevalence of epilepsy decreases with the age [figure 1 ] and intelligence quotient [figure 2 ] of intellectual disability children. spearman correlation rho (p) of prevalence of epilepsy with age and intelligence quotient in children with intellectual disability graph showing prevalence of epilepsy with the age of intellectual disability children graph showing prevalence of epilepsy with the intelligence quotient of intellectual disability children spearman 's p demonstrates that lower iq scores are correlated with epilepsy in children with i d. a case - control study conducted in north india demonstrated that children with generalized epilepsy have lower iq scores than their controls with not epilepsy. a linear decline in iq epilepsy also found more associated with male gender and that was consistent with our study. children with i d those had family history of mental illness, mental retardation and epilepsy shown higher chances of having epilepsy. it is already known that people with i d have higher rates of epilepsy. further inferring from findings, we can say that people with i d are on higher risk than non - id and this risk of epilepsy goes further higher among i d as their iq score lowers. thus, professionals should use extra caution to detect epilepsy as early as person with i d come in their contact so that person can receive early diagnosis and treatment in order to protect from diminishing their cognitive abilities further. i d person those do not have epilepsy also need regular attention by professionals in order to check for any symptom of it, so that immediate care can be offered. this is a cross - sectional study that demonstrates the relationship between iq scores, age, and the occurrence of epilepsy in our study group. however, because of its limited design, the study could not be used to determine a causative relationship. in addition, epilepsy has several categories, which were not differentiated in this study. this is a cross - sectional study that demonstrates the relationship between iq scores, age, and the occurrence of epilepsy in our study group. however, because of its limited design, the study could not be used to determine a causative relationship. in addition, epilepsy has several categories, which were not differentiated in this study. despite the limitations, this study adds knowledge to the existing scientific literature by describing that a person 's likelihood of suffering from epilepsy increases as iq scores drop in children with i d. thus, we can say that children with low iq have a higher chance of displaying epilepsy and, therefore, should be routinely tested for the condition. | background : epilepsy is a disorder that is commonly found in people with intellectual disability (i d). the prevalence of epilepsy increases with the severity of i d. the objective of this study was to determine if there is an association between intelligence quotient (iq) and epilepsy in children with id.materials and methods : a total of 262 children, aged 3 - 18 years, with i d were identified as part of a community - based rehabilitation project. these children were examined for epilepsy and diagnosed by a psychiatrist and physicians based on results of electroencephalogram tests. a spearman 's correlation () was used to determine if there was an association between iq scores and the occurrence of epilepsy. x2 statistics used to examine the relationship of epilepsy with gender, socioeconomic status, population type, severity of i d, family history of mental illness, mental retardation, epilepsy, and coexisting disorder.results:spearman's rho 0.605 demonstrates inverse association of iq with epilepsy. x2 demonstrates statistically significant association (p < 0.05) with gender, severity of i d, cerebral palsy, behavior problems, and family history of mental illness, mental retardation, and epilepsy.conclusions:lower iq score in children with i d has association with occurrence of epilepsy. epilepsy is also found highly associated with male gender and lower age. |
gingival - periodontal diseases include a set of pathologies that affect the protective and insertion tissues of teeth. a wide range of clinical pictures arises from a complex interrelationship among bacterial aggression, intrinsic host response, and risk factors or disease modifiers. traditional diagnosis is done by measuring different clinical and radiographic indices : probing depth (pd), attachment level (al), and bleeding on probing (bop) and/or spontaneous bleeding, ulceration, exudate, and suppuration ; however, these methods only provide information on past destruction. given the severity of aggressive periodontitis (ap) and its tendency to advance, early detection is a major concern. variations in the onset, severity, and clinical characteristics can be used to recognize and describe different forms of periodontitis including early - onset periodontitis now called ap, and adult or chronic periodontitis (cp). both can be sub - classified as laser periodontitis (lp), moderate periodontitis (mp), and severe periodontitis (sp). aggressive periodontitis usually appears early in life, which means that the etiological agents were able to produce clinically detectable levels of disease within a fairly short time. this is crucial for understanding these diseases because it implies infection by highly virulent microflora or a high level of susceptibility of the subject to periodontal disease. its diagnosis requires the exclusion of systemic diseases that can severely deteriorate host defenses and lead to premature loss of teeth. chronic periodontitis is usually more prevalent in adults but may also be found in children and youths. the progression of cp is usually slow or moderate, but there may be short periods of rapid destruction. it may be modified by systemic conditions such as diabetes, smoking, and stress. given the severity of ap and its tendency to advance, early detection is a major concern. during the migration process of epithelial cells of the gum from deep layers toward more superficial layers, the formation of a layer of keratin which protects the gingival from direct mechanical, chemical, and bacteriological stimuli, thus preserving its integrity, is a phenomenon that translates into cytological changes which can reflect the degree of periodontal health. oral exfoliative cytology includes the study and interpretation of the features of naturally or artificially exfoliated cells from the oral mucosa. it consists of observing the morphology of superficial epithelial cells under the microscope after sampling, fixing, and staining.., 1990, suggest that these quantitative techniques based on evaluating parameters such as variations in the size of the nucleus and the cytoplasm and alterations in the nucleus / cytoplasm ratio can increase the diagnostic sensitivity of exfoliative cytology. the use of exfoliative cytology could be used as a complementary diagnosis method because it is easy to perform and allows repeated sampling without destroying the integrity of the tissue. cytological study of healthy gum shows exfoliated surface cells and a smaller proportion of intermediate cells from the stratum spinosum. there are also a few leukocyte - type transitory cells with defense functions, red blood cells, and microbial flora. the aim of this study was to analyze cytological changes of the periodontal pocket in patients with ap and cp in their clinical sub - stages lp, mp, and sp. we worked with patients aged 2454 years, of whom 41 had ap (ages 24.0 9.0) (21 male and 20 female), and 40 had cp (ages 58.3 7.0) (13 male and 27 female). the control or clinically healthy group was made up of 40 healthy individuals (ages 44.0 10.0) (18 male and 22 female). clinical and dental records were prepared for all subjects, and they signed informed consent for cytological sampling. an ethical clearance was obtained before the study by the ethical committee of the institution in compliance with the declaration of helsinki of 1975 and the who standards for observational studies. it included : plaque index (pi), gingival index (gi), pd, al, and bop using a marquis - type periodontal probe (hu - friedy, nc, usa). patients were classified according to clinical and radiographic criteria into four categories, for both ap and cp : clinically healthy : pd 3 mm. exclusion criteria for both the patients and the control group were : smoking, systemic diseases, chirurgic and nonchirurgic periodontal therapy, use of antibiotics, steroidal or nonsteroidal anti - inflammatory agents, all in the 6 months prior to the study. although smoking, medication, and systemic diseases may affect in different ways the development of the different periodontitis and are frequently associated with chronic disease, these factors were excluded. in this way in order to the cytological founds may be exclusively related to the periodontal diagnose. samples were collected from the soft wall of the periodontal pocket and gingival crevice of patients and control subjects, respectively, using specific curettes (hu - friedy, nc, usa), after removing supra - gingival bacterial plaque. the following parameters were observed under a trinocular microscope (carl zeiss axiostar, gttingen, germany) : desquamation of surface and intermediate cells, polymorphonuclear neutrophils (pmn), histiocytes, and microbial flora. wallis test for qualitative variables was used to determine the differences between the ap and cp diagnoses. when there were differences between the groups, the anova one - way test was applied, and when the differences were significant, tukey 's test was used. we worked with patients aged 2454 years, of whom 41 had ap (ages 24.0 9.0) (21 male and 20 female), and 40 had cp (ages 58.3 7.0) (13 male and 27 female). the control or clinically healthy group was made up of 40 healthy individuals (ages 44.0 10.0) (18 male and 22 female). clinical and dental records were prepared for all subjects, and they signed informed consent for cytological sampling. an ethical clearance was obtained before the study by the ethical committee of the institution in compliance with the declaration of helsinki of 1975 and the who standards for observational studies. it included : plaque index (pi), gingival index (gi), pd, al, and bop using a marquis - type periodontal probe (hu - friedy, nc, usa). patients were classified according to clinical and radiographic criteria into four categories, for both ap and cp : clinically healthy : pd 3 mm. exclusion criteria for both the patients and the control group were : smoking, systemic diseases, chirurgic and nonchirurgic periodontal therapy, use of antibiotics, steroidal or nonsteroidal anti - inflammatory agents, all in the 6 months prior to the study. although smoking, medication, and systemic diseases may affect in different ways the development of the different periodontitis and are frequently associated with chronic disease, these factors were excluded. in this way in order to the cytological founds may be exclusively related to the periodontal diagnose. samples were collected from the soft wall of the periodontal pocket and gingival crevice of patients and control subjects, respectively, using specific curettes (hu - friedy, nc, usa), after removing supra - gingival bacterial plaque. the following parameters were observed under a trinocular microscope (carl zeiss axiostar, gttingen, germany) : desquamation of surface and intermediate cells, polymorphonuclear neutrophils (pmn), histiocytes, and microbial flora. the kruskal wallis test for qualitative variables was used to determine the differences between the ap and cp diagnoses. when there were differences between the groups, the anova one - way test was applied, and when the differences were significant, tukey 's test was used. among the patients selected, those with ap were on average much younger than those with cp, confirming the influence of age on the characteristics of the two types of periodontitis. for the control group, we selected individuals of ages between those of the patients with ap and cp [table 1 ]. of all the patients with ap (n = 41), 25% (n = 10) were diagnosed with lp, 27% (n = 11) with mp and 48% (n = 20) with sp. for cp (n = 40), 25% (n = 10) were diagnosed with lp, 25% (n = 10) with mp, and 50% (n = 20) with sp. characteristics of patients with periodontitis and control subjects regarding the cytological study, figure 1 shows the surface cells and the exfoliated cells of the intermediate layer of patients with ap [figure 1a ], pc [figure 1b ] and a control individual [figure 1c ]. the first (surface cells) show increased cell volume, imprecise edges and blurry appearance of the nucleus, in a patient with periodontitis compared to the control group. they are rounded or polygonal, some with folded edges, slightly thicker cytoplasm than in surface cells ; the nucleus is central and round. the cells in the intermediate layer are slightly enlarged in ap [figure 1a ], and cp [figure 1b ] compared to the control group [figure 1c ]. surface cells and intermediate cells in patients with aggressive periodontitis (a) chronic periodontitis (b) and control group (c) 400 pap the semi - quantitative difference for surface cells was significant (p 0.05) between mp and the control group. semi - quantitative analysis of surface cells in patients with aggressive periodontitis, chronic periodontitis, and control group (c) the semi - quantitative study of intermediate cells showed statistically significant differences (p 0.001) in the quantity among groups lp, mp, and sp for patients diagnosed with ap and cp. semi - quantitative analysis of intermediate cells in patients with aggressive periodontitis and chronic periodontitis and control group (c) figures 4a c show pmns and histiocytes of the soft wall of the periodontal pocket in patients with ap, cp, and control individual, respectively. the pmns have a polymorphic nucleus and numerous granules in their cytoplasm with differential staining. more pmns and histiocytes are observed in ap [figure 4a ] than in cp [figure 4b ] and control [figure 4c ]. neutrophil polimorhphonucleate leukocytes and histiocytes in patients with aggressive periodontitis (a) chronic periodontitis (b), and control group (c) 400 pap the statistical analysis of pmns showed no significant difference (p > 0.001) between ac and cp, although there were differences (p 0.001) was found in the number and type of exfoliated cells or microbes (results not shown). the extent to which the tissues are affected determines the lp, mp, and sp clinical pictures. these pictures, as well as gingivitis, can be studied biochemically, cytologically, and histologically using specific markers. exfoliative cytology, which is a straightforward noninvasive diagnostic method that can be applied during inflammation, can be considered as a more practical technique to evaluate the oral mucosa. desquamation of gingival epithelium depends on mitotic activity of basal cells, their enzyme processes, and mechanical irritations of gingival tissue. in recent years, exfoliative biopsy has been introduced with good results in specification and diagnosis of the lesions buccales. a few studies have used exfoliative cytology to evaluate changes in the oral mucosa in gingival inflammation and periodontal disease. in periodontal inflammatory processes lange 1965 suggests an increase in the young cells in the deepest layers of the epithelium such as intermediate and prepyknotic cells, reducing the number of cells containing keratin particles. according to that study, there is a direct relationship between the degree of inflammation and reduction in keratinization. endo. 2008, found a reduction of keratinization in superficial inflammations of the gingival. our study found more superficial and intermediate cells in patients with ap than in patients with pc and the control group. these results differ from those of other authors, who found fewer of these cells in patients with chronic periodontal disease. the presence of intermediate cells in the proportion in which they appear in these studies might indicate the onset of periodontal disease in the preclinical, as yet asymptomatic stage. many authors conclude that exfoliative cytology has a certain value in the diagnosis of precancerous lesions, and particularly in determining their prognosis. there is currently no cytological study on chronic and aggressive periodontal disease ; nevertheless, our research has shown that the cytological study of the soft wall of the pocket shows tissue damage as an increase in inflammatory cells, cells from deep layers of the epithelium, and disease - related microorganisms. some studies have shown cell changes in the oral mucosa of patients with systemic diseases such as endocrine disorders and respiratory diseases. other researchers have found cytomorphometric changes in the cells of the oral mucosa of smokers, similar to those found in diabetics, caused by the chronic inflammation of the oral mucosa. changes in the oral mucosa similar to those appearing in individuals with type 2 diabetes have also been found in patients with vitamin b12 and folic acid nutritional deficiencies. histological studies on tissue samples show significant differences in the percentage of collagen fibers and type of cell population such as histiocytes and pmns, in patients with periodontal disease and gingivitis compared to healthy individuals, and also between both groups of patients. our study showed a high level of histiocytes in mp and sp for cp compared to the control group, as expected, since they are immune cell types that protect the body against infection. our results showed higher values for pmns and microbial flora in patients with cp and ap than in the group of healthy patients, providing evidence of the cellular immune system defense in these groups of patients. although the control group was made up of clinically healthy individuals, cytological examination showed the presence of pmns, histiocytes and microbial flora, due to the fact that under normal conditions, the gingival have an inflammatory infiltrate that makes up about 5% of the volume of the connective tissue. the analysis considering sex and age showed no significant difference in exfoliated cells, coinciding with the results of sguier. 2000. due to the presence of ulceration and mucosal erosion present in stomatitis and gingivitis, these cells may vary in size and shape, being mostly increased in their nuclear size, with multiple ovoid nuclei and poorly conserved cytoplasm. the use of exfoliative cytology for obtaining samples on which to apply sophisticated diagnosis techniques, cytomorphometry, analysis of dna content, and molecular analysis seems to be gaining ground as a reliable method for the diagnosis of oral cancer in its earliest stages. the main advantages of exfoliative cytology are that the technique is rapid, relatively painless, easy to perform allows repeated sampling of biological material without destroying tissue integrity, which means it can be performed repeatedly in preventive screening programs and during routine dental examinations. cytological studies of the periodontal pocket of patients with clinically diagnosed ap and cp had greater tissue damage as shown by the increase in cells from the deep epithelium layers, inflammatory cells, and microbial flora compared to the group of healthy subjects. patients with ap had more surface and intermediate epithelium cells from the periodontal pocket than cp patients and the group of healthy subjects. in patients with ap, surface cells increased with the severity of the disease. the cytological study showed that patients with ap had greater tissue damage, shown by the increase in intermediate and superficial cells of the epithelium of the periodontal pocket compared to the group of healthy subjects and to a lesser extent, to patients with cp. only superficial cells made it possible to differentiate the sub - stages of the disease. | background : oral exfoliative cytology includes the study and interpretation of the features cells exfoliated from the oral mucosa. the aim of this study was to analyze cytological changes in the periodontal pocket of patients with different clinical stages of aggressive periodontitis (ap) and chronic periodontitis (cp).materials and methods : patients aged 2454 years, of whom 41 were diagnosed with ap, 40 with cp, sub - classified as mild, moderate and severe periodontitis, and 40 healthy individuals who were the control group. samples of the epithelium of the periodontal pocket were taken for the cytological study.results:superficial and intermediate cell values were significantly greater in patients with ap than in patients with cp or the control group. histiocyte number was higher in patients with cp than in those with ap, and differed significantly in both types of periodontitis compared to the control group. there were significant differences in polymorphonuclear neutrophil leukocytes when both types of periodontitis were compared to the control group. microbial flora was statistically higher in patients with cp, and there were differences between patients with periodontitis and the control group.conclusions:the cytological study demonstrated that patients with ap had greater tissue damage, shown by the increase in intermediate and superficial cells of the epithelium of the periodontal pocket compared to the group of healthy subjects and to a lesser extent, to patients with cp. only superficial cells made it possible to differentiate the sub - stages of the disease. |
in 1996, mazzaferro.1 documented excellent survival results after liver transplantation (lt) in a highly selected group of patients with hepatocellular carcinoma (hcc) with a single tumor 329 (19%)bilobar32 (21%)0.15size median (range ; cm)2.6 (.116)0.040.47 (0.14,1.61) 5 cm15 (10%)stage i31 (20%)0.0071.17 (0.39,3.50) ii69 (44%) iii26 (17%) iv29 (19%)positive lymph nodes3 (2.0%)0.31vascular invasion microscopic33 (21%)0.0013.16 (0.92,10.93) macroscopic6 (4%) 5 cm (p = 0.04), pathological tmn stage (p = 0.007), microvi (p = 0.001), and macrovi (p 329 (19%)bilobar32 (21%)0.15size median (range ; cm)2.6 (.116)0.040.47 (0.14,1.61) 5 cm15 (10%)stage i31 (20%)0.0071.17 (0.39,3.50) ii69 (44%) iii26 (17%) iv29 (19%)positive lymph nodes3 (2.0%)0.31vascular invasion microscopic33 (21%)0.0013.16 (0.92,10.93) macroscopic6 (4%) 5 cm (p = 0.04), pathological tmn stage (p = 0.007), microvi (p = 0.001), and macrovi (p 4 cm) and multiple tumors (3) correlate with an increased incidence of vascular invasion and may provide a surrogate marker for entities that are often difficult to detect before lt. recurrence rates after lt may not simply reflect only size and number as suggested in the initial milan series, but may be a complex interplay of host- and tumor - related factors, which are still largely unknown.20 this report suggests that tumor grade, size, number, and microvi do not influence outcome after lt for hcc ; only the presence of macrovi appears to be associated with poor outcomes on multivariate analysis. macrovi and microvi were more commonly found in multicentric (three or more) and large (> 4 cm) hcc. furthermore, 21 of 22 recurrences had evidence of either microvi or macrovi on pathologic examination. because we are unable to identify these biologic factors preoperatively, markers of histopathologic or biologic variables that predict poor outcomes are extremely important.14,2022 macrovi was shown to be an independent predictor of tumor recurrence after lt in some studies.4,9,10,14 the pittsburgh group found that microvi and major vascular invasion was associated with increased risk of recurrence by multivariate analysis.4,8 in a report of 344 patients with hcc treated by lt, microvi and macrovi were associated with 4.4- and 15-fold increased risk of recurrence, respectively.8 shetty.14 found that macrovi, but not microvi, was a significant predictor of dsf and os after lt for hcc. in this study, microvi is associated with higher stage and recurrent tumors, but does not appear to be an independent factor for survival. the role of microvi on posttransplant recurrence and survival outcomes for hcc still remains unclear. several published studies have found poorly differentiated histological grade or microvi to be independent predictors of impaired survival after lt.11,1517 jonas.11 found vascular invasion and histological grade to be the only statistically significant independent predictors of poor survival after lt in 120 patients. in their study, only poorly differentiated tumors larger than 5 cm predicted the presence of microvi. but other studies involving lt and hcc were not able to corroborate poor results regarding microvi.3,14,23,24 the close relationship between histological grade and microvi may explain why microvi is often eliminated in multivariate models for analyzing tumor recurrence prognostic factors that include histological grading and vice versa.4,8,11,15,23,25,26 multiple tumors, larger tumors, and higher grade of differentiation have all been shown to be associated with microvi after resection for hcc. esnaola.13 reported that tumor size greater than 4 cm and poorly differentiated / undifferentiated histopathologic grade increased the odds of microvi by 3 and 6.3-fold, respectively, but these tumors were primarily in child class a cirrhotic. the degree of fibrosis and scarring of the liver may play a significant role in the biological behavior and significance of microvi and macrovi. we have previously shown that microvi was a significant predictor of early recurrence and death after resection for hcc in cirrhotic patients.5 most recurrences were intrahepatic and away from the staple line, suggesting that liver mobilization and manipulation may cause progression of microvi or a new tumor has developed in the presence of ongoing oncogenic stimulus from cirrhosis. for these reasons, prognostic factors for lt from resection studies should be interpreted with caution and a possible rationale why microvi is so important after resection but not after lt.12,13 lack of manipulation of the liver and intrahepatic dissection may be a potential explanation for the lack of importance of microvi with lt. because of the importance of histological features of hcc, some have advocated pre - lt biopsy to examine grade, vascular invasion, and genetic typing.13,15,20 complications of needle biopsy such as tumor tract seeding and lack of sensitivity have made routine biopsy unfavorable.27 in our experience, needle biopsy was a poor predictor of microvi or macrovi, and therefore was not considered in our pre - lt work - up for hcc (data not shown). there are several limitations to this study and therefore some of the results should be interpreted with caution. the need to standardize grading systems for hcc has long been recognized and would allow us to determine if tumor grade is indeed an important prognostic marker for recurrence and survival. few tumors in this study were graded as poorly differentiated ; moreover, in 27 patients, their grade was not determined. results from histopathological analysis are often met with inherent biases from the pathologist and comprehensive evaluation of the whole liver explant may vary among pathologists and institutions. finally, with very few tumors containing macrovi, strong conclusions about prognostic characteristics concerning macrovi can not be made in this report. whether microvi is a harbinger of macrovi or in some way correlated with a more aggressive form of hcc remains unclear. the use of pathological and biological features of the tumor may allow us to identify those patients who are at increased risk of recurrence ; then these patients should be considered for adjuvant therapy before evidence of a recurrence. because vascular invasion is more common in multicentric (> 3) hcc or large tumors (> 4 cm), we propose shortening the interval of pre - lt imaging in patients with these tumors to identify vascular invasion or rapid growth of the tumor before lt. genetic testing of tumors before lt may be a novel method to predict other prognostic factors affecting recurrence.20 several studies have reported improved posttransplant survival in hcc patients with transcatheter arterial chemoembolization (tace) or systemic therapy,2831 but overall results for adjuvant chemotherapy post - lt were disappointing.32 because of small numbers, this would be best done in a multicenter - randomized trial. we are currently evaluating the role of tumor size and number in our allocation system in lt for hcc to determine their respective predictive value for prognosis. in summary, lt for hcc can be performed with acceptable survival outcomes. a single tumor characteristic alone macrovi alone was associated with very poor outcomes after lt. extending criteria of lt for advanced hcc is possible only with better patient selection using improved pre - lt staging and identification of histopathological biological markers such as macrovi that would preclude lt. | macroscopic vascular invasion (macrovi) is associated with poor outcomes after liver transplantation (lt) for hepatocellular carcinoma (hcc). whether microvascular invasion (microvi) is associated with the same adverse prognosis is unclear. one hundred and fifty - five consecutive patients with confirmed hcc after lt from march 1991 to 2004 at our institution were reviewed. patients had to satisfy milan criteria to be accepted for lt. they were followed with surveillance images every 3 months while on the waiting list. disease - free survival (dfs) and overall survival (os) were evaluated by kaplan meier analysis. demographic, tumor, and histopathologic characteristics were tested for their prognostic significance. median follow - up after lt was 30 months. overall graft survival rates were 87, 74, and 65% at 1, 3, and 5 years, respectively. all recurrences (22/155, 14%) developed within 4 years after lt with an overall 5-year dfs of 79%. vascular invasion, either microvi or macrovi, was more likely in patients with multicentric hcc (n 3, p 4 cm (p = 0.04). tumor size > 5 cm (p = 0.04), advanced pathological tmn stage (p = 0.007), microvi (p = 0.001), and macrovi (p < 0.001) predicted poor tumor - free survival on univariate analysis, but only macrovi was significant in multivariate analysis (hazard ratio 54.2, 95% confidence interval 11, 266). furthermore, only macrovi was a significant predictor of mortality after lt (p = 0.01). macrovascular invasion is strongly associated with high rates of recurrence and diminished survival after lt whereas microvi is not an independent risk factor. |
unstable distal radius fractures requiring surgery have long been treated with diverse techniques including pinning, external fixation, nailing, and plating.12 open reduction and internal fixation with locking distal radius plates have become very popular over the last 10 years.345 however, controversy still exists regarding the complications associated with current implants and fracture patterns that are not amenable to those surgical techniques.678 low - profile dorsal plates were developed to address these problems by providing bicolumnar fixation and minimizing extensor tendon complications.910 to date, few studies have described the effectiveness of or the complications associated with minimally invasive plate osteosynthesis (mipo) techniques using these new implants.11 we studied the radiographic and functional outcomes of bicolumnar plating osteosynthesis through a minimally invasive dorsal approach in unstable distal radius fractures. thirty patients with unilateral distal radius fracture who underwent bicolumnar plate fixation with a minimally invasive dorsal approach between september 2008 and december 2010 were included in this retrospective study. in the end, 24 of 30 patients underwent clinical followup at 1 year or more were finally included in study [table 1 ]. there were 8 men and 16 women, with an average age of 53 years (range 1885 years). ten fractures were attributed to a simple ground fall and 14 fractures were due to either a fall from more than standing height or a traffic collision ; two were work - related fractures. the eligibility criteria were dorsally unstable fracture of the distal part of the radius [figure 1 ]. fracture patterns were categorized according to the mller - ao comprehensive classification12 and were confirmed by an independent orthopedic specialist. fractures of ao type a1 and b3 were intentionally excluded from our study since neither of these fracture patterns indicated dorsal plating fixation. according to the ao classification, 3 of the fractures were type a, 5 were type b, and 16 were type c. the type c fractures included 9 c1 fractures, 5 c2 fractures, and 2 c3 fractures. the average time interval between injury and surgery was 2 days (range 16 days). we used the gartland and werley scoring system13 for assessment of functional outcome with modification based on sarmiento. 's criteria of the minimum in range of motion (rom) for normal function, which consisted of extension (45), flexion (30), radial deviation (15), ulnar deviation (15), pronation (50), and supination (50).14 standard preoperative and postoperative posteroanterior and lateral wrist radiographs were obtained for each patient. we recorded radiographic parameters from the electronic images (centricity enterprise web 3.0 ; ge medical systems, paris, france) such as the degree of volar angulation, radial inclination (ri) angle, and radial height (rh) [figure 2 ]. routine followup radiographs were taken in the clinic at 6 weeks, 3 months, and 1 year after surgery. statistical analysis with a two - sample t - test was used to determine the significance of differences in radiographic parameters between postoperative radiographs and those of the 1-year followup. radiographs of the wrist joint anteroposterior and lateral views of an 18-year - old male showing a dorsally displaced, ao type c1 fracture of the distal radius with displaced fracture of the ulnar styloid radiographic parameters were measured from the electronic images including radial inclination (ri) angle, radial height (rh) and volar tilt (vt) angle all operations were performed by the single surgeon (chen, ac - y). under general anesthesia, distal radius fractures were reduced and fixed temporarily by kirschner - wire (k - wire) after realignment was confirmed using a mini c - arm image intensifier. two k - wires were used to secure the dorsal lunate facet fragment for correction of dorsal angulation. a 1 cm skin incision was made distal to the fracture side along the intermediate column and was directly deepened to enter the fourth compartment. the extensor tendons were then protected and retracted toward the ulna to explore the dorsal lunate fragment. blunt dissection was performed using a freer elevator underneath the extensor tendons until it met the second incision proximal to the fracture site. the extensor retinaculum was partially released in the distal half, and the dorsal locking plate (synthes ltd., paoli, pa, usa) was inserted along the ulnar border of the distal radius. in most cases, the dorsal locking plate was precontoured at the distal end to allow caudocephalic placement of locking screws in the subchondral area of the distal fragment. the radial sensory nerve was identified and protected. an anatomical radial locking plate (synthes ltd., paoli, pa, usa) was inserted subcutaneously along the interval between the first and second compartments. the radial styloid fragment was fixed with one or two locking screws, and the plate was secured with two screws through another incision proximally [figure 3 ]. for intraarticular fractures involving the scaphoid or lunate fossa, dorsal capsulotomy was performed through the same incision to allow direct visualization and facilitate restoration of articular congruity. k - wire was used as a joystick to mobilize the articular fragment [figure 4 ]. in cases with metaphyseal comminution, injectable bone graft substitutes were used through an 11-gauge spinal needle to fill the osseous defect. radiographs anteroposterior and lateral views of distal forearm and wrist taken 3 months after operation showing bicolumnar plate fixation for the distal radius, suture anchor fixation for the ulnar styloid, and osseous union peroperative arthroscopic views showing kirschner - wire was used as a joystick to mobilize the articular fragment. all operations were performed by the single surgeon (chen, ac - y). under general anesthesia, distal radius fractures were reduced and fixed temporarily by kirschner - wire (k - wire) after realignment was confirmed using a mini c - arm image intensifier. two k - wires were used to secure the dorsal lunate facet fragment for correction of dorsal angulation. a 1 cm skin incision was made distal to the fracture side along the intermediate column and was directly deepened to enter the fourth compartment. the extensor tendons were then protected and retracted toward the ulna to explore the dorsal lunate fragment. blunt dissection was performed using a freer elevator underneath the extensor tendons until it met the second incision proximal to the fracture site. the extensor retinaculum was partially released in the distal half, and the dorsal locking plate (synthes ltd., paoli, pa, usa) was inserted along the ulnar border of the distal radius. in most cases, the dorsal locking plate was precontoured at the distal end to allow caudocephalic placement of locking screws in the subchondral area of the distal fragment. the radial sensory nerve was identified and protected. an anatomical radial locking plate (synthes ltd., paoli, pa, usa) was inserted subcutaneously along the interval between the first and second compartments. the radial styloid fragment was fixed with one or two locking screws, and the plate was secured with two screws through another incision proximally [figure 3 ]. for intraarticular fractures involving the scaphoid or lunate fossa, dorsal capsulotomy was performed through the same incision to allow direct visualization and facilitate restoration of articular congruity. k - wire was used as a joystick to mobilize the articular fragment [figure 4 ]. in cases with metaphyseal comminution, injectable bone graft substitutes were used through an 11-gauge spinal needle to fill the osseous defect. radiographs anteroposterior and lateral views of distal forearm and wrist taken 3 months after operation showing bicolumnar plate fixation for the distal radius, suture anchor fixation for the ulnar styloid, and osseous union peroperative arthroscopic views showing kirschner - wire was used as a joystick to mobilize the articular fragment. the average final correction of deformity was 4.1 mm (range 09 mm) for rh, 7.6 (range 018) for ri, and 20.7 (range 539) for volar tilt (vt) [table 2 ]. no significant differences were found between postoperative and final rh, ri, and vt (p > 0.05). the rom of the wrist compared to the other side at 1 year followup averaged 85% of extension, 75% of flexion, 83% of radial deviation, 85% of ulnar deviation, 93% of pronation, and 85% of supination [table 3 ]. grip strength measured using an electronic dynamometer (tkk 5401 ; takei scientific instruments co., ltd., niigata, japan) was restored to 83% (range 65100%) of the other hand at the final followup evaluation. functional scores, according to the gartland and werley scoring system, were 3.7 3.6 (range 012) ; there were 14 excellent, 7 good, and 3 fair results. range of motion and grip strength three of 24 patients (13%) had surgical complications : one was osseous and two were soft tissue related. this occurred in an elderly and very osteoporotic patient of ao type c2 fracture with metaphyseal comminution. progressive vt and loss of rh with loose proximal screws were noted after 3 months of followup. this patient acquired osseous union with pain - free motion of the wrist at 6 months. as for soft tissue complications, there were no wound infections or extensor tendon ruptures. another patient asked us to remove the radial styloid locking plate because of an uncomfortable sensation when the patient played volleyball. traditionally, the dorsal approach in the distal radius may have some shortcomings including wide dissection, excursion of tendons after rerouting (especially the extensor pollicis longus), and tendon attrition caused by the dorsal implants.1516 a modified dorsal approach with separate incisions was designed to alleviate the above complications, and it proved to be an efficacious option for managing distal radius fractures with limited complications.17 in our series, good or excellent results were achieved in 21 of 24 patients. in the remaining three patients with fair results, one had proximal screw loosening and delayed union until 6 months. the overall outcomes were comparable to those of other studies that used conventional open and mini - open procedures.61819 in a successful mipo surgery, the quality of indirect reduction and fixation stability should not be compromised by smaller skin incisions and less exposure. we performed radiographic analysis in our series ; rh, ri, and vt were all perfect. in addition, there were no significant differences between postoperative and 1-year radiographic parameters, confirming that fixation was adequately secured to maintain realignment until osseous healing.20 moreover, general concerns regarding soft tissue complication after dorsal plating in the distal radius could thus be lessened by minimizing tissue dissection through separate skin incisions. although mipo techniques for distal radius fractures have also been successfully performed through a volar approach by preserving the pronator quadratus muscle,521 they may require either downsized implants or shifting to transverse skin incision distally to fit the minimal exposure involved in such techniques. a recent study compared the traditional volar approach with a volar mipo approach.22 the clinical and radiographic results were not statistically different although patient 's satisfaction was higher and pain score was lower in the mipo group. however, neurovascular complications were concerned with mipo through volar approach. whereas dorsal approach allows a direct and quick access to the distal radius. in index surgery, mipo techniques have been introduced through a modified dorsal approach using metal implants that are commonly used in conventional osteosynthesis procedures. additional surgical procedures, for example, bone grafting or open reduction of articular fragment, can also be introduced through the same skin incision over the dorsal wrist when necessary.20 several limitations of this study were noted. first, it is a retrospective study with relatively high number of patients (six patients, 20%) lost at the followup. further prospective and larger outcome studies with direct comparison to other osteosynthesis techniques are necessary to better evaluate the long term effect and superior efficacy of the dorsal mipo technique. the clinical results of a modified dorsal approach for distal radius fracture achieved in the current study were encouraging. bicolumnar double plating could provide sufficiently secure fixation and yield a low rate of complications. both functional survey and radiographic analysis confirmed that the index procedure is a feasible option for the management of dorsally unstable distal radius fractures and can result in favorable surgical outcomes. no benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article. no benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article. | background : controversy still exists regarding the current treatment modalities for unstable distal radius fractures. there are yet few articles investigating the efficacy of bicolumnar dorsal plating technique, which is designed to minimize tissue dissection while providing sufficiently secure fixation. a clinical study was performed to evaluate the feasibility of the minimally invasive plate osteosynthesis (mipo) technique using a modified dorsal approach for the treatment of distal radius fractures.materials and methods : thirty patients with unilateral distal radius fracture who underwent bicolumnar plate fixation with a minimally invasive dorsal approach between september 2008 and december 2010 were included in this retrospective study. twenty four patients (8 men and 16 women) with a mean age of 53 years (range 18 - 85 years) were available for followup of at least 1 year or more were included in final study. herein, we report the functional radiological outcomes of the study. there were three cases of ao type a fracture, five cases of ao type b fracture, and 16 cases of ao type c fracture.results:the union was achieved in all the patients. the functional results at one - year followup, assessed using the modified gartland and werley scoring system, were excellent in 14 patients, good in seven patients, and fair in three patients. the average correction of deformity was 4.1 mm for radial height, 7.6 for radial inclination, and 20.7 for volar tilt.conclusions:mipo with a dorsal approach is a feasible option for the management of displaced distal radius fractures and can result in favorable surgical outcomes. |
informed consents were signed by each patient before cardiac electrophysiology procedure. after ceasing of antiarrhythmic drugs for more than five halflife periods, paul, mn 551179983, usa or japan lifeline co., ltd, tokyo 1400002, japan) were, respectively, inserted into right ventricular apex and coronary sinus (cs) through right femoral vein and left subclavian vein. programmed cs stimulation with extrastimuli was used for inducing tachycardia. the selection of the target sites for ablation was determined by the shortest av / va interval during sinus rhythm, avrt, or right ventricular apex pacing with a 4mm standard tip ablation catheter. the energy of rfca was delivered by power of 35w and maximum temperature of 65c. a 62yearold man without structural heart disease was referred to our institution because of the longstanding episodes of palpitation. the standard 12lead electrocardiogram showed frequent pvcs, and no other baseline abnormality was found (fig. 1b), and ventricularatrial infusion was located at cs 56 which was also confirmed by rv pacing (fig. delivery of radiofrequency energy with an ablation catheter (35 w, 5060c, 60 sec) at the target site resulted in ventricularatrial disassociation with right ventricular apex pacing, and no avrt could be induced after ablation. surprisingly, the spontaneous pvcs also disappeared. to confirm the origination of the pvcs, mechanical stimulation (fig. 1d) the ablation catheter a little further into the left ventricle were used to compare the morphology of spontaneous and induced pvcs. as shown in the figures, the patient has been free from the same pvcs without any medications during 6month followup. (a) premature ventricular contractions shown by the standard 12lead electrocardiogram ; (b) tachycardia ; (c) intracardiac electrogram of ablation target ; (d) premature ventricular contractions with mechanical stimulation at ablation site (a) and pacing at ablation site (b). a 54yearold man without structural heart disease presented a history of recurrent palpitations of 2 years was admitted to our laboratory. the morphology of pvcs was very similar with the qrs waves within preexcitation electrocardiogram. during electrophysiology examination, it was revealed that the ventricular insertion was located at left posterior septum where local potential advanced surface qrs waves for about 20 ms with obvious qspattern unipolar potentials (fig. ablation at this site blocked the accessory pathway with preexciting conduction and also eliminated the pvcs at the same time. the patient has been free from the same pvcs without any medications during a followup period of 9 months. (a) preexcitation syndrome and frequent premature ventricular contractions shown by the standard 12lead electrocardiogram ; (b) intracardiac electrogram of ablation target. radiofrequency catheter ablation has been established as an effective and reliable treatment of pvcs for decades 1. in general, most of the pvcs originate from the ventricular outflow tract or left ventricular inferoseptal site 2. less commonly, pvcs can originate from the mitral annulus 3, the tricuspid annulus 4, purkinjefascicular network, left ventricular papillary muscles, and the moderator band in the right ventricle 5. usually, the pvcs and ventricular tachycardia originating from the valve annulus can be located by the accessory pathway 's algorithm based on their electrocardiogram characteristics and be compared with the target site pacing after ablation 6. the anatomic relationships of accessory pathways and ventricular muscle may cause disturbance of electrical activity of focal cardiac muscle cells in specific patients. during the accessory pathways ablating, the abnormal ventricular insertion which caused focal electrical abnormality the intracardiac electrogram confirmed that both accessory pathways and pvcs originating from the same site of the heart. as report above, the morphology of pacing induced pvcs was almost the same as spontaneous ones. however, the exact mechanism of this phenomenon is still unknown. however, reports of individuals with pvcs originating from the same anatomic sites as accessory pathways were rare. possibly there are some underlining mechanism and network between pvcs and accessory pathway which could not be recognized at this time. | key clinical messageradiofrequency catheter ablation has been used for treating cardiac arrhythmias, such as premature ventricular contractions and accessory pathway. we report two cases with successful ablation of leftsided accessory pathways and premature ventricular contractions from mitral annulus with one ablation. to our knowledge, no similar reports have been found so far. |
pro - apoptotic signaling is mediated by specific ligands and surface death receptors which are capable of transmitting it from an extra- to an intracellular environment and activating the execution of apoptosis. death receptors belong to the tumor necrosis factor (tnf) family, whose most important members are tnf - r1 and fas. the ligands for these receptors form a group of related cytokines consisting, inter alia, of tnf-, lymphotoxin (lt), fas - ligand (fasl) and trail. these ligands function in an autocrine or paracrine manner and their binding to the respective cell membrane receptors is essential for apoptotic signaling. fasl is normally synthesized as a membrane protein, but can also be released from the cell surface by proteolytic cleavage [2, 3 ]. both the fas receptor and fasl are predominantly expressed on activated cytotoxic t and nk lymphocytes (ctls). binding of fasl to its receptor is reflected in the apoptosis of ctls, consequently participating in the maintenance of immunological homeostasis. fasl expressed in some structures, such as in the testis or the anterior eye chamber, protects them from autoimmune cytotoxic lymphocyte attacks. the tumor - specific immune response, executed by ctls, is however also the key host reaction against cancer. it was revealed that the tumor expression of fasl, inducing the apoptosis of ctls, might enable the neoplasm to evade immune destruction by these cytotoxic cells. the detection of fasl and its mrna in the variety of human malignancies with different histogenesis supports the concept of fasl - mediated immune escape of the tumor cells [614 ]. in some cases, fasl and its mrna many further findings seem to confirm the hypothesis that a deregulated fas / fasl system can result in the immune escape of the tumor [16, 17 ]. resistance to apoptosis and alterations in fas signaling were observed first in breast carcinoma cell lines. several further studies on breast cancer patients indicated that fas / fasl status may have a significant impact on patient survival [12, 14, 19, 20 ]. their results, together with the evidence obtained during experiments on other solid malignancies [2127 ], suggest that the tumor levels of fas / fasl possibly will influence the prognosis of oncological patients. surprisingly, recently both fas and its ligand have gained less scientific interest. it is hardly justifiable particularly in the case of breast cancer where there is a need for new markers, enabling more precise prognosis and identification of patients who might benefit from aggressive treatment. the therapeutic decisions for breast cancer patients are still based mostly on tumor histological type and grade, its clinical stage, patient age, hormone receptor status and, recently, also on her2-neu expression. consequently, the aim of this short review is to analyze the current knowledge of the prognostic value of fas / fasl in breast cancer patients and the possibilities of clinical implementation of these molecule determinations. results of various authors suggest that the phenotype of fas - deficient primary breast tumor is more aggressive and usually reflects a worse prognosis.. revealed that the disease - free survival was significantly longer in patients with fas - positive tumors compared to the ones with fas - negative breast cancer tissues. the aforementioned results were further confirmed by reimer. and botti., who found that the fasl : fas ratio > 1 was related to significantly shorter disease - free survival. the aforementioned data were further completed with the results of our studies on breast cancer patients. they have proved significant associations between the lack of fas expression and lymph node involvement or the number of recurrences. fas expression in the primary tumor was also considerably less frequent among breast cancer patients with bone metastases compared to women without skeletal spread. moreover, negative staining for fas proved to be a significant predictor for survival free from bone metastases under univariate analysis. finally, the expression of fas was significantly less frequent in breast cancer patients in whom malignant cells infiltrated through the perilymphatic fat. simultaneously, the infiltration of paranodal fatty tissue occurred more often in cases of ductal carcinomas, larger primary tumors (pt 2) and regional lymph node involvement (pn 1), and shortened overall survival in breast cancer patients under univariate analysis [bbenek, du, kolak unpublished ]. consequently, the latter experiment also confirmed the negative prognostic value of fas deficiency in primary tumors. searching through the available literature, however, we have found markedly less information on the prognostic value of fas ligand in breast cancer patients. our studies to date revealed that the presence of fasl is characteristic for poorly differentiated breast cancer specimens the role of fasl in the skeletal spread of breast cancer seems to be similar to the role of fas. we have demonstrated that fas ligand expression in the primary tumor was considerably less frequent among breast cancer patients with bone metastases compared to women without skeletal spread. interestingly, conversely to fas, a similar association was not observed between the expression of fasl and the neoplastic infiltration of perilymphatic fat. we have previously shown that lymph node involvement was associated with the lack of fas in primary breast tumors, while it was independent from the occurrence of fas ligand. consequently, the molecular background of nodal and paranodal invasion of breast cancer seems to be similar in terms of fas / fas ligand expression. studying the influence of the tumor expression of fas ligand on the outcome of breast cancer patients, sjstrm. demonstrated that among a small panel of apoptosis - related molecules fasl was the most significant predictor of overall survival. that relationship, however, was not further confirmed by other authors. also our experiences do not support the impact of fas ligand expression on the outcome, in terms of both overall and disease - free survival. nevertheless, the lack of fas ligand expression proved to be a significant predictor for survival free from bone metastases. in conclusion, the aforementioned data indicate the considerable prognostic potential of the fas / fasl system in breast cancer patients. both the results of other authors and our own experiences indicate that the lack of these molecules is related to a significantly worse prognosis. it probably results from the resistance of fas - deficient breast tumors to the mechanisms of apoptosis. nevertheless, still many questions dealing with the direct association between expression of these molecules and the survival of breast cancer patients need to be understood. such a relationship was not demonstrated in all the analyses of survival published to date. its probability is relatively high, however, in view of significant associations found between the lack of fas / fas ligand in primary tumors and the spread of breast cancer to various locations [28, 29 ]. it is very likely that studies with longer follow - up are necessary to confirm definitively the prognostic value of the molecules studied. it was proven that some factors, insignificant in the analyses of 5- or even 10-year survival, gained their prognostic value in the context of longer follow - ups of breast cancer patients. moreover, our results suggest that the fas / fasl - dependent mechanisms of spread may be different for various target tissues [29, bbenek, du, kolak unpublished ]. also this hypothesis needs to be verified by further experiments. concluding, the expression of the fas / fas - ligand system has potential prognostic application in view of current knowledge and consequently it should be considered as an additional prognostic factor in breast cancer patients. | fas and its ligand (fasl) are known to play a crucial role in the genetically controlled mechanism of cell death, and their deregulation in cancer cells is involved in the immune escape of the tumor. the aim of this review is to analyze the current knowledge on the prognostic value of fas / fasl in breast cancer patients. both the results of other authors and our own experiences indicate that the lack of fas ligand, and particularly fas, is related to a significantly worse prognosis. it probably results from the resistance of fas - deficient breast tumors to the mechanisms of apoptosis. on the other hand, some results suggest that the fas / fasl - dependent mechanisms of tumor spread may be different for various target tissues. the expression of the fas / fas - ligand system has potential prognostic application in view of current knowledge, and consequently should be considered as an additional prognostic factor in breast cancer patients. |
by exploiting the ability of retroviruses to move genes into random sites of mammalian genomes and by exploiting some features of their replication, retrovirus vectors have been developed that select for instances in which the virus integrates into expressed genes. since integrated proviruses tag transcriptionally active sites, the vectors provide a means to identify and isolate promoters active in different cell types. furthermore, the viruses may be useful as insertional mutagens, since they select for instances in which integration occurs into expressed sites. this reduces the number of integrants needed to screen for loss of gene function and may enable genes controlling phenotypes in mammalian cells to be isolated.imagesfigure 3.figure 4. |
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the brachial plexus consists of nerve roots c5t1, which mainly innervate the muscles of the upper arm, and control the normal activities of these muscles. brachial plexus injury, particularly total root avulsion, is one of the most serious disabilities of the extremities. it has an unfavorable prognosis and may cause paralysis of part of or even the entire upper arm. therefore, treatment of brachial plexus injury is a major topic of study in the field of peripheral nerve injury. nerve transfer is currently the optimal clinic strategy for the treatment of brachial plexus injury. to achieve good results from treatment, one major goal of this literature review is to provide a comprehensive survey on the numerous intra and extra - plexal nerves that have been used in transfer procedures, both within china and overseas, to repair brachial plexus injury. the other goal is to discuss the role of candidate nerves for transfer in the surgical management of common severe brachial plexus problems encountered clinically. nerve transfer involves the reconstruction of a distal de - innervated nerve by using a proximal foreign nerve as the donor of neurons and axons to re - innervate the distal targets (chen., 1994). two types of nerves can be used : extra - plexal donor nerves and intra - plexal donor nerves (addas and midha, 2009). extra - plexal donor nerves include the intercostal nerve (icn), spinal accessory nerve, cervical plexus, phrenic nerve and contralateral c7 root. intra - plexal donor nerves include the ipsilateral nerve trunk and the bundle branch of the medial or ulnar nerves. nerve transfer is indicated in the following situations (hems, 2011) : (1) brachial plexus root avulsion or proximal intra - foraminal injury close to the spinal cord with no, or poor, nerve stump available for nerve grafting ; (2) proximal injury with a long distance to the target muscle ; (3) significant vascular or bony injuries in the region of the brachial plexus ; (4) previously failed attempts at brachial plexus or proximal nerve repair. a review of the historical precedents as well as the anatomical basis and rationale for nerve transfers in brachial plexus surgery was clearly presented 30 years ago (gu, 2005). since then, nerve transfer procedures have been increasingly performed to treat severe brachial plexus injury, and the methods of treatment for total brachial plexus root avulsion have continued to improve (mcguiness and kay, 2002 ; bertelli and ghizoni, 2003). according to the site of injury, lesions of the brachial plexus are classified as either preganglionic or postganglionic. for a postganglionic lesion, conservative treatment or nerve grafting, including nerve suture or neurolysis, can often achieve good results. extra - plexal or intra - plexal donor nerve transfer or an alternative reconstructive procedure should be considered without delay. the former involves the following nerve transfers : icn transfer to the axillary nerves, and accessory nerve transfer to the suprascapular nerves, phrenic nerve transfer to the musculocutaneous nerves, and contralateral c7 nerve transfer to the median nerves or radial nerves. in intra - plexal nerve transfer, part of the bundle branch of the ulnar or median nerves is transferred to the musculocutaneous nerves, or the branch of the radial nerve supplying the long head of the triceps brachii is transferred to the axillary nerves. the first is to re - innervate the recipient nerve as closely as possible to the target organ. the outcome of nerve transfers is enhanced when the donor and recipient are properly selected, as cortical re - adaptation is the physiological basis of functional recovery. the concept of icn transfer for repairing brachial plexus injury can be credited to seddon, tsuyama and nagano. he used the ulnar nerve as a nerve graft to connect the icns to the musculocutaneous nerve. six years later, tsuyama improved seddon 's method by directly splicing icns to the musculocutaneous nerve, thus achieving satisfactory results. after reviewing 159 cases, nagano. (1989) concluded that using the fish - mouth technique during suturing achieved the highest efficacy in nerve transfer procedures. based on these principles, gu (2005) made the following conclusions based on his own clinical practice : (1) when cutting out icns, the fourth icn should be used as the center ; (2) when transferring to the musculocutaneous nerve, at least two icns should be sutured ; (3) when cutting out three icns, transverse incisions should be made along the intercostal spaces ; (4) while cutting out at least four icns, a longitudinal incision should be made along the anterior axillary line. with innovations based on these principles, the efficacy of nerve transfer has increased to 67.39%. since then, icns have been commonly used as extra - plexal donor nerves. they are commonly transferred to the thoracodorsal nerves and the branch of the radial nerve supplying the long head of the triceps brachii to restore shoulder abduction and external rotation (terzis and kokkalis, 2008). this is of great significance for patients who have severe brachial plexus injury involving the c5t1 roots, where the sources of the donor nerves are limited (midha, 2004). better results can be achieved in patients younger than 30 years old who receive the operation within 6 months of injury (nagona, 1998). classified as an extra - plexal nerve, sans are rarely injured in patients who have brachial plexus injury (samardzi., 2000). when they were first used in 1972 since then, sans have been transferred to suprascapular and musculocutaneous nerves to restore shoulder abduction and elbow flexion, respectively. brunelli and brunelli (1991) showed that san transfer to the suprascapular nerves was the best solution because the supraspinatus and infraspinatus muscles are supplied by similar nerves. in 1987, gu proved that the efficacy of directly suturing sans to suprascapular nerves was higher than that of using a nerve graft. the closer the transfer is to the target muscle, the shorter the distance the regenerating axons have to travel and the better the functional re - innervation (addas and midha, 2009). traditionally, accessory to suprascapular nerve transfer has been accomplished through an anterior supraclavicular approach. however, there are some disadvantages of this method, such as scarring, nerve retraction or displacement, and nerve tumor. each of these complications may cause difficulties when searching for the suprascapular and accessory nerves. consequently, clinicians have started to use a posterior approach, particularly when transfer of the triceps branch of the axillary nerve is considered in combination to accessory nerve transfer (rui., 2013). colbert and mackinnon (2006) used the posterior approach, and achieved better protection to the nerves supplying the trapezius as well as better recovery of shoulder abduction in comparison to the anterior approach. plate. (2011) came to the same conclusion after treating nine patients using combined nerve transfer. the posterior approach offers the following advantages : (1) it is better for functional recovery as the anastomosis lies next to the target muscles ; (2) anatomically, the scapular nerve and accessory nerve can be easily tracked, maintaining a fixed location when descending to shoulder level ; (3) this operation relieves nerve compression syndrome of the suprascapular nerve caused by the transverse ligament. a larger number of cases will be needed to evaluate its efficacy compared with the traditional method, because the number of distal fibers of the accessory nerve is substantially low (lao, 2010). the phrenic nerve originates mainly from the fourth cervical root, while both c3 and c5 roots contribute to and augment the nerve. as a result, it contains more pure motor axons than other nerves, allowing for the possibility of partial or complete nerve transfer with great success (viterbo., 1995). injury to both c4 and c5 is uncommon as both nerve roots are strongly bound by fibrous tissue forming a chute - like structure. liu. (2014) compared the vascularized phrenic nerve transfers of 14 patients with the non - vascularized phrenic nerve transfers of 19 patients. no statistically significant difference was found between the two groups after a 3-year follow up. this is most likely because the nerve itself has a small diameter and a well - vascularized bed. in recent years, the thoracoscope has been widely used in cutting out the phrenic nerve. in 1999, preliminary results showed that this method was used to transfer the phrenic nerve to the ulnar nerve, successfully restoring thumb flexion in 20 patients. compared with traditional methods used in cutting out the phrenic nerve, the length of the excised nerve 2002) successfully cut out the full length of the phrenic nerve with the aid of a thoracoscope and transferred it to the musculocutaneous nerve. this method reduced the recovery time for elbow flexion to an average of just 5 months. (1995) showed that the lower icns of rats also participated in the innervation of the diaphragm. when the phrenic nerve is sacrificed for nerve transfer, respiratory function decreases by an average of 10% (luedemann., 2002). this can produce symptoms in high oxygen demand situations, particularly in infants and children with respiratory infections. in 2006, jiang. (2006) found a statistically significant drop in vital capacity when using the right phrenic nerve compared with the left. he demonstrated that transposition of the left phrenic nerve does not lead to severe respiratory dysfunction. consequently, clinicians should be cautious when using the right phrenic nerve if maximal inspiratory pressure is low preoperatively. until a long - term follow - up study is completed the contralateral c7 root has a large number of nerve fibers (approximately 420,00), and it was first used for nerve transfer in 1986 (gu., 1992). since then, many clinical cases and experiments have demonstrated that transfer using this root can achieve very good outcomes (kim., 2003). also, there is little risk to function by harvesting the c7 spinal nerve, providing additional sources of donor nerves (gu., contralateral c7 nerve transfer has been widely used, both within china and overseas, for shoulder and elbow restoration. this method returns grasp function and achieves sensory recovery, and is considered a major breakthrough in the treatment of brachial plexus injury. contralateral c7 nerve transfer is an optimal strategy for treating brachial plexus injury (dubuisson and kline, 2002 ; friedman., 1990), especially when root avulsion and phrenic and accessory nerve damage is encountered simultaneously. in this situation, contralateral c7 transfer is the only solution (terzis and kokkalis, 2010). the procedure for contralateral c7 nerve transfer can be divided into two stages ; stage i and stage ii (gu, 1992). in stage i, the contralateral c7 root is transferred to the nerve graft (ulnar nerve with vessel pedicle is frequently used), and in stage ii, the other part of the nerve graft is transferred to the recipient nerve, including the median nerve and radial nerve. there are numerous follow - up patient reports, providing evidence of the importance of such a division. (1999) reported that only 20% of the 96 patients who underwent contralateral c7 nerve root transfer regained forearm and hand flexor muscle strength to grade 3 or 4, measured using the modified mrc scale, within period i. lao (2010) concluded that different methods contributed to the large disparity in the proportion of patients who achieved strength recovery during periods i and ii36% and 63%, respectively. cortical plasticity appears to play an important physiological role in the functional recovery of the re - innervated muscles. recently, an increasing number of investigators have focused their attention on cerebral plasticity following contralateral cervical nerve transfer in humans. 2001) have shown that the brain is capable of extensive reorganization after the peripheral nervous system is injured (wang., 2010). contralateral c7 nerve transfer provides an ideal opportunity to study the relationship between brachial plexus injury and cortical reorganization (florence., 1996). peripheral nerve conduction testing shows that degeneration and regeneration after peripheral nerve injury are complex processes. physiological changes occur not only at the site of injury, but also upstream (brain and spinal cord) and downstream (muscles and effectors) of the damage (kemp., 2010). peripheral nerve injuries block the flow of output from the motor cortex to the denervated muscles, thereby resulting in paralysis. however, with the gradual maturation of regenerating axons, the dominant functions of the muscles eventually recover (muetn - gmez., 2004 ; (2004, 2005) showed that plastic changes indeed take place in the contralateral motor cortex of adult rats with total brachial plexus root avulsion. (2005) demonstrated changes within the primary motor cortex of patients who underwent contralateral c7 nerve translocation following brachial plexus injury using functional mri. he also proposed that earlier rehabilitation of the affected limb results in better and more extensive cortical reorganization. in 2012, liu. (2012) used contralateral c7 nerve transfer to treat six patients with central spastic hemiplegia. after a 2-year follow - up, they concluded that functional recovery of the affected limb, particularly of the wrist and elbow, was satisfactory. in patients with total root avulsion of the brachial plexus, elbow flexion is restored by icn transfer, and shoulder function is restored by shoulder arthrodesis. hand function is restored by an icn transfer for the median nerve, or by free muscle transplantation in combination with nerve transfer (nagano, 1998). different neural transplantation approaches for the treatment of brachial plexus injury provide differential outcomes, but direct transplantation of collateral branches are associated with better outcome compared with the use of other nerve plexuses, especially in patients with delayed treatment (bertelli and ghizoni, 2010). the concept of icn transfer for repairing brachial plexus injury can be credited to seddon, tsuyama and nagano. he used the ulnar nerve as a nerve graft to connect the icns to the musculocutaneous nerve. six years later, tsuyama improved seddon 's method by directly splicing icns to the musculocutaneous nerve, thus achieving satisfactory results. after reviewing 159 cases, nagano. (1989) concluded that using the fish - mouth technique during suturing achieved the highest efficacy in nerve transfer procedures. based on these principles, gu (2005) made the following conclusions based on his own clinical practice : (1) when cutting out icns, the fourth icn should be used as the center ; (2) when transferring to the musculocutaneous nerve, at least two icns should be sutured ; (3) when cutting out three icns, transverse incisions should be made along the intercostal spaces ; (4) while cutting out at least four icns, a longitudinal incision should be made along the anterior axillary line. with innovations based on these principles, the efficacy of nerve transfer has increased to 67.39%. since then, icns have been commonly used as extra - plexal donor nerves. they are commonly transferred to the thoracodorsal nerves and the branch of the radial nerve supplying the long head of the triceps brachii to restore shoulder abduction and external rotation (terzis and kokkalis, 2008). this is of great significance for patients who have severe brachial plexus injury involving the c5t1 roots, where the sources of the donor nerves are limited (midha, 2004). better results can be achieved in patients younger than 30 years old who receive the operation within 6 months of injury (nagona, 1998). classified as an extra - plexal nerve, sans are rarely injured in patients who have brachial plexus injury (samardzi., 2000). when they were first used in 1972 since then, sans have been transferred to suprascapular and musculocutaneous nerves to restore shoulder abduction and elbow flexion, respectively. brunelli and brunelli (1991) showed that san transfer to the suprascapular nerves was the best solution because the supraspinatus and infraspinatus muscles are supplied by similar nerves. in 1987, gu proved that the efficacy of directly suturing sans to suprascapular nerves was higher than that of using a nerve graft. the closer the transfer is to the target muscle, the shorter the distance the regenerating axons have to travel and the better the functional re - innervation (addas and midha, 2009). traditionally, accessory to suprascapular nerve transfer has been accomplished through an anterior supraclavicular approach. however, there are some disadvantages of this method, such as scarring, nerve retraction or displacement, and nerve tumor. each of these complications may cause difficulties when searching for the suprascapular and accessory nerves. consequently, clinicians have started to use a posterior approach, particularly when transfer of the triceps branch of the axillary nerve is considered in combination to accessory nerve transfer (rui. colbert and mackinnon (2006) used the posterior approach, and achieved better protection to the nerves supplying the trapezius as well as better recovery of shoulder abduction in comparison to the anterior approach. plate. (2011) came to the same conclusion after treating nine patients using combined nerve transfer. the posterior approach offers the following advantages : (1) it is better for functional recovery as the anastomosis lies next to the target muscles ; (2) anatomically, the scapular nerve and accessory nerve can be easily tracked, maintaining a fixed location when descending to shoulder level ; (3) this operation relieves nerve compression syndrome of the suprascapular nerve caused by the transverse ligament. a larger number of cases will be needed to evaluate its efficacy compared with the traditional method, because the number of distal fibers of the accessory nerve is substantially low (lao, 2010). the phrenic nerve originates mainly from the fourth cervical root, while both c3 and c5 roots contribute to and augment the nerve. as a result, it contains more pure motor axons than other nerves, allowing for the possibility of partial or complete nerve transfer with great success (viterbo., 1995). for a complete phrenic nerve, injury to both c4 and c5 is uncommon as both nerve roots are strongly bound by fibrous tissue forming a chute - like structure. liu. (2014) compared the vascularized phrenic nerve transfers of 14 patients with the non - vascularized phrenic nerve transfers of 19 patients. no statistically significant difference was found between the two groups after a 3-year follow up. this is most likely because the nerve itself has a small diameter and a well - vascularized bed. in recent years, the thoracoscope has been widely used in cutting out the phrenic nerve. in 1999, preliminary results showed that this method was used to transfer the phrenic nerve to the ulnar nerve, successfully restoring thumb flexion in 20 patients. compared with traditional methods used in cutting out the phrenic nerve, the length of the excised nerve can be increased by using a thoracoscope. in 2002, xu. (2002) successfully cut out the full length of the phrenic nerve with the aid of a thoracoscope and transferred it to the musculocutaneous nerve. this method reduced the recovery time for elbow flexion to an average of just 5 months. (1995) showed that the lower icns of rats also participated in the innervation of the diaphragm. when the phrenic nerve is sacrificed for nerve transfer, respiratory function decreases by an average of 10% (luedemann., 2002). this can produce symptoms in high oxygen demand situations, particularly in infants and children with respiratory infections. in 2006, jiang. (2006) found a statistically significant drop in vital capacity when using the right phrenic nerve compared with the left. he demonstrated that transposition of the left phrenic nerve does not lead to severe respiratory dysfunction. consequently, clinicians should be cautious when using the right phrenic nerve if maximal inspiratory pressure is low preoperatively. until a long - term follow - up study is completed the contralateral c7 root has a large number of nerve fibers (approximately 420,00), and it was first used for nerve transfer in 1986 (gu., 1992). since then, many clinical cases and experiments have demonstrated that transfer using this root can achieve very good outcomes (kim., 2003). also, there is little risk to function by harvesting the c7 spinal nerve, providing additional sources of donor nerves (gu., 2003). contralateral c7 nerve transfer has been widely used, both within china and overseas, for shoulder and elbow restoration. this method returns grasp function and achieves sensory recovery, and is considered a major breakthrough in the treatment of brachial plexus injury. contralateral c7 nerve transfer is an optimal strategy for treating brachial plexus injury (dubuisson and kline, 2002 ; friedman., 1990), especially when root avulsion and phrenic and accessory nerve damage is encountered simultaneously. in this situation, contralateral c7 transfer is the only solution (terzis and kokkalis, 2010). the procedure for contralateral c7 nerve transfer can be divided into two stages ; stage i and stage ii (gu, 1992). in stage i, the contralateral c7 root is transferred to the nerve graft (ulnar nerve with vessel pedicle is frequently used), and in stage ii, the other part of the nerve graft is transferred to the recipient nerve, including the median nerve and radial nerve. there are numerous follow - up patient reports, providing evidence of the importance of such a division. (1999) reported that only 20% of the 96 patients who underwent contralateral c7 nerve root transfer regained forearm and hand flexor muscle strength to grade 3 or 4, measured using the modified mrc scale, within period i. lao (2010) concluded that different methods contributed to the large disparity in the proportion of patients who achieved strength recovery during periods i and ii36% and 63%, respectively. cortical plasticity appears to play an important physiological role in the functional recovery of the re - innervated muscles. recently, an increasing number of investigators have focused their attention on cerebral plasticity following contralateral cervical nerve transfer in humans. 2001) have shown that the brain is capable of extensive reorganization after the peripheral nervous system is injured (wang., 2010). contralateral c7 nerve transfer provides an ideal opportunity to study the relationship between brachial plexus injury and cortical reorganization (florence., 1996). peripheral nerve conduction testing shows that degeneration and regeneration after peripheral nerve injury are complex processes. physiological changes occur not only at the site of injury, but also upstream (brain and spinal cord) and downstream (muscles and effectors) of the damage (kemp., 2010). peripheral nerve injuries block the flow of output from the motor cortex to the denervated muscles, thereby resulting in paralysis. however, with the gradual maturation of regenerating axons, the dominant functions of the muscles eventually recover (muetn - gmez. (2004, 2005) showed that plastic changes indeed take place in the contralateral motor cortex of adult rats with total brachial plexus root avulsion. (2005) demonstrated changes within the primary motor cortex of patients who underwent contralateral c7 nerve translocation following brachial plexus injury using functional mri. he also proposed that earlier rehabilitation of the affected limb results in better and more extensive cortical reorganization. in 2012, liu. (2012) used contralateral c7 nerve transfer to treat six patients with central spastic hemiplegia. after a 2-year follow - up, they concluded that functional recovery of the affected limb, particularly of the wrist and elbow, was satisfactory. in patients with total root avulsion of the brachial plexus, elbow flexion is restored by icn transfer, and shoulder function is restored by shoulder arthrodesis. hand function is restored by an icn transfer for the median nerve, or by free muscle transplantation in combination with nerve transfer (nagano, 1998). different neural transplantation approaches for the treatment of brachial plexus injury provide differential outcomes, but direct transplantation of collateral branches are associated with better outcome compared with the use of other nerve plexuses, especially in patients with delayed treatment (bertelli and ghizoni, 2010). the use of nerve transfers has been a major advancement in the field of brachial plexus nerve reconstructive surgery. many different and ingenious transfer strategies have been developed and have helped enhance functional recovery. root avulsion of the brachial plexus should be treated with comprehensive treatment and rehabilitation (korak., 2004). the efficacy of treatment is strongly impacted by the following factors : (1) type of involved roots ; (2) time period between injury and operation ; (3) degree of damage ; (4) restorative exercises post - operation ; and (5) co - therapy of the central nerves. a large number of clinical and animal experiments have shown that a combined treatment approach involving the brain, spinal cord and effectors that control muscle movement is required to obtain satisfactory clinical results in the rehabilitation of brachial plexus root avulsion. | in the treatment of brachial plexus injury, nerves that are functionally less important are transferred onto the distal ends of damaged crucial nerves to help recover neuromuscular function in the target region. for example, intercostal nerves are transferred onto axillary nerves, and accessory nerves are transferred onto suprascapular nerves, the phrenic nerve is transferred onto the musculocutaneous nerves, and the contralateral c7 nerve is transferred onto the median or radial nerves. nerve transfer has become a major method for reconstructing the brachial plexus after avulsion injury. many experiments have shown that nerve transfers for treatment of brachial plexus injury can help reconstruct cerebral cortical function and increase cortical plasticity. in this review article, we summarize the recent progress in the use of diverse nerve transfer methods for the repair of brachial plexus injury, and we discuss the impact of nerve transfer on cerebral cortical plasticity after brachial plexus injury. |
its average annual incidence has been estimated at 48 per 100 000 for deep venous thrombosis and 23 per 100 000 for pulmonary embolism. the clinical significance of venous thromboembolism is not only because of the risk of death from pulmonary embolism, but also because of the high risk of recurrent events, the occurrence of subsequent morbidity such as the post - thrombotic syndrome, and the consequent economic impact caused by the high rate of hospitalization. since the early 1960s, anticoagulant therapy has been proven to be pivotal in the treatment of venous thromboembolism. with the exception of massive pulmonary emboli (occurring in < 5% of patients who present with pulmonary embolism, in whom thrombolytic therapy should be considered), treatment of established deep venous thrombosis and pulmonary embolism is essentially identical, and includes the administration of heparin for 5 - 10 days, and oral anticoagulants for at least 3 months. until recently, it can be administered by continuous intravenous infusion, starting with a bolus of 5000 international units (iu) followed by 30 000 - 35 000 iu / day, adjusted to achieve an activated partial thromboplastin time (aptt) of 1.5 - 2.5 times the control. alternatively, after the intravenous bolus injection, unfractionated heparin can be administered subcutaneously, with twice daily injections of 15 000 - 20 000 iu, which are aimed at maintaining therapeutic levels of aptt. in fact, inadequate anticoagulation may be responsible for a high number of recurrent venous thromboembolic events. it has been reported that patients who receive intravenous heparin without reaching the therapeutic range within the first 24 h may have a rate of recurrence as much as 15 times higher than that in patients who had heparin levels within the therapeutic range. its anticoagulant effect is unpredictable and varies considerably among patients, depending on age, sex, body weight, smoking status and renal function. this wide variability is caused by heparin binding to acute - phase reactant proteins, levels of which vary among normal individuals and disease states. before therapeutic plasma levels are achieved, the binding to plasma proteins and to receptor sites on the endothelium must be saturated. moreover, unfractionated heparin has further effects on haemostasis, such as inhibition of platelet aggregation and augmentation of vessel wall permeability, which can significantly enhance its potential to cause bleeding complications. finally, as many as 3% of treated patients will develop heparin - induced thrombocytopenia. their reduced molecular weight (and thus their reduced number of saccharide units) as compared with unfractionated heparin leads to potential pharmacological and pharmacokinetic advantages over the parent compound, which result in a greater clinical utility. their antithrombotic activity is mainly based on inactivation of factor - xa because of a reduced ability to inactivate factor iia when compared with unfractionated heparin. moreover, the lmwhs do not bind to the endothelium and have a lower affinity for plasma proteins. this results in a more predictable bioavailability, a substantially longer half - life, a stable dose / response relationship when injected subcutaneously, and potentially a more antithrombotic than haemorrhagic activity in comparison with unfractionated heparin. lmwhs also have minimal interaction with platelets, and a reduced incidence of heparin - induced thrombocytopenia has been observed. because of their properties, the lmwhs can be administered subcutaneously in weight - adjusted, once or twice daily doses without the need for laboratory monitoring. in the early 1990s, two randomized trials assessed the efficacy and safety of the lmwhs in the treatment of deep venous thrombosis. in a multicenter, double - blind clinical trial, hull randomized 219 patients to receive unfractionated heparin administered intravenously with an initial bolus of 5000 iu followed by 30 000 iu or 40 000 iu every 24 h (depending on the presence or absence of risk factors for bleeding), and 213 patients to receive a once daily, weight - adjusted dose of the lmwh tinzaparin (175 iu / kg). a nonsignificant reduction in the incidence of recurrent venous thromboembolism (6.9% in the standard heparin - treated group versus 2.8% in the lmwh - treated group), a significant reduction in the rate of major bleeding (5.0% versus 0.5%) and a significant reduction in the rate of deaths (9.6% versus 4.7%) in favour of the lmwh were observed. in the same year, prandoni randomized 170 patients to a similar regimen of unfractionated heparin (a bolus of 100 iu / kg followed by a continous infusion of 35 000 iu every 24 h, adjusted to achieve a target aptt of 1.5 - 2.0 times control), or to a twice daily, weight - adjusted, subcutaneous administration of the lmwh nadroparin. the results were similar to those reported by hull : in the lmwh group there was a nonsignificant reduction in the rate of recurrent venous thromboembolism (14% in the standard heparin - treated group versus 7% in the lmwh - treated group), a nonsignificant reduction in major bleeding (3.5% versus 1%) and a nonsignificant reduction in deaths after 6 months of follow up (14% versus 7%). many other randomized trials subsequently compared the lmwhs with standard heparin in the initial treatment of acute proximal venous thrombosis, and their results were evaluated in three different meta - analyses, which concluded that lmwhs have greater efficacy and safety. the most recent meta - analysis, including the most recent studies, found no difference between the unfractionated heparin and lmwh (table 1). the authors of that report concluded that lmwhs have equal effectiveness to that of unfractionated heparin in the prevention of recurrent episodes of venous thromboembolism, and equal safety with respect to the occurrence of major bleeding. interestingly, a statistically significant reduction in total mortality in patients treated with lmwhs was also found in this meta - analysis. also, a pooled analysis from the selected trials showed no substantial differences among the different lmwh products used in the studies, but, as correctly recognized by those authors, no direct comparisons of the different lmwhs are actually available. finally, it is important to note that in all of the above - mentioned trials patients younger than 18 years, pregnant patients and patients with severe renal failure were excluded, and consequently the results of these studies can not be extrapolated to these specific patient groups. the simple, unmonitored dosing system and the practical administration of the lmwhs mean that these agents could facilitate outpatient management of deep venous thrombosis. two large trials published in 1996 have clearly demonstrated the safety and efficacy of such an approach. in the tasman study, conducted in europe, australia and new zealand, 198 patients were randomized to receive an adjusted - dose of intravenous unfractionated heparin in the hospital, and 202 patients to receive a fixed, weight - adjusted subcutaneous dose of the lmwh nadroparin, which was administered at home, if it was considered possible. the rate of events was comparable in the two groups with regard to recurrent venous thromboembolism (8.6 and 6.9%, respectively), major bleeding (2.0 and 0.5%, respectively) and death (8.1 and 6.9%, respectively). importantly, the duration of hospitalization was reduced by 67% from 8.1 days in the unfractionated heparin group to 2.7 days in the lmwh group. in the lmwh group, 75% of the patients the second study was carried out in canada by levine (table 2). the design of the trial and the sample size were similar to those of the tasman study. there were 253 patients randomized to the intravenous unfractionated heparin group, and 247 patients to the lmwh enoxaparin group. the event rates of recurrent venous thromboembolism and major bleeding did not reach statistical significance and therefore it was concluded that the lmwh enoxaparine was not inferior to unfractionated heparin in the treatment of deep venous thrombosis. as in the previous study, the time spent in the hospital was remarkably reduced, from 6.5 days to 1.1 days, and 120 out of the 247 patients randomized to the lmwh were never admitted to the hospital. this study and the tasman study suggest the feasibility of a significant change in the clinical management of patients presenting with deep venous thrombosis, ie treating them at home with lmwh. two recently published reports from canada and one from the netherlands suggest that more than 80% of patients with proximal deep venous thrombosis can safely be treated from the first day with lmwh at home without the need for hospitalization. from pharmacokinetic data it has been observed that therapeutic anti - xa levels could be achieved over 24 h when lmwhs are administered once daily. once daily dosing is attractive because it may be more acceptable to the patient and involves less nursing time. in one controlled study, once daily nadroparin was shown to have equal efficacy and safety as twice daily nadroparin. the primary end - point of a combination of venous thromboembolism and mortality was reported in 13 patients (4.1%) in the group that received daily nadroparin and in 24 patients (7.2%) who received twice daily nadroparin. thus, an absolute difference of 3.1% (95% confidence interval -6.6% to 0.5%) was observed in favour of the once daily therapy. major bleeding occurred in four patients both in the once daily group (1.3%) and in the twice daily group (1.2%). because deep venous thrombosis and pulmonary embolism are considered two manifestations of the same disease - venous thromboembolism - the lmwhs have also recently been tested in the setting of patients with haemodynamically stable pulmonary embolism. three trials were published between 1997 and 2000 : one was carried out in a population of patients presenting with venous thromboembolism, including submassive pulmonary embolism ; one specifically in patients with submassive pulmonary embolism ; and a third was conducted in patients admitted for deep venous thrombosis who also had objectively documented pulmonary embolism. the columbus trial was an international, randomized, open - label study that enrolled 1021 patients presenting with acute symptomatic deep venous thrombosis, pulmonary embolism, or both. one group receive the lmwh reviparin administered subcutaneously twice daily at fixed, weight - adjusted doses (6300 anti - xa units if body weight was more than 60 kg, 4200 anti - xa units if body weight was between 46 and 60 kg, and 3500 units for a body weight between 35 and 45 kg). the other group received unfractionated heparin administered intravenously in a 5000 iu bolus followed by a dose of 1250 iu / h, adjusted to achieve an aptt range between 60 and 85 s. home treatment was encouraged for patients assigned to reviparin. only 12 patients were excluded because thrombolytic therapy was planned, and 271 patients with pulmonary embolism were included in the study. the total rate of recurrences in the subgroup with pulmonary embolism was comparable to that of the group of patients with deep venous thrombosis (5.9% versus 4.8%, respectively), and was also similar in the two treatment groups (5.8% in the reviparin group versus 6.0% in the unfractionated heparin group). there were six episodes of fatal pulmonary embolism (2.2%) during the 3 months of follow up, all of which occurred in the subgroup of 271 patients enrolled with pulmonary embolism. a total of 612 patients presenting with symptomatic pulmonary embolism were assigned to receive the lmwh tinzaparin administered subcutaneously at a once daily dose of 175 anti - xa units / kg body weight, or to receive unfractionated heparin administered intravenously with an initial bolus of 50 iu / kg followed by an initial dose of 500 iu / kg, adjusted to achieve therapeutic levels of the aptt between 2 and 3 times the control value. after 8 days, the occurrence of events included in the primary outcome (recurrent venous thromboembolism, major bleeding or death) was similar in the two treatment groups : 2.9% in the unfractionated heparin group and 3.0% in the tinzaparin group. after 90 days this similarity remained, with a 7.1% and 5.9% incidence in the unfractionated heparin and tinzaparin groups, respectively. the study by hull included a subpopulation of an earlier trial with objectively documented pulmonary embolism with underlying deep venous thrombosis. patients were assigned to the lmwh tinzaparin, administered, as in the previous study, subcutaneously in a once daily fixed dose of 175 anti - xa units / kg of body weight, or to unfractionated heparin administered intravenously with an initial bolus of 5000 iu followed by a continuous infusion of 40 320 iu/24 h (or 29 760 iu/24 h for those patients with risk factors for bleeding), which was adjusted to achieve a therapeutic aptt range between 1.5 and 2.5 times the control value. of the originally included 432 patients with deep venous thrombosis, 200 had high - probability perfusion lung scan findings and there were no recurrent episodes of venous thromboembolism in the group of patients treated with tinzaparin, and seven new episodes (four pulmonary embolisms) in the unfractionated heparin group (95% confidence interval for the difference 1.9% to 11.7% ; p = 0.01). death occurred in 6.2% and 8.7% of patients, respectively ; only in one patient (in the unfractionated heparin group) was death related to pulmonary embolism. the lmwhs are currently considered a potentially valid alternative to unfractionated heparin in the treatment of pulmonary embolism in patients whose clinical condition is stable. one might argue that there is a need for more clinical studies, involving large groups of unselected patients, presenting with clinically suspected pulmonary embolism, who were treated with lmwhs from the moment of diagnosis. a meta - analysis comparing lmwh in the treatment of venous thromboembolism ci, confidence interval ; rr, relative risk lmwh in the outpatient treatment of deep venous thrombosis pooled analysis of the studies of koopman and levine. in the early 1990s, two randomized trials assessed the efficacy and safety of the lmwhs in the treatment of deep venous thrombosis. in a multicenter, double - blind clinical trial, hull randomized 219 patients to receive unfractionated heparin administered intravenously with an initial bolus of 5000 iu followed by 30 000 iu or 40 000 iu every 24 h (depending on the presence or absence of risk factors for bleeding), and 213 patients to receive a once daily, weight - adjusted dose of the lmwh tinzaparin (175 iu / kg). a nonsignificant reduction in the incidence of recurrent venous thromboembolism (6.9% in the standard heparin - treated group versus 2.8% in the lmwh - treated group), a significant reduction in the rate of major bleeding (5.0% versus 0.5%) and a significant reduction in the rate of deaths (9.6% versus 4.7%) in favour of the lmwh were observed. in the same year, prandoni randomized 170 patients to a similar regimen of unfractionated heparin (a bolus of 100 iu / kg followed by a continous infusion of 35 000 iu every 24 h, adjusted to achieve a target aptt of 1.5 - 2.0 times control), or to a twice daily, weight - adjusted, subcutaneous administration of the lmwh nadroparin. the results were similar to those reported by hull : in the lmwh group there was a nonsignificant reduction in the rate of recurrent venous thromboembolism (14% in the standard heparin - treated group versus 7% in the lmwh - treated group), a nonsignificant reduction in major bleeding (3.5% versus 1%) and a nonsignificant reduction in deaths after 6 months of follow up (14% versus 7%). many other randomized trials subsequently compared the lmwhs with standard heparin in the initial treatment of acute proximal venous thrombosis, and their results were evaluated in three different meta - analyses, which concluded that lmwhs have greater efficacy and safety. the most recent meta - analysis, including the most recent studies, found no difference between the unfractionated heparin and lmwh (table 1). the authors of that report concluded that lmwhs have equal effectiveness to that of unfractionated heparin in the prevention of recurrent episodes of venous thromboembolism, and equal safety with respect to the occurrence of major bleeding. interestingly, a statistically significant reduction in total mortality in patients treated with lmwhs was also found in this meta - analysis. also, a pooled analysis from the selected trials showed no substantial differences among the different lmwh products used in the studies, but, as correctly recognized by those authors, no direct comparisons of the different lmwhs are actually available. finally, it is important to note that in all of the above - mentioned trials patients younger than 18 years, pregnant patients and patients with severe renal failure were excluded, and consequently the results of these studies can not be extrapolated to these specific patient groups. the simple, unmonitored dosing system and the practical administration of the lmwhs mean that these agents could facilitate outpatient management of deep venous thrombosis. two large trials published in 1996 have clearly demonstrated the safety and efficacy of such an approach. in the tasman study, conducted in europe, australia and new zealand, 198 patients were randomized to receive an adjusted - dose of intravenous unfractionated heparin in the hospital, and 202 patients to receive a fixed, weight - adjusted subcutaneous dose of the lmwh nadroparin, which was administered at home, if it was considered possible. the rate of events was comparable in the two groups with regard to recurrent venous thromboembolism (8.6 and 6.9%, respectively), major bleeding (2.0 and 0.5%, respectively) and death (8.1 and 6.9%, respectively). importantly, the duration of hospitalization was reduced by 67% from 8.1 days in the unfractionated heparin group to 2.7 days in the lmwh group. in the lmwh group, 75% of the patients the second study was carried out in canada by levine (table 2). the design of the trial and the sample size were similar to those of the tasman study. there were 253 patients randomized to the intravenous unfractionated heparin group, and 247 patients to the lmwh enoxaparin group. the event rates of recurrent venous thromboembolism and major bleeding did not reach statistical significance and therefore it was concluded that the lmwh enoxaparine was not inferior to unfractionated heparin in the treatment of deep venous thrombosis. as in the previous study, the time spent in the hospital was remarkably reduced, from 6.5 days to 1.1 days, and 120 out of the 247 patients randomized to the lmwh were never admitted to the hospital. this study and the tasman study suggest the feasibility of a significant change in the clinical management of patients presenting with deep venous thrombosis, ie treating them at home with lmwh. two recently published reports from canada and one from the netherlands suggest that more than 80% of patients with proximal deep venous thrombosis can safely be treated from the first day with lmwh at home without the need for hospitalization. from pharmacokinetic data it has been observed that therapeutic anti - xa levels could be achieved over 24 h when lmwhs are administered once daily. once daily dosing is attractive because it may be more acceptable to the patient and involves less nursing time. in one controlled study, once daily nadroparin was shown to have equal efficacy and safety as twice daily nadroparin. the primary end - point of a combination of venous thromboembolism and mortality was reported in 13 patients (4.1%) in the group that received daily nadroparin and in 24 patients (7.2%) who received twice daily nadroparin. thus, an absolute difference of 3.1% (95% confidence interval -6.6% to 0.5%) was observed in favour of the once daily therapy. major bleeding occurred in four patients both in the once daily group (1.3%) and in the twice daily group (1.2%). because deep venous thrombosis and pulmonary embolism are considered two manifestations of the same disease - venous thromboembolism - the lmwhs have also recently been tested in the setting of patients with haemodynamically stable pulmonary embolism. three trials were published between 1997 and 2000 : one was carried out in a population of patients presenting with venous thromboembolism, including submassive pulmonary embolism ; one specifically in patients with submassive pulmonary embolism ; and a third was conducted in patients admitted for deep venous thrombosis who also had objectively documented pulmonary embolism. the columbus trial was an international, randomized, open - label study that enrolled 1021 patients presenting with acute symptomatic deep venous thrombosis, pulmonary embolism, or both. one group receive the lmwh reviparin administered subcutaneously twice daily at fixed, weight - adjusted doses (6300 anti - xa units if body weight was more than 60 kg, 4200 anti - xa units if body weight was between 46 and 60 kg, and 3500 units for a body weight between 35 and 45 kg). the other group received unfractionated heparin administered intravenously in a 5000 iu bolus followed by a dose of 1250 iu / h, adjusted to achieve an aptt range between 60 and 85 s. home treatment was encouraged for patients assigned to reviparin. only 12 patients were excluded because thrombolytic therapy was planned, and 271 patients with pulmonary embolism were included in the study. the total rate of recurrences in the subgroup with pulmonary embolism was comparable to that of the group of patients with deep venous thrombosis (5.9% versus 4.8%, respectively), and was also similar in the two treatment groups (5.8% in the reviparin group versus 6.0% in the unfractionated heparin group). there were six episodes of fatal pulmonary embolism (2.2%) during the 3 months of follow up, all of which occurred in the subgroup of 271 patients enrolled with pulmonary embolism. a total of 612 patients presenting with symptomatic pulmonary embolism were assigned to receive the lmwh tinzaparin administered subcutaneously at a once daily dose of 175 anti - xa units / kg body weight, or to receive unfractionated heparin administered intravenously with an initial bolus of 50 iu / kg followed by an initial dose of 500 iu / kg, adjusted to achieve therapeutic levels of the aptt between 2 and 3 times the control value. after 8 days, the occurrence of events included in the primary outcome (recurrent venous thromboembolism, major bleeding or death) was similar in the two treatment groups : 2.9% in the unfractionated heparin group and 3.0% in the tinzaparin group. after 90 days this similarity remained, with a 7.1% and 5.9% incidence in the unfractionated heparin and tinzaparin groups, respectively. the study by hull included a subpopulation of an earlier trial with objectively documented pulmonary embolism with underlying deep venous thrombosis. patients were assigned to the lmwh tinzaparin, administered, as in the previous study, subcutaneously in a once daily fixed dose of 175 anti - xa units / kg of body weight, or to unfractionated heparin administered intravenously with an initial bolus of 5000 iu followed by a continuous infusion of 40 320 iu/24 h (or 29 760 iu/24 h for those patients with risk factors for bleeding), which was adjusted to achieve a therapeutic aptt range between 1.5 and 2.5 times the control value. of the originally included 432 patients with deep venous thrombosis, 200 had high - probability perfusion lung scan findings and were included in this study. only 28 (14%) had presented with symptoms of pulmonary embolism. there were no recurrent episodes of venous thromboembolism in the group of patients treated with tinzaparin, and seven new episodes (four pulmonary embolisms) in the unfractionated heparin group (95% confidence interval for the difference 1.9% to 11.7% ; p = 0.01). death occurred in 6.2% and 8.7% of patients, respectively ; only in one patient (in the unfractionated heparin group) was death related to pulmonary embolism. the lmwhs are currently considered a potentially valid alternative to unfractionated heparin in the treatment of pulmonary embolism in patients whose clinical condition is stable. one might argue that there is a need for more clinical studies, involving large groups of unselected patients, presenting with clinically suspected pulmonary embolism, who were treated with lmwhs from the moment of diagnosis. a meta - analysis comparing lmwh in the treatment of venous thromboembolism ci, confidence interval ; rr, relative risk. lmwh in the outpatient treatment of deep venous thrombosis pooled analysis of the studies of koopman and levine. lmwhs have replaced unfractionated heparin in the initial treatment of patients with deep venous thrombosis. numerous well - designed clinical trials have demonstrated that lmwhs are as effective and safe as unfractionated heparin and, because no laboratory control is needed, they are the initial treatment of choice for initiating out - of - hospital anticoagulant treatment in patients with acute deep venous thrombosis. for patients with pulmonary embolism, lmwhs are a potential alternative for unfractionated intravenous heparin. whether all patients with pulmonary embolism can be treated at home from the first day with lmwhs | venous thromboembolism is a common disease that is associated with considerable morbidity if left untreated. recently, low - molecular - weight heparins (lmwhs) have been evaluated for use in acute treatment of deep venous thrombosis and pulmonary embolism. randomized studies have shown that lmwhs are as effective as unfractionated heparin in the prevention of recurrent venous thromboembolism, and are as safe with respect to the occurrence of major bleeding. a pooled analysis did not show substantial differences among different lmwh compounds used, but no direct comparison of the different lmwhs is currently available. finally, in patients with pulmonary embolism, there is a relative lack of large studies of daily practice. it could be argued that large prospective studies, in patients who were treated with lmwhs from the moment of diagnosis, are needed. |
pmn are innate immune cells that migrate from the circulation to sites of infection, where they are responsible for antimicrobial functions. pmn use phagocytosis, degranulation, and formation of neutrophil extracellular traps (nets) to kill microbes [2, 3 ]. nets are formed through a unique cell death program named netosis that involves activation in most cases of nicotinamide adenine dinucleotide phosphate- (nadph-) oxidase, which produces reactive oxygen species (ros) [46 ]. during netosis, the characteristic lobular nucleus of neutrophils disappears, and the chromatin expands in the cytosol, while the cell membrane remains intact. three or four hours after stimulation, the cell membrane breaks and the chromatin fibers get released forming a netlike structure outside the cell. net fibers are composed of chromatin covered with histones and antimicrobial proteins derived from the neutrophil granules, such as the bactericidal / permeability - increasing protein (bpi), elastase, myeloperoxidase, lactoferrin, and metalloprotease 9 [2, 4 ]. the requirements for nadph - oxidase and myeloperoxidase in net formation differ depending on the stimulus [8, 9 ]. besides their antimicrobial capacity, nets seem to act as a physical barrier where microorganisms get trapped and consequently prevent further spread of pathogens. thus, nets bind, block, and kill microorganisms extracellularly and independently of phagocytosis. binding of receptors for the fc portion of antibodies (fc receptors) to opsonized microorganisms is one of the most important mechanisms for pathogen recognition and activation of neutrophils. human neutrophils express constitutively two antibody receptors that are members of the fc receptor family for igg molecules, namely, fcriia (cd32a) and fcriiib (cd16b). fcriia is composed of a single polypeptide chain bearing an itam on its cytoplasmic domain. this itam confers on fcriia the ability to initiate signaling events that regulate cell responses, including phagocytosis, cytokine production, antibody - dependent cell - mediated cytotoxicity, and the respiratory burst. fcriiib is present exclusively on neutrophils and it is a glycophosphatidylinositol- (gpi-) linked receptor, lacking transmembrane and cytoplasmic domains. although signaling molecules associated with fcriiib are still unknown and its signaling mechanism remains unidentified, several reports show that fcriiib can initiate signaling events leading to calcium transients, actin polymerization, activation of integrins, and nf-b activation. several pathogens, including virus, bacteria, fungi, and parasites, have all been reported to be inducers of net formation. in addition, proinflammatory stimuli such as lipopolysaccharide (lps), interleukin- (il-) 8, and tumor necrosis factor (tnf) [20, 21 ] and also some pharmacological stimuli such as phorbol 12-myristate 13-acetate (pma), an activator of protein kinase c (pkc), are efficient inducers of nets. some reports indicate that net formation was increased when microorganisms were opsonized with autologous serum. recent reports indicated that antigen - antibody complexes seem capable of inducing net formation [22, 23 ]. in one report, a b cell - deficient mouse was used to show nets could not be formed, thus suggesting a direct role for fc receptors in this function. in this study, however no particular receptor was identified. in another report it was determined that fcriiib promoted endocytosis of soluble immune complexes and that fcriia promoted net formation in vivo. however, more recently, it was reported that fcriiib is the receptor responsible for net formation in response to immobilized immune complexes. thus, in order to provide some insight into this controversy, each of the two human fc receptors was stimulated individually by specific monoclonal antibodies and net formation was evaluated. net formation was dependent on nadph - oxidase, pkc, and extracellular signal - regulated kinase (erk) activation. neutrophils (pmn) were purified from blood collected from adult healthy volunteers exactly as previously described. the informed consent form and all experimental procedures were approved by the bioethics committee at instituto de investigaciones biomdicas, unam. piceatannol, a spleen tyrosine kinase (syk) inhibitor, was from acros organics (new jersey, usa). wortmannin, a phosphatidylinositol 3-kinase (pi-3k) inhibitor ; g6976, a protein kinase c (pkc) inhibitor ; g6983, another pkc inhibitor ; 3-(1-methyl-1h - indol-3-yl - methylene)-2-oxo-2,3-dihydro-1h - indole-5-sulfonamide (isyk), another syk inhibitor (catalog number 574711) ; and the antibleaching mounting medium fluorsave (catalog number 345789) were from calbiochem / emd millipore (billerica, ma). uo126, a mek (erk kinase) inhibitor, was from promega (madison, wi, usa). diphenyleneiodonium (dpi), an nadph - oxidase inhibitor ; (e)-3-[4-methylphenylsulfonyl]-2-propenenitrile (bay 117082), an nf-b inhibitor ; phorbol 12-myristate 13-acetate (pma) ; and all other chemicals were from sigma aldrich (st. louis, mo). the following antibodies were used : anti - human fcriia (cd32) mab iv.3 (atcc hb-217) was from american type culture collection (manassas, va). anti-1 integrin mab ts2/16 was donated by martin hemler (dana farber cancer research institute, boston, ma). anti-2 integrin (cd18) mab ib4 and anti - human fcriiib (cd16) mab 3g8 were donated by dr. eric j. brown (university of california in san francisco, san francisco, ca). mouse monoclonal anti - neutrophil elastase (d-7 ; catalog number sc-365950), rabbit polyclonal anti - histone h2b (fl-126 ; catalog number sc-10808), rabbit polyclonal anti - erk 1 (catalog number sc-94), and mouse monoclonal anti - phospho - erk 1 (ptyr204) (catalog number sc-7383) were from santa cruz biotechnology (santa cruz, ca). alexa fluor 555-conjugated donkey anti - rabbit igg (catalog number a-31572), alexa fluor 488-conjugated donkey anti - mouse igg (catalog number a-21202), and fitc - conjugated f(ab)2 goat anti - mouse igg (catalog number a-10683) were from invitrogen molecular probes (eugene, or). f(ab)2 goat anti - mouse igg (catalog number 0855468), hrp - conjugated f(ab)2 goat anti - mouse igg (catalog number 0855572), and hrp - conjugated f(ab)2 goat anti - rabbit igg (catalog number 0855686) were from mp biomedicals (santa ana, ca). hybridoma cells were grown in dmem (gibco ; grand island, ny) containing 10% fetal bovine serum (fbs) also from gibco (grand island, ny). antibodies were purified from saturated (8-day - old) tissue culture supernatants with protein - g sepharose 4 fast flow from ge healthcare bio - sciences ab (uppsala, sweden). after elution from the sepharose with 0.1 m glycine - hcl, ph 2.7, antibodies were dialyzed against pbs, adjusted to 1 mg / ml, and filter - sterilized. the functionality of antibodies was confirmed by their binding to neutrophil receptors (supplemental figure 1s in supplementary material available online at http://dx.doi.org/10.1155/2016/2908034). for receptor stimulation, pmn (1 10) in 500 l pbs were placed into a 1.5 ml eppendorf tube, and 10 g / ml of the corresponding anti - receptor monoclonal antibody was added. cells were incubated at 4c for 20 minutes and then washed twice with 1 ml of cold pbs (4c) centrifuged at 1,743 g, 1 minute each time. this centrifugation protocol did not preactivate cells as long as they were maintained cold. next, pmn were resuspended in 500 l cold (4c) rpmi-1640 medium (gibco ; grand island, ny) containing 5% fetal bovine serum (fbs) also from gibco (grand island, ny). neutrophils were left untreated for pma stimulation or previously treated with anti - receptor antibodies (as described above) for receptor stimulation. neutrophils (1 10) in 500 l rpmi-1640 medium were added to each well of a 24-well plate, containing a 12 mm coverslip, and then incubated in a humidified incubator with 5% co2 at 37c for 30 minutes. then 100 l of 120 nm pma in pbs or 100 l of 450 g / ml f(ab)2 goat anti - mouse igg (for receptor stimulation) was added to each well. next, 600 l of 2% paraformaldehyde in pbs was added to each well, and the plates were incubated overnight in 5% co2 at 37c. in selected experiments, pmn were incubated for 30 minutes before stimulation, with the inhibitors piceatannol (50 m), wortmannin (50 nm), uo126 (50 m), g6983 (1 m), g6976 (1 m), dpi (10 m), bay 117082 (2.5 m), or the vehicle dimethyl sulfoxide (dmso) alone. all washes and incubations were done at room temperature by placing the coverslip upside down over a 250 l drop of each solution formed on a well of parafilm placed on a tube rack, exactly as previously described. coverslips were taken out of the 24-well plate one at a time and washed four times with water for 5 minutes each. next, they were placed over 0.1% triton x-100 in 4% paraformaldehyde for 10 minutes, then on pbs for 5 minutes, and then on 10 g / ml of the corresponding primary antibody (anti - neutrophil elastase or anti - histone) in 5% bsa in pbs for 60 minutes. coverslips were then washed four times with pbs for 5 minutes each and placed on 8 g / ml of the corresponding secondary antibody (alexa fluor - conjugated anti - rabbit igg or anti - mouse igg) in 5% bsa in pbs containing 300 nm dapi. slides were observed with a fluorescence inverted microscope model ix-70 from olympus (center valley, pa). images were captured with an evolution - vf cooled color camera from media cybernetics (rockville, md) and the computer program qcapture pro 6.0 from qimaging, surrey (british columbia, canada). images were processed with the computer program imagej 1.47v from the national institutes of health (bethesda, md). opsonization of 4.8 m fluorescent (catalog number 16592) or nonfluorescent (catalog number 17135) latex particles from polysciences, inc. these particles were used in phagocytosis assays as described or in net formation assays as follows. pmn (1 10 cells) in 500 l were centrifuged in a 1.5 ml eppendorf tube at 1,743 g for 1 min. after removing the supernatant, the cell pellet was disaggregated by tapping the tube against a rack, and 80 l of opsonized latex particles (1.25 10 particles / ml) resuspended in rpmi-1640 medium with 5% fbs was added., 1 ml of cold pbs was added, the tube was centrifuged at 1,743 g for 1 min, and the cell pellet was resuspended in 500 l rpmi-1640 medium with 5% fbs. cell suspension was transferred into a well of a 24-well plate, containing a 12 mm coverslip, and then incubated in a humidified incubator with 5% co2 at 37c for 4 h. then 500 l of 2% paraformaldehyde in pbs was added to each well, and the plate was kept in the incubator overnight. finally, the coverslip was used for net visualization as described above. a 96-well plate was previously covered with 25 g / ml poly - d - lysine for three hours at room temperature. each well was then washed three times with 50 l pbs for 5 minutes each time, and the plate was allowed to air dry inside a flow laminar hood for two hours. neutrophils were resuspended at 1.25 10 cells / ml in rpmi-1640 medium, containing 500 nm sytox green (molecular probes, inc. ; eugene, or), and 80 l of this cell suspension (1 10 pmn) was added to each well of the 96-well plate. next, the plate was incubated at 37c in a 5% co2 incubator for 20 minutes. for fcr stimulation, the supernatant was removed gently with a micropippetor and 50 l of 10 g / ml of the corresponding anti - fc receptor antibody was added to each well. the plate was placed in a 35c prewarmed microplate reader model synergy ht from biotek instruments (winooski, vt) and incubated there for 20 minutes. next, the supernatant was removed gently with a micropippetor and 100 l of 75 g / ml of f(ab)2 goat anti - mouse igg containing 500 nm sytox green was added to each corresponding well. at this time, for pma stimulation 20 l of 100 nm pma was added to each corresponding well. finally the fluorescence from the bottom of the plate was read, using the 485 nm excitation and 528 emission filters. for net formation induced with opsonized latex particles, pmn (1 10 cells) in 500 l rpmi-1640 medium with 5% fbs and 500 nm sytox green were mixed with 80 l of opsonized latex particles (1.25 10 particles / ml). then 100 l of the pmn - particle mixture was transferred into a well of a 96-well plate and incubated in a 35c prewarmed microplate reader for 4 hours. fluorescence from the bottom of the plate was read using the 485 nm excitation and 528 emission filters. cells were lysed in ripa buffer (150 mm nacl, 5 mm edta, 50 mm hepes, 0.5% sodium deoxycholate, 1% nonidet p-40, and 10 mm 2-mercaptoethanol, ph 7.5) containing complete protease inhibitor cocktail from roche (basel, switzerland), for 15 minutes at 4c. proteins were then electrotransferred onto polyvinylidene fluoride (pvdf) membranes (immobilon - p ; millipore, bedford, ma). membranes were incubated in blocking buffer (1% bsa and 5% nonfat dry milk (carnation ; nestle, glendale, ca) and 0.1% tween 20 in pbs) overnight at 4c. membranes were subsequently probed with the corresponding antibody in blocking buffer, for 1 hour at room temperature, anti - erk 1 (1/1000 dilution) or anti - phospho - erk 1 (1/500 dilution). membranes were washed with pbs six times and incubated with a 1/3000 dilution of hrp - conjugated f(ab)2 goat anti - rabbit igg or hrp - conjugated f(ab)2 goat anti - mouse igg for 1 hour at room temperature. after washing six more times, the membrane was developed with a chemiluminescence substrate (supersignal ; pierce, rockford, il) according to the manufacturer 's instructions. apoptosis was assayed with the fitc annexin v and propidium iodide (pi) dead cell apoptosis kit for flow cytometry (catalog number v13242) from molecular probes, inc. briefly, pmn were treated with nothing, pma, or the antibodies against each of the fc receptors as described above. after a two - hour incubation at 37c, pmn (1 10) were washed in pbs and resuspended in annexin - binding buffer (10 mm hepes, 140 mm nacl, and 2.5 mm ca, ph 7.4), and 2 l of fitc annexin v and 1 l of 1.5 mm pi were added. cells were incubated for 15 min at room temperature and then 400 l of annexin - binding buffer was added. pmn apoptosis (positive control) was induced by uv - light irradiation as previously described. ros production was assessed with the dcfda - cellular reactive oxygen species detection assay kit (catalog number ab113851) from abcam, inc. briefly, pmn were treated with mab iv.3 or mab 3g8 at 10 g / ml for 20 minutes on ice. pmn were washed with 1x buffer and then incubated with 15 m dcfda in 1x buffer for 30 minutes at 37c. after one wash in 1x buffer, 5 10 pmn were placed in each well of a 96-well clear - bottom black plate from corning inc. (new york, ny) and incubated for 20 minutes at 35c in a plate reader, model synergy ht from biotek instruments (winooski, vt). then, for antibody treatment 50 l of goat anti - mouse igg (150 g / ml) was added and for pma treatment, 50 l of pma (40 nm) was added. fluorescence was read for two hours at excitation 485 nm and emission 535 nm. briefly, pmn (1 10 cells) were resuspended in 100 l cold phagocytosis buffer (2 mm calcium chloride, 1.5 mm magnesium chloride, and 1% human serum albumin in pbs) and mixed with 3.5 l of a suspension (1 10 beads / ml) of iv.3-opsonized, or 3g8-opsonized or control - opsonized (no antibody) fluorescent latex beads. pmn and beads were incubated at 37c for 30 min, centrifuged at 6000 rpm for 1 min, and resuspended in 100 l of ice - cold trypsin - edta solution (0.05% trypsin, 1 mm edta in pbs) to detach uninternalized beads from the cells. after a 15 min incubation on ice, pmn were washed with 1 ml cold pbs plus 0.5% bsa plus 2 mm edta and resuspended in 500 l cold 1% paraformaldehyde in pbs. to analyze phagocytosis by flow cytometry, latex particles were gated out during sample acquisition and 10,000 cells were acquired per sample. phagocytosis was reported as percent of fluorescence - positive cells (cells internalizing at least one fluorescent particle). phagocytosis was also analyzed by microscopy and reported as phagocytic index, the number of beads internalized by 100 cells. single variable data were compared by unpaired - sample student 's t - tests using the computer program kaleidagraph version 3.6.2 for mac (synergy software ; reading, pa). also, variance homogeneity was checked by using levene 's test, and multiple pair - comparisons were performed using tukey 's test after ordinary one - way analysis of variance (anova). analyses were done using the sas software version 9.0 (2012) from sas institute inc. several types of pathogens have been reported to induce net formation, but there are not reports on particular receptors used by neutrophils to recognize these pathogens and to induce netosis. most studies on nets have used pma, a potent activator of pkc, and efficient inducer of nets. in this case, no receptor is involved since pma directly activates intracellular signaling. some reports indicated that net formation was increased when microorganisms were opsonized with autologous serum and also that antigen - antibody complexes seemed to be capable of inducing net formation. these studies suggested a possible role for igg fc receptors (fcr) in net formation. however the particular fc receptor involved in triggering this function is a matter of controversy. thus, in order to explore what particular fc receptor could induce net formation, pmn were stimulated by cross - linking individual receptors with specific monoclonal antibodies. when pmn were stained with dapi, the typical lobular nuclei were clearly seen (figure 1(a)). immunolabeling of histones also showed the localization of these proteins within the pmn nucleus (figure 1(b)). when pmn were treated with pma, nuclei lost their typical morphology and long nets were formed (figure 1(d)). also, the cell morphology was altered ; pmn appeared larger and diffuse (supplemental figure 2s). cross - linking fcriia with the specific mab iv.3 did not induce net formation and pmn retained intact nuclei with typical lobular morphology (figures 1(g) and 1(h)). similarly, cross - linking 1 integrins (figures 1(m) and 1(n)) or 2 integrins (figures 1(p) and 1(q)) did not induce any net formation (supplemental figure 2s). in contrast, cross - linking fcriiib with the specific mab 3g8 induced strong net formation (figure 1(j)) similar to the one induced by pma (figure 2). these fcriiib - induced extracellular dna fibers were also covered with histones (figure 1(k) and supplemental figure 3s). cross - linking of fcriia together with fcriiib or 2 integrins together with fcriiib did not induce changes in the amount of net formation induced by fcriiib alone (not shown). an important characteristic of nets is that they are covered with antimicrobial proteins from the pmn granules. the presence of neutrophil elastase on nets was confirmed for nets induced both by pma and by fcriiib (figure 3 and supplemental figure 4s). these data indicated that cross - linking fcriiib is an efficient stimulus for net formation., apoptosis appears spontaneously when these cells get older or after they have activated their proinflammatory functions. because the net quantification method is related to detection of extracellular dna, it was important to determine whether pmn were in apoptosis after stimulation of fc receptors. after pma stimulation or fc receptor cross - linking, pmn did not have an increase in annexin v - binding, indicating that pmn were not in apoptosis [34, 36 ] (supplemental figure 5s). because pma is an activator of pkc, the involvement of this kinase in net formation induced by fcriiib was tested with two specific pkc inhibitors. however, when pmn were treated previously with g6983, an inhibitor of pkc, pkc, and pkc isozymes (figure 4), or with g6976, a conventional pkc inhibitor (figure 4), nets were not formed after pma stimulation. similarly, net formation after fcriiib cross - linking (figure 4) was inhibited by these pkc inhibitors (figure 4). in addition, downstream of pkc, the mek, erk pathway has been reported to participate in net formation after pma stimulation. when pmn were treated with uo126, a potent specific mek inhibitor, nets were not formed after pma stimulation (figure 5). also, uo126 treatment blocked net formation after fcriiib stimulation (figure 5). these data suggested that fcriiib stimulation led to net formation using pkc and erk. to confirm that erk 1 was activated after pma or fc receptor stimulation as previously reported, pmn were stimulated in the presence or absence of the mek inhibitor and erk 1 activation was detected by western blotting. pma induced erk phosphorylation in pmn (figure 6(a)), and this erk activation was completely blocked by the mek inhibitor uo126 (figure 6(a)). similarly, fcriia cross - linking (figure 6(b)) or fcriiib cross - linking (figure 6(c)) resulted in efficient erk 1 phosphorylation. this erk 1 activation was completely blocked in both cases by the mek inhibitor (figures 6(b) and 6(c)). these data suggested that both fc receptors can induce erk activation, but this enzyme is not sufficient for net formation, since only fcriiib led to release of nets. other signaling molecules that are important for fc receptor signaling via itam are syk and pi-3k. although, fcriiib does not have an itam, it has been suggested that fcriiib might signal in cooperation with fcriia. thus, to explore the possible involvement of these molecules in fcriiib - induced net formation, pmn were treated with piceatannol (figure 7) or isyk (supplemental figure 6s), selective inhibitors of syk, or with wortmannin (figure 7), a selective inhibitor of pi-3k, before stimulation. these inhibitors prevented net formation when pmn were stimulated by pma (figure 7). similarly, net formation was inhibited after cross - linking of fcriiib in the presence of piceatannol (figure 7) but proceeded normally in the presence of wortmannin (figure 7). interestingly, isyk only caused small but statistically significant inhibition of net formation after pma stimulation, while it did not block fcriiib - induced net formation (supplemental figure 6s). these data suggested that fcriiib - induced net formation involves syk, but it is independent of pi-3k. nets formed after pma stimulation require activation of nadph - oxidase and formation of ros and also activation of nf-b. thus, we explored the involvement of these molecules in fcriiib - induced net formation. pmn treated with diphenyleneiodonium (dpi), an nadph - oxidase inhibitor, were not able to form nets after pma stimulation (figure 8). similarly, dpi - treated pmn could not form nets after cross - linking of fcriiib (figure 8). pma treatment, as well as cross - linking of both fcriia and fcriiib, indeed induced ros production that was completely blocked by dpi (supplemental figure 7s). in addition, pmn treated with bay 117082, an nf-b inhibitor, were not able to form nets after pma stimulation (figure 9). in contrast, pmn treated with bay 117082 at two different concentrations formed nets efficiently after cross - linking of fcriiib (figure 9). these data suggested that fcriiib could indeed induce the formation of nets via nadph - oxidase activation, but independently of nf-b activation. clearly, selective activation of fcriiib on the pmn membrane was enough to induce the formation of nets. in order to explore whether cross - linking of fc receptors by a more natural stimulus could also induce net formation, pmn were mixed with opsonized latex particles. these particles covered with protein a and then opsonized with selective anti - fc receptor antibodies can be recognized by only one or the other of the fc receptors. as shown previously, pmn were capable of efficient phagocytosis of latex beads opsonized with mab iv.3 (anti - fcriia) and of very poor phagocytosis of latex beads opsonized with mab 3g8 (anti - fcriiib) (figure 10). these beads were opsonized at similar levels with both anti - fc receptor antibodies (supplemental figure 8s). pmn and fluorescent beads can be easily separated as two distinct populations in a flow cytometer. thus, by gating on cells an increase in fluorescence indicates efficient phagocytosis (supplemental figure 9s). the efficient fcriia - mediated phagocytosis was dependent on erk activation, since the mek inhibitor uo126 prevented it (figure 10), and it was independent of nf-b activation, since the inhibitor bay 117082 did not affect it (figure 10). these data were also confirmed by evaluating phagocytosis by microscopy (supplemental figure 10s). in contrast, the poor phagocytic response of fcriiib was independent of both mek and nf-b (figure 11). these beads when not opsonized (figure 11(a)(a)) or when opsonized with anti - fcriia antibodies (figure 11(a)(b)) could not induce the formation of nets. however, beads opsonized with anti - fcriiib antibodies efficiently induced the formation of nets (figure 11(a)(c and d)). in addition, a mixture of beads opsonized with either anti - fcriia antibodies or anti - fcriiib antibodies also induced net formation to the same level as anti - fcriiib beads alone (figure 11(b)). these data strongly suggested that fcriia can efficiently promote phagocytosis, while it can not induce the formation of nets. in contrast, fcriiib poorly promotes phagocytosis, but it can efficiently induce the formation of nets. neutrophils are the most abundant circulating leukocytes in mammals and they are rapidly recruited to sites of infection, where they act as the first line of defense against invading pathogens. neutrophil activation, through various membrane receptors, is also required for the initiation of the several defense mechanisms displayed by these cells, including phagocytosis, respiratory burst, release of various microbicidal molecules by degranulation, and the recently described formation of neutrophil extracellular traps (nets). nets are extracellular fibers formed by chromatin covered with histones and antimicrobial proteins derived from neutrophil granules. nets seem to act as a physical barrier where microorganisms get trapped and display antimicrobial activity that is independent of phagocytosis. despite the fact that many pathogens, including virus, bacteria, fungi, and parasites, have all been reported to induce net formation, no particular receptors on the neutrophil membrane leading to release of nets have been identified until very recently. iga - opsonized bacteria or iga - opsonized beads activated the fcri (cd89) leading to release of nets. other previous reports indicated that net formation was increased when microorganisms were opsonized with autologous serum, and also antigen - antibody complexes seemed capable of inducing net formation [22, 23 ]. these reports thus suggested a role for igg fc receptors (fcrs) in net formation. in the neutrophil two types of fcr are constitutively expressed, namely, fcriia and fcriiib [12, 13 ]. this fact has made it difficult to establish which functions are initiated by each of these two fcrs. for phagocytosis, there is no doubt that fcriia is an important receptor. in contrast, fcriiib is an important receptor for signaling to the nucleus. in the case of net formation, it is not clear which fcr is preferentially responsible for this function. it was previously reported that fcriiib promoted endocytosis of soluble immune complexes and that fcriia promoted net formation in vivo. however, more recently, it was reported that fcriiib is the receptor responsible for net formation in response to immobilized immune complexes. in addition, neutrophil stimulation by igg antineutrophil cytoplasmic antibodies (anca) led to degranulation and neutrophil extracellular trap formation in an fcriiib allele - specific manner. here, we have found that indeed fcriiib, but not fcriia, induced significant amounts of nets. selective fcriiib cross - linking with specific monoclonal antibodies on human neutrophils induced net formation. the release of these nets was detected 3 - 4 hours after stimulation and was dependent on ros, since the nadph - oxidase inhibitor dpi abrogated trap release. this net release was similar to the one induced by cross - linking fcri or by phorbol 12-myristate 13-acetate (pma) stimulation but different from the rapid, oxidant - independent net release recently described. fcriiib - induced nets consisted of long dna fibers decorated with histones and neutrophil elastase showing a bona fide neutrophil extracellular trap structure. although both fcriia and fcriiib induced a strong respiratory burst as shown by activated ros production, cross - linking of fcriia alone did not induce net formation. ros are required for net formation in most cases [2, 4, 8 ], but they are not sufficient, since ros production induced by phagocytosis can not initiate net formation. fcriiib - induced nets are similar in shape and molecular structure to those induced by pma, and the molecular mechanism leading to their release seems to be also similar. pma is a direct activator of protein kinase c (pkc) ; thus any possible receptor involved in net formation is bypassed. several inhibitors of pkc have been shown to block net formation. in agreement with those reports, we found that two different inhibitors of pkc indeed blocked net formation after pma and fcriiib stimulation. in addition, inhibition of syk with piceatannol blocked the release of nets induced either by pma or by fcriiib (figure 7). however, inhibition of syk with isyk slightly reduced only pma - induced netosis (figure 6s). the differential inhibition of net formation with two reported syk inhibitors suggests that some of the discrepancies in the literature regarding signaling pathways regulating netosis may be due to the use of various pharmacological inhibitors. it is necessary to revise these pathways more carefully in future studies. despite this caveat, inhibition of syk with piceatannol points to an important role for this kinase in net formation induced by specific cross - linking of fcriiib. syk was also found to participate in net formation induced by soluble immune complexes [22, 50 ], by insoluble immune complexes, and by pma. syk is normally associated with initial signaling events at the level of cell surface receptors, but pma can bypass these receptors to directly activate pkc. yet activation of syk by pma has been previously described in neutrophils. pma induced pkc - dependent phosphorylation of syk, and piceatannol reduced ros production in response to pma. together these reports and our data support the idea that syk activation is involved in both pma- and also fcriiib - induced ros - dependent netosis. downstream of pkc, a role for the mek, erk pathway and for nf-b in pma - induced net formation has been suggested. mek inhibition blocked pma- and fcriiib - induced netosis indicating that erk activation is required in this process. erk was also found to be required for net formation in response to soluble immune complexes and immobilized immune complexes. a previous report indicated that erk is required for nadph - oxidase activation, placing erk upstream of ros production, while another report suggested that ros are downstream of erk activation. therefore, it seems that nadph - oxidase activation for net formation may proceed not only through an erk pathway, but also independently of erk activation, depending on the stimulus [19, 20 ]. however, fcriiib - induced netosis was unaffected when neutrophils were treated with the same inhibitor for nf-b (figure 9). similarly, inhibition of pi-3k by wortmannin reduced net formation by pma but had no effect on fcriiib - induced net formation (figure 7). a possible role for pi-3k involvement in net formation induced by immobilized immune complexes we did not find the same result, but as mentioned above for syk the particular inhibitor used may be responsible for these different results. it was proposed that pi-3k could influence nf-b activation via phosphatidylinositol - trisphosphate and in turn nf-b activate genes important for signaling to net formation. these ideas, however, have not been proven experimentally and the role of pi-3k and nf-b in fcriiib - mediated netosis needs further exploration. fcriiib has been suggested to signal in cooperation with other molecules such as integrin mac-1 (cd11b / cd18), also known as complement receptor 3. however, complement receptor ligands are not sufficient to induce net formation in isolated neutrophils. similarly, in our case selective cross - linking of 2 integrins with mab ib4 also did not induce any net formation. in contrast, blocking mac-1 with antibodies against both cd11b and cd18 chains prevented net formation by lps, by -glucan, and by immobilized immune complexes. these reports and our data suggest that 2 integrins cooperate with other receptors to induce netosis, but they can not by themselves cause net formation. the involvement of 2 integrins in net formation might be more related to the adhesion requirement of neutrophils to form nets than to a signaling capacity of the integrin. along the same line of thought, cross - linking of other receptors such as 1 integrins also did not promote any net formation (figure 1), although the same procedure was capable of activating nf-b in neutrophils. recently, it was also reported that net formation in response to candida albicans required fibronectin via 1 integrins. similarly, the adhesive protein invasin from yersinia pseudotuberculosis promotes bacteria crossing the intestine epithelium by binding to 1 integrins on m - cells. however, invasin - mediated triggering of 1 integrin was essential but not sufficient for net production. clearly, integrins cooperate in different scenarios to activate netosis after various stimuli including immune complexes, but the exact role they play in this process remains elusive. moreover, as mentioned above, selective cross - linking of fcriia also did not promote net formation. this was not due to a defect in fcriia signaling because the same cross - linking procedure led to robust activation of erk (figure 6). in addition, latex beads opsonized with the specific anti - fcriia mab iv.3 were efficiently phagocytosed by neutrophils (figure 10). in contrast, latex beads opsonized with the specific anti - fcriiib mab 3g8 were poorly phagocytosed by neutrophils (figures 10 and 10s) but efficiently induced net formation. our results support the idea presented in a recent report showing that neutrophils sensed microbe size and selectively released nets in response to large pathogens, such as candida albicans hyphae and extracellular aggregates of mycobacterium bovis, but not in response to small yeast or single bacteria. in this study, phagocytosis via the receptor dectin-1 acted as a sensor of microbe size and prevented net release by downregulating the translocation of neutrophil elastase to the nucleus. similarly, we present here that neutrophils responded via fcriia with efficient phagocytosis ; however net formation was absent. in contrast, stimulation via fcriiib led to poor phagocytosis but to significant net formation. thus we conclude that nets are not formed when an opsonized target can be efficiently phagocytosed via fcriia. however, upon inefficient phagocytosis via fcriiib engagement, net formation is induced strongly. together, these data support the idea that indeed each fcr on the human neutrophil is capable of triggering specific responses. the inflammatory environment may be responsible for what receptor fcriia or fcriiib may predominate and initiate a particular cell response. fcriiib is expressed 4- to 5-fold more abundantly and has a higher affinity for igg than fcriia, thus probably becoming the preferred receptor to first engage immune complexes. at the same time, inflammatory stimuli can lead to fcriiib shedding from the cell, favoring now immune complex interactions with fcriia to induce phagocytosis and cytotoxicity. we believe that when a strong activating threshold is achieved by cross - linking fcriiib an efficient induction of net formation takes place. in conclusion, our data show that fcriiib governs fc receptor - induced net formation in human neutrophils. the signaling pathway used by fcriiib to induce nets involves pkc, pkc, and erk 1. | neutrophils (pmn) are the most abundant leukocytes in the blood. pmn migrate from the circulation to sites of infection, where they are responsible for antimicrobial functions. pmn use phagocytosis, degranulation, and formation of neutrophil extracellular traps (nets) to kill microbes. nets are fibers composed of chromatin and neutrophil - granule proteins. several pathogens, including bacteria, fungi, and parasites, and also some pharmacological stimuli such as phorbol 12-myristate 13-acetate (pma) are efficient inducers of nets. antigen - antibody complexes are also capable of inducing net formation. however the particular fc receptor involved in triggering this function is a matter of controversy. in order to provide some insight into what fc receptor is responsible for net formation, each of the two human fc receptors was stimulated individually by specific monoclonal antibodies and net formation was evaluated. fcriia cross - linking did not promote net formation. cross - linking other receptors such as integrins also did not promote net formation. in contrast fcriiib cross - linking induced net formation similarly to pma stimulation. net formation was dependent on nadph - oxidase, pkc, and erk activation. these data show that cross - linking fcriiib is responsible for net formation by the human neutrophil. |
minimally invasive gynecologic surgery (migs) for gynecologic malignancies was initially challenged regarding safety and efficacy. discussions of port - site metastasis (psm) questioned whether surgeon technical skill, port - site tumor extraction, local surgical seeding of port - sites, or pneumoperitoneum contributed to psms (curet, 2004). the lap2 trial reported a 0.24% (4/1696) psm incidence (walker. while uncommon, psm remains troubling as some experts consider psm indicative of advanced disease and poor prognosis (zivanovic. many psms are present in the setting of disseminated disease (zivanovic., 2008, national comprehensive cancer network guidelines recommend surgical excision of resectable, isolated metastases and consideration of adjuvant chemotherapy or radiation therapy (national comprehensive cancer network, 2014). therefore, administration of radiation or chemotherapy after surgical treatment of an isolated psm is currently individualized based on expert opinion. here we present an unusual case of a clinically isolated psm diagnosed after a long 40 month disease free interval following migs staging of an early stage uterine carcinosarcoma. ten months after management of the initial isolated psm, the patient developed obvious disseminated disease. increased reporting of outcomes associated with clinically isolated psm may influence post - resection management by increasing utilization of systemic chemotherapy to treat suspected underlying disseminated disease. a 71 year old woman with body mass index of 21 kg / m was presented with post - menopausal uterine bleeding and underwent endometrial biopsy, which revealed a high - grade endometrial serous adenocarcinoma. computed tomography of the abdomen and pelvis demonstrated a 3.5 cm uterine mass, and was without evidence of lymphadenopathy, ascites, or metastasis. robotic - assisted total laparoscopic hysterectomy, bilateral salpingo - oophorectomy, pelvic and para - aortic lymph node dissection, and infracolic omentectomy was performed without complication. five abdominal incisions were made for the robotic camera and instruments, as well as an accessory port. electrocautery bilateral tubal ligation was first performed to prevent retrograde cancer cell spillage into the peritoneum. port sites were closed using a gore suture passer and the skin was closed with 40 vicryl. pathologic diagnosis was stage ii uterine carcinosarcoma with components of serous adenocarcinoma, endometrioid adenocarcinoma, undifferentiated carcinoma, and undifferentiated spindle cell sarcoma. the tumor was 2.5 cm in maximum dimension in the uterine fundus with maximum depth of invasion of 15% of total myometrial thickness and had superficial cervical stromal invasion. she received adjuvant high - dose - rate vaginal cuff brachytherapy (2100 cgy total dose in 3 weekly fractions) between six cycles of paclitaxel (135 mg / m) and ifosfamide (1.6 g / m) every three weeks, with three cycles of chemotherapy administered before brachytherapy and an additional three cycles after brachytherapy. she remained without evidence of disease for 40 months, when she presented with a subcutaneous mass on the right lower quadrant at the location of a previous port - site. computed tomography of the abdomen and pelvis demonstrated a 3.5 cm heterogeneous mass in the deep subcutaneous tissues of the right lower quadrant and was without evidence of other disease (fig. she underwent surgical resection of the mass, which extended to the fascia but not the peritoneum, which was not entered. pathology confirmed metastasis (high grade adenocarcinoma) consistent with her previous uterine primary (fig. she received external beam radiotherapy of 5990 cgy total dose to the right lower quadrant recurrence site. five months later, she had a new palpable lump at the right flank, 3 cm superior to the previous mass. positron emission tomography (pet / ct) was performed and revealed a solitary focus of activity in the superficial right abdominal wall distinct from the previous recurrence site (fig. the mass was resected with clean margins and pathology again confirmed metastasis consistent with the primary diagnosis. in the presence of severe back pain, she underwent another pet / ct two months after resection, which revealed new pulmonary nodules concerning for metastasis and l4 vertebral metastasis. she began palliative oral etoposide (50 mg alternating with 100 mg for days 121, then 7 days off, every 28 days) chemotherapy. many hypotheses have been proposed to explain the development of psm including direct wound seeding by specimen removal or contaminated instruments, aerosolization of exfoliated cancer cells, the chimney effect caused by insufflation, and pneumoperitoneum, carbon dioxide, or tissue trauma altering the normal immunologic defenses at port sites (curet, 2004). there is little evidence to support any practice to reduce psm risk (curet, 2004). the lap2 trial reported a low psm incidence of 0.24%, which is acceptable compared to laparotomy (walker., 2012, gcer., 2005). furthermore, the lap2 trial concluded that the use of mis was safe regardless of pathological subtype (walker., 2012). reviewed published data of psm after staging for endometrial cancer (palomba., 2012). isolated psm were all endometrioid histology, high grade, and stage i or ii at initial diagnosis. one patient declined excision and underwent palliative radiation plus hormone therapy before succumbing to disease 5 months later. one patient had excision followed by radiation and was disease free for 30 months before recurring and dying of disease 42 months after first recurrence. also concluded that apparently isolated psm may represent occult, disseminated metastatic disease (palomba., 2012). carlson. reported an interesting psm at a right upper quadrant (ruq) port - site used for laparoscopic cholecystectomy that was performed 6 months prior to the patient 's staging and diagnosis of stage iiic serous ovarian cancer (carlson., 2002). twenty - seven months after laparoscopic surgical staging and cytoreduction of the ovarian malignancy, she developed a psm at a ruq port - site from her prior laparoscopic cholecystectomy. six months later she developed a pelvic recurrence and was treated with additional platinum chemotherapy. she subsequently remained disease free for three years prior to the report of her case. the apparently isolated ruq psm also preceded the development of additional metastatic disease despite treatment of the psm with resection and systemic chemotherapy. uterine carcinosarcoma, although initially considered to be a sarcoma, is now recognized as an advanced carcinoma that undergoes an epithelial - to - mesenchymal transformation (cantrell., it is one of the most aggressive types of uterine pathologies, associated with high recurrence rates and a 5-year survival of 3339% (cantrell., 2015). in our case, the patient developed an isolated uterine carcinosarcoma psm after a 40 month disease free interval but before any indication of systemic disease. later, she developed an additional abdominal wall metastasis, as well as suspected pulmonary and bone metastases. we propose that when patients develop an apparently isolated psm, although additional metastases may not yet be evident by imaging, the presence of psm indicates high risk for progression of disease and warrants systemic treatment in additional to local excision and/or radiation. we propose that patients with apparently isolated psm without evidence of other metastatic disease require systemic chemotherapy in addition to excision and/or radiation therapy to the site of recurrence. psm of carcinosarcoma indicates a poor prognostic condition for the patient and palliative discussions should be initiated. case reports, case series and literature reviews informing expert opinion will likely remain the highest level of evidence for the clinical dilemma of isolated psm. written informed consent was obtained from the patient for publication of this case report and accompanying images. a copy of the written consent is available for review by the editor - in - chief of this journal on request. | a 71-year - old woman with suspected endometrial cancer underwent robotic - assisted hysterectomy, bilateral salpingo - oophorectomy, pelvic and para - aortic lymph node dissection, and infracolic omentectomy revealing a stage ii uterine carcinosarcoma with components of serous adenocarcinoma and undifferentiated spindle cell sarcoma. there was no evidence of distant metastasis at the time of surgery. however pelvic washings were positive for malignant cells. she received adjuvant chemotherapy and vaginal cuff brachytherapy. forty months later she developed a subcutaneous mass at the location of previous port site which was confirmed to be recurrence of the uterine primary. she subsequently developed additional distant metastases to the abdominal wall, lungs, and bone. port site metastasis (psm) was the earliest indicator of disseminated metastatic disease in this patient. we review challenges in the management of patients with psm and propose that psm be considered as a sign of systemic disease even when presenting as an apparently isolated recurrence. |
malnutrition from loss of appetite, indigestion, malabsorption, and metabolic problems is a common condition in cancer patients undergoing treatment. side effects of chemotherapy include anemia, appetite loss, dysgeusia, changes in sensitivity to food temperature, constipation, diarrhea, dysphagia, xerostomia, fatigue, and early satiation that can decrease food intake and lead to extreme weight loss. the goal of nutritional intervention for such patients is to improve their nutritional condition by mitigating treatment side effects, offering individualized patient care, and improving food intake while respecting patients ' food habit with regard to administering more progressive interventions. additionally, as the main method of nutritional intervention, patients are recommended to eat small and frequent meals 5 - 6 times a day to increase their overall intake since most patients experience appetite loss resulting from metabolic problems and chemotherapy treatment. if the amount of food does not exceed 60% of the daily recommended intake, tube feeding or intravenous alimentation is performed. if diets are appropriated to each patient 's condition, oral food intake is recommended whenever possible. hot or cold foods and drinks are offered depending on patient 's receptivity and variations are made according to patient 's condition. when a low bacteria diet is needed, appropriate foods are given and the patient is educated about the dietary practices. the purpose of the current case study is to record the process of nutritional diagnosis and intervention conducted on malnourished cancer patients with chemotherapy for treatment at a hospital. in this case, after admission to the hospital for general weakness, the patient was identified as being at risk of malnutrition according to an early nutritional assessment program by the nutrition team at hospital. approval of the institutional review board at (khnmc irb 2013 - 116) the kyung hee university hospital at gangdong was obtained. on october 21, 2013, a 61-year - old man was admitted to the gastroenterology ward. to measure the patient 's nutritional condition, medical history, physical measurements, biochemical data, medical examinations and treatments, nutritional physical examination data, and food / nutrition - related diet history the patient was diagnosed with diabetes mellitus 10 years ago, hypertension (10 years ago), and rectal cancer 3 years ago, for which the patient underwent an ileostomy on november 14, 2011 and received leucovorin plus fluorouracil (lv5fu2) as preop # 1 and postop # 8 chemotherapy. afterward, the patient underwent ileostomy repair on december 3, 2012, and then a liver wedge resection on july 18, 2013 for liver s4 metastasis during the follow - up (f / u). afterward the patient received chemotherapy # 4 from september 2 through october 14, 2013 using oxaliplatin, folinic acid, and 5-fluorouracil (flofox4). the patient 's current drug prescriptions include lantus 10 u / day, apidra 6 u # 3/day, and nutriflex lipid peri [+ humulin - r (regular insulin) 15 u ] every other day (eod), and chemotherapy was paused due to general weakness. the first screening visit was conducted on october 22, 2013. at the time of hospital admission (october 21, 2013), the physical measurements were : height, 167 cm ; body weight, 53.2 kg ; ideal body weight (ibw), 61.4 kg ; % ibw, 86.6% ; body mass index (bmi), 19.1 kg / m ; usual weight, 64.8 kg (july 18, 2013) ; and weight change of -11.6 kg (17.9% loss) over the most recent 3 months. examinations upon admission showed the following : total protein / albumin, 5.6/3.2 g / dl ; hemoglobin / hematocrit (hgb / hct), 8.4 g / dl/25.4% ; total lymphocyte count (tlc), 741.2 cells / mm ; cholesterol, 162 mg / dl ; blood urea nitrogen / creatinine (bun / cr), 20/1.0 mg / dl ; ca / p, 8.3/2.3 mg / dl ; na / k / cl, 132/4.2/98 meq / l ; c - reactive protein, 6.28 mg / dl ; hemoglobin a1c, 9.7% ; and casual glucose, 470 mg / dl. the nutritional and physical examination data showed that the patient had stomatitis, nausea, and vomiting as side effects of chemotherapy and the blood pressure was 142/83 mmhg. in terms of food / nutrition - related diet history, the dietary prescription and diet - related experiences were such that the patient experienced a loss of appetite after chemotherapy, did not receive education intended for cancer patients (due to being too tired). the patient 's food and beverage consumption over the most recent week (after the fourth round of chemotherapy) includes six glasses of cola (480 kcal / day ; 120 g of sugar) due to stomatitis, nausea, and vomiting. at the hospital, the administration of other foods was not attempted since the patient experienced pain and vomiting after consuming soft foods, particularly gruel (not eaten) and intravenous alimentation (nutriflex lipid peri eod ; energy, 465 kcal ; c : p : f ratio, 34.4:17.2:48.4 ; carbohydrate, 40 g ; protein, 20 g ; fat, 25 g). the patient stayed in the bed whole day. in the patient - generated subjective global assessment conducted for nutritional assessment, the patient scored 15 points, indicating the need for improved symptom and focused nutrition management. approximately 1600 - 1860 kcal of energy (baseline weight, 53.2 kg ; based on calculation, 30 - 35 kcal / kg needed) and 64 - 85 g of protein (baseline weight, 53.2 kg ; based on calculation, 1.2 - 1.6 g / kg needed) were set as the required nutritional intake. two nutritional diagnoses were made : frist, malnutrition arising from a decrease in food intake related to the side effects of chemotherapy (stomatitis, nausea, vomiting) as well as a lack of food / nutrition - related knowledge regarding food types and quantities. the evidence for this diagnosis being that the patient experienced a 17.9% weight loss over the most recent 3 months and consumed only 28% of the required nutrients during the most recent week (after the fourth round of chemotherapy) ; second, the patient 's lack of knowledge with regard to foods and nutrients which is possibly attributable to fear for food intake and this was observed through refusal to ingest food and tendency to ingest only cola after chemotherapy. as a nutritional intervention for the first diagnosis, the medical team was advised to change the patient 's diet to diabetic thin rice gruel (figure 1) (1,700 kcal and 72 g of protein) and supply any intravenous aliments according to the amount of oral intake, which would provide 30% of the hospital food and the intravenous aliment maintenance to the patients. the hospital - supplied food consisted of soft and non - irradiated foods considering the patient 's stomatitis, and in the case of this particular patient, it was decided that close monitoring after dietary changes would be required. as a nutritional intervention for the second diagnosis, the patient was consulted for the need to consume a diverse range of foods and the hospital diet (diabetic thin rice gruel and consider the patient 's preferences and lactose intolerance). this intervention would stop for the patient consuming cola, which could worsen the nausea, and try the hospital - supplied foods. the second screening visit was conducted on october 24, 2013, and physical measurements, biochemical data, medical examinations and treatments, nutritional physical examination data, and food / nutrition - related diet history were examined. on follow - up, there was no change in physical measurements, and examinations revealed the following : total protein / albumin, 5.1/3.1 g / dl ; hgb / hct, 8.5 g / dl/25.7% ; tlc, 712 cells / mm ; cholesterol, 135 mg / dl ; bun / cr, 19/1.0 mg / dl ; ca / p, 7.8/1.6 mg / dl ; na / k / cl, 138/50/107 meq / l ; blood glucose (fasting, postprandial 2 hour glucose # 3), 177/208/363/309 mg / dl (10/22), 270/206/206/120 mg / dl (10/23), and 126/133/260/246 mg / dl (10/24). nutritional and physical examination data, included gastroenterological symptoms such as nausea and vomiting after consuming certain foods. for example, the patient had a vomit after drinking orange juice while apple juice did not cause any gastrointestinal trouble. vital signs were 135/80 mmhg. in terms of food / nutrition - related diet history, the patient 's dietary prescription and diet - related experiences were such that he was informed about diabetic thin rice gruels, non - irritating foods, and nutrient supplement drinks. the patient 's food and beverage consumption consisted of the supplied hospital diet : 20% of rice gruel, 50% of the steamed eggs and nutritious beverages, and 100% of the soup and apple juice (energy, 1,050 kcal ; c : p : f ratio, 56.0 : 14.4 : 29.6 ; carbohydrate, 147 g ; protein, 37.2 g ; fat, 34.5 g) along with intravenous aliments (hepasol inj 500 ml / day ; 200 kcal, 50 g protein). after the first screening, the patient tried various foods and started learning to move in a wheelchair. the patient 's nutrient requirements were the same as at the first screening. upon nutrition monitoring and assessment, the goal set after the first screening with regard to nutritional intervention was partially achieved considering that the hospital food consumption was at 62% of the overall consumption and the intravenous aliment had changed (nutriflex lipid peri eod to hepasol / d). after the second nutritional intervention, the patient stopped consuming cola and began to consume a diverse range of hospital foods ; thus, the goal of the intervention was reached. after the second screening, the patient again had two nutritional diagnoses : first, excessive protein supplementation, because extra protein was administered via the intravenous aliments outside oral intake, and the patient was consuming 125% the required protein quantity ; second, the patient 's lack of knowledge regarding food and nutrition due to absence of education about chemotherapy and food preparation. with regard to nutritional interventions, that for the first nutritional diagnosis involved advising the medical team to stop supplying protein via the intravenous aliments. for the second nutritional diagnosis, advising the medical team educate the patient about food choices and preparation which are appropriate for managing side effects of chemotherapy. the goal of the intervention was consumption of energy and protein > 70% of the requirement and maintenance or increase of body weight. continuous monitoring was required, and even after discharged, the patient required proper outpatient care. in this case, due to side effects following chemotherapy, the patient experienced significant and unintentional weight loss as a result of stomatitis, nausea, and vomiting and malnourished with poor ingestion. afterward, through nutritional interventions via the nutrition care process, the patient 's quantity of food intake was increased and further increases were expected under continuous care (figure 2). similarly, in earlier studies, individualized nutritional education effectively increased caloric and protein intake in cancer patients. thus, it is speculated that, had this patient also received a more active education regarding chemotherapy side effects and food preparation before and after his colon cancer surgery, poor ingestion caused by stomatitis and prevent weight loss after the second round of chemotherapy could have been treated easily. hereafter, to address this issue, all patients undergoing chemotherapy should be educated about practices that could enhance response to treatment and undergo continuous observation during treatment. additionally, with regard to this patient, it is regrettable that, despite the fact that repeated malnutrition was diagnosed during several admissions to the hospital during his second round of chemotherapy, the patient was not administered active care for malnutrition. | in this case study, the process of nutritional diagnosis and intervention conducted at a hospital on a malnourished patient who underwent treatment for a chronic illness (chemotherapy for cancer treatment) was recorded. the patient received his first round of chemotherapy for colorectal cancer, and then a second round after the cancer metastasized to the liver. the patient was malnourished and had experienced weight loss (17% loss in the most recent 3 months) due to side effects of chemotherapy including stomatitis, nausea, and vomiting. nutritional diagnosis and intervention via the nutrition care process were implemented through two screening rounds, and the quantity of oral intake increased from 28% to 62% of the recommended daily intake. the patient required continuous monitoring and outpatient care after hospital discharge. it is speculated that if a more active patient education and dietary regimen with respect to chemotherapy side effects had been offered after the patient 's first chemotherapy cycle, it might have been possible to treat ingestion problems due to stomatitis during the second cycle of chemotherapy and prevent the weight loss. henceforth, patients receiving chemotherapy should be educated about nutrition management methods and monitored continuously to prevent malnutrition. |
trata - se de uma prtica antiga e teve seu primeiro relato em 1913, mas, por conta dos eventos adversos decorridos de sua utilizao inadequada, como o uso de solues hipertnicas, a prtica passou a ser inutilizada. essa pratica tem sido utilizada em pacientes que apresentam diagnsticos de desidratao moderada em razo de quadros de disfagias severas, demncias, obstruo do intestino por conta de neoplasias, sonolncia. h ainda a possibilidade de administrao de medicamentos para aqueles pacientes que no apresentam condies para se puncionar um acesso venoso perifrico. a hipodermclise descrita tambm como uma prtica simples de ser realizada e mais barata que as demais tcnicas. os medicamentos e fluidos administrados por meio da hipodermclise tm sua absoro por meio do mecanismo da difuso capilar.. a farmacocintica semelhante a dos medicamentos administrados pela via intramuscular, mas apresenta tempo de ao prolongado, alm de melhor tolerabilidade para aqueles medicamentos cujo ph prximo da neutralidade e que sejam hidrossolveis. para facilitar a administrao dos medicamentos por meio da hipodermclise, algumas literaturas sugerem o uso da hialuronidase, pois esta uma enzima que degrada o cido hialurnico presente no tecido, levando diminuio de sua viscosidade e aumentando, assim, a taxa de absoro dos medicamentos administrados. existem locais (stios de puno) que so mais adequados para a terapia, como regio deltoide, regio anterior do trax, regio escapular, regio abdominal, e nas faces anterior e lateral das coxas (figura 1). figura 1stios de puno no brasil, essa tcnica vem ganhando seu espao para uso em pacientes que se encontram em cuidados paliativos ou naqueles bastante idosos e debilitados. a terapia subcutnea abrange no s os fluidos de reposio, mas tambm medicamentos que passaram a serem prescritos para essa via, como antimicrobianos e analgsicos, entre outros. parte desses medicamentos no apresenta descrio em bula sobre a possibilidade de serem administrados por essa tcnica ; dessa forma, quando prescritos, consideramos seu uso dessa maneira como off - label. para os profissionais prescritores, de um modo geral, a indicao da via baseada na literatura internacional ou em prprias experincias clinicas, que no esto relatadas em artigos oficiais. a falta de informaes fidedignas acaba gerando, para o servio de farmcia local, certa dificuldade no momento de se fazer a avaliao de uma prescrio mdica, bem como na maneira de orientar a equipe de enfermagem sobre os cuidados necessrios para a utilizao da droga prescrita, pois cada medicamento apresenta uma caracterstica exclusiva, como ph, estabilidade, alm de volumes de diluio e diluentes apropriados. pelo fato de existirem poucas informaes na literatura sobre este assunto, surgiu o interesse de analisar, por meio deste trabalho, o que as literaturas nacionais e internacionais disponibilizam sobre a hipodermclise e o quanto tais informaes podem ser valiosas para o farmacutico, uma vez que esse profissional o responsvel pelos medicamentos dentro de uma instituio mdico - hospitalar. foram consultados livros e manuais sobre cuidados paliativos, e foram realizadas buscas por artigos e guidelines nos bancos de dados scientific eletronic library online (scielo), medline e google acadmico. os descritores utilizados foram : hipodermclise (hypodermolysis), cuidados paliativos (palliative care) e via subcutnea (subctuaneous), publicados em lngua portuguesa e inglesa, em um recorte temporal abrangendo o perodo entre os anos de 1999 a 2012. os trabalhos foram analisados conforme o objetivo proposto, dessa forma, foi utilizado os trabalhos que traziam informaes relacionados com o uso de medicamentos e o modo para sua utilizao, solues para hidratao (cloreto de sdio 0,9%, cloreto de sdio 0,45%, glicose 5%) alm de vantagens e desvantagens da tcnica e possveis reaes adversas. ainda assim, no ofereceram um grande nmero de informaes a respeito, principalmente sobre o uso de drogas. as informaes foram tabuladas em uma planilha eletrnica (excel) e apresentadas na forma de quadros e tabelas. para a formulao do quadro relacionado s compatibilidades, foi utilizado como ferramenta o banco de dados eletrnico micromedex. o quadro 1 apresenta informaes sobre os medicamentos citados nos trabalhos. quadro 1tabela de medicamentos mais utilizados pela via subcutneadrogaindicaodosesdiluente mais indicadotempo de infuso indicadocomentriosampicilina infeces500mg / diaatropina1,2mg/1 vez ao dia cefepima infeces1g / diasfcefotaxima infeces500mg / diasf30 minutosceftazidima infeces500mg / diasf30 minutosceftriaxona infeces1g / diasf30mimcetorolacodor intensa30 - 90mg / diasfvia exclusivaciclizinanusea e vmito25 - 50 mg a cada 8 horas (mximo de 150mg / dia)inf. continua = adincompatvel com sfclonazepamagitao e ansiedade5 - 8mg / diasf ou d irritante, diluir o mximo toleradodexametasona1. 8 - 20mg / diadiclofenacodor75 - 150mg / diasf irritante, diluir o mximo toleradodipironador1 g at a cada 6 horassfvia exclusivaescopolaminaclicas intestinais60 - 180mg / diaadmximo de 40mg / dia em infuso contnuafamotidinaprotetor gstricofenobarbitalconfuso200mg / diaadvia exclusiva, mesmo compatvel com morfinafentanildorusual : 100 -1.000mcg / dia resgate 10mcg a cada 1 horasf1ml / h=5mcg / h (soluo de 500mcg em 100ml de diluente)furosemidadispneia devido congesto pulmonar20 - 40mgsfgranisetronanusea e vmito3 - 9mg / dia50ml sf>10 minutos haloperidolnusea e vmito sedao, agitao2,5 - 10mg / diaadconcentrao mxima 2mg / ml. sf pode precipitarhidromorfonador50% da dose oralhidroxizinaantialrgicolevomepromazinanusea e vmitos intensos5 - 100mg / diasf irritante, diluir o mximo toleradodose mxima de 200mgmetadonador intensa50% da dose oralsf60ml / h irritante, variar o local da puno a cada 24 horasmetoclopramidanuseas e vmitos30 - 120mg / diaad irritante, diluir o mximo toleradomidazolam1. iniciar com 1mg / h e aumentar at 4mg / hmorfinador e dispneia50% da dose oralsf ou ada dose de 10mg / ml pode ser administrada a cada 4 horasnaproxenodor550 - 600mg / diaincompatvel com a morfinaoctrotdeo1. reduo de secreo gstrica, motilidade, vmitos e diarreia1. 50 - 500mcg(mximo de 1.500mcg)ondansetronanusea e vmito8 - 24mg / diasf ou adprometazina nusea antialrgico12,5 - 25mg / diaranitidinaprotetor gstrico50 - 150mg / dia (mximo de 300mg)ad tobramicina infeces75mg / diatramadoldor100 - 600mg / diasf adaptado de : ferreira ka e santos ac.pereira i. cuidado paliativo. ad : gua destilada ; sf : soro fisiolgico ; : sem informao disponvel. ad : gua destilada ; sf : soro fisiolgico ; : sem informao disponvel. conforme a metodologia citada para a busca das informaes, foram selecionadas 17 literaturas (quadro 2) e um banco de dados eletrnicos. quadro 2literaturas selecionadas nas bases de dadosautortituloanomedicamentos/ hidratao / ambos / no citavantagens e desvantagensreaes adversasmodo de preparo e administrao dos medicamentospereira i cuidado paliativo. ? 1999ambossimsimnoconselho regional de enfermagem de so paulo hipodermclise2009ambossimsimnotakaki cy. hipodermclise e administrao de medicamentos por via subcutnea : uma tcnica do passado com futuro2009ambosnosimsimgriffithis a clinical guideline for subcutaneous infusion (hypodermoclysis). hypodermoclysis (subcutaneous infusion) effective mode of treatment of dehydration in long - term care patients2004hidrataonosimnofrasca d,. pharmacokinetics of ertapenem following intravonous and subcutaneous infusions in patients2010medicamentosnonosimnhs greater glasgow and clyde guideline for the use of subcutaneous medications in palliative care for adults2010medicamentossimsimnoazevedo hipodermclise : um mtodo alternativo para infuso de fluidos e medicamentos pela via subcutnea2009ambossimsimsimfonzo - christe c,. subcutaneous administration of drugs in the elderly : survey of practice and systematic literature review2005ambosnosimsim aps a seleo das literaturas, tivemos os seguintes achados : 5 literaturas abrangeram apenas informaes relacionadas ao procedimento de hidratao, ou seja, no foram citados medicamentos, 4 apenas focaram o uso de medicamentos e 8 literaturas apresentam ambas informaes. apenas 5 trabalhos citaram informaes relacionadas com a forma de preparo e administrao dos medicamentos. dos trabalhos selecionados, 10 deles trazem informaes relacionadas s vantagens e desvantagens do mtodo, (quadro 3), e apenas 2 referncias no citaram quaisquer reaes adversas. quadro 3vantagens e desvantagens da hipodermclise(1 - 4,8 - 11,14,16)vantagensdesvantagensbaixo custotempo de infuso usual de 1ml / minutomais confortvel que administrao intravenosaapenas 3.000ml no perodo de 24 horas podem ser infundidos e devem ser fracionados em stios distintosmais fcil de obter novos locais de administraopode levar a edemas locaispode ser feito em homecare limitada a administrao de eletrlitosreduo de hospitalizaessuplementos nutricionais e solues hipertnicas no so indicadospoucos relatos de casos de tromboflebitespossibilidade de reaes locaisno tem sido relacionada com infeces e sepsesno indicado em casos de desidratao gravepode ser instalada e interrompida facilmente, abrindo e fechando o sistema de infusoem casos de urgncias e emergnciasno tem sido associada formao de cogulosnos casos de infeces bacterianas gravesno exige materiais complexos as reaes adversas mais citadas foram dor, inflamao no local da puno e, at mesmo, edemas e necroses teciduais. em um dos trabalhos localizados, foram acompanhados 57 pacientes em uma instituio de longa permanncia para idosos (ilpi) que recebiam hidratao por hipodermclise ; 88% deles apresentaram melhoras no estado clinico geral e 84% tiveram melhora do status cognitivo aps o uso da hipodermclise. com relao s informaes relacionadas a medicamentos, pouco se tem descrito, pois poucas drogas foram at o momento estudadas para essa via e poucas apresentam licena para o uso em infuso subcutnea. conforme um dos artigos analisados, no qual um questionrio aberto respondido por mdicos, sobre que tipos de medicamentos seriam mais comumente utilizados, a morfina foi a droga mais prescrita (98%), seguida por haloperidol (90%), furosemida (69%) e metoclopramida (44%), entre outras. ainda nesse trabalho, os mdicos foram questionados sobre qual a maneira utilizada para validar as informaes, e 70% responderam que prescreviam e validavam a prescrio com outros colegas mdicos, 32% validavam com o servio de farmcia do hospital e apenas 22% consultavam a literatura. a maioria das classes de medicamentos j utilizados para essa via, so os opiides, antibiticos, antemticos e sedativos. o quadro 3 traz algumas informaes relacionadas exclusivamente aos medicamentos que j tiveram relatos em literatura de utilizao pela hipodermclise. alm dessas drogas citadas na tabela, j existem trabalhos que relatam o uso de outros antimicrobianos, dentre eles ertapenen, amicacina, gentamicina e teicoplamina, mas so literaturas ainda com informaes limitadas e que aparentemente demonstraram equivalncia em comparao com as via usuais, mas o nmero de pacientes utilizados foi bem pequeno. grande parte das reaes adversas citadas nos trabalhos ocorreram em decorrncia do uso inadequado, como, por exemplo : locais inadequados de puno, medicamento inapropriado para a via, diluio inadequada e falta de rodzio da puno (trocar a cada 96 horas). por meio desta pesquisa, notamos que as informaes relacionadas com a forma de preparo e administrao de medicamentos ainda no esto padronizadas. assim, devemos avaliar as condies do paciente antes de indicar a via, e se existem outras drogas ou at mesmo solues que j estejam sendo administradas pela via subcutnea, como uma soluo fisiolgica, por exemplo. 24 horas no pode ultrapassar 3.000ml divididos em dois locais de puno diferentes (1.500ml em cada puno a cada 24 horas). essa questo pode ser minimizada uma vez que duas ou mais drogas podem ser administradas em um nico sistema de infuso. por meio dos resultados encontrados, observamos que a tcnica da hipodermclise uma metodologia segura, eficaz, barata e de fcil aplicabilidade e aparenta trazer alguns benefcios. entretanto so poucos os estudos originais disponveis sobre esse tema, principalmente aqueles que incluem a administrao de medicamentos, as amostras dos trabalhos localizados foram pequenas, sendo assim, difcil chegar a uma definio sobre a eficcia na utilizao desses medicamentos, o que tambm foi evidenciado por alguns autores em seus trabalhos. os estudos originais utilizados para compor esse trabalho, foram focados em questes relacionadas com a hidratao, principalmente em idosos. dos medicamentos j indicados e comumente prescritos para via subcutnea, como relatado em um dos trabalhos, so prescritos na sua maioria, mais baseados em prtica clinica, do que nas literaturas propriamente. alm disso, sua forma de ser administrada acaba sendo conforme seu uso intravenoso, pois por enquanto no se tem estabelecido qual a forma mais adequada para o preparo e a administrao para os pacientes ; contudo, na literatura, h informaes que preconizam que a diluio deva ser de 1ml de medicamento para 1ml de diluente. no entanto, essa informao ainda no um consenso para todas as drogas prescritas, pois cada uma delas apresenta seu perfil de diluio, estabilidade e, principalmente, ph. e essas questes podem ser fundamentais para que se evitem eventos adversos. se a tcnica no for aplicada adequadamente, pode sim trazer problemas para o paciente, conforme descrito nos resultados desse trabalho. o que de certa forma, ao invs de trazer os benefcios possveis pela tcnica, acabe sendo mais prejudicial. dos 17 trabalhos utilizados, apenas um nico artigo no abordou informaes especificas sobre a tcnica. esse por sua vez, buscou saber qual o conhecimento de uma equipe de enfermagem sobre a tcnica, o que de fato chamou a ateno, pois grande parte dos enfermeiros que responderam o questionrio (71%) no conheciam a tcnica. o que refora a idia de que devido a pouca disponibilidade de informaes em literatura, ou a disponibilidade de informaes repetidas, torna a tcnica pouco divulgada e, alm disso, exista uma grande dificuldade para o farmacutico e para a equipe que acompanha esse perfil de prescrio mdica em proporcionar uma orientao de qualidade para que haja o manejo seguro da tcnica de preparo e administrao de drogas atravs da via subcutnea. o quadro elaborado sobre as compatibilidades mostra algumas possibilidades de se otimizar stios de puno, bem como volumes de administrao, podendo levar assim, um conforto maior para o paciente devido a diminuio na manipulao do paciente. a maior dificuldade neste trabalho foi localizar informaes relacionadas especificamente ao modo de preparo e ao tempo de administrao dos medicamentos, como citado nos resultados, apenas 5 descreveram de alguma forma a maneira de preparo e administrao de medicamentos. a indstria farmacutica deveria, por sua vez, investir e elaborar estudos voltados para essa tcnica de administrao. isso pode vir a ser um diferencial de mercado, uma vez que tal tcnica voltada para pacientes em cuidados paliativos e em idosos, pois estes, por sua vez, apresentam reduo de massa muscular, dificuldades em se puncionar acessos perifricos e dificuldades de deglutio. novos estudos poderiam ser elaborados em uma parcela considervel de uma populao especifica, para tambm podermos construir um perfil de segurana para o paciente e para o prprio medicamento. a compilao dessas informaes pode direcionar o farmacutico bem como a equipe mdica e de enfermagem na avaliao dos medicamentos a serem administrados pela hipodermclise, podendo garantir, dessa maneira, o sucesso da terapia e a segurana do paciente, alm de diminuir os riscos de eventos adversos relacionados administrao por tal via. | the aim of this study was to analyze the information available in the literature about the drugs that can be administered through hypodermoclysis and the resulting impact that this information may have on the routine of the pharmacist working at a hospital. the study was based on a review of the literature. the results showed positive points of the procedure, but little specific information about medications such as routes of administration, standard dilutions, optimal doses, etc. thus, it was possible to verify that there is no definite information as to the correct way to administer the drugs in this route, even though this is an effective and safe option, according to the literature. the lack of information has a negative impact on the support provided by the pharmacist to the nursing staff to ensure that the drug actually reaches its therapeutic goals safely. |
secondary amyloidosis (aa) can occur during the dynamic progression of chronic rheumatic diseases such as rheumatoid arthritis (ra), juvenile idiopathic arthritis (jia), ankylosing spondylitis and psoriatic arthritis [15 ]. despite recent progress in the treatment of aa the prospective disease progression of aa often depends on a number of factors such as the presence of comorbidities and the degree of organ involvement pathology. it is generally accepted that the early diagnosis of aa may influence both disease prognosis and progression. the clinical condition of ra patients with advanced aa is usually poor and the possibility of effective intensive treatment in such patients diminishes over time. in addition, a significant proportion of patients with ra display pathological amyloid deposits. due to these issues, clinical screening on a regular basis and at a relatively early period of disease progression remains essential in overcoming aa - associated clinical complications. laboratory tests and imaging techniques are generally used for the clinical diagnosis of suspected amyloidosis. gold standard. amyloid deposits are generally detected with the use of the congo red dye test and subsequent viewing of the treated specimen under a polarized light microscope. amyloid deposits in the treated tissue sample display optical birefringence and appear green in color under plane - polarized light. over the previous 2 decades progress has been made in clinical laboratory tests as well as diagnostic imaging techniques such as ultrasound, mri and ct. nonetheless, the early diagnosis of connective tissue disorders (ctd) and other diseases remain problematic, most probably due to the sensitivity of available instruments. fluorescence spectroscopy is a technique previously used for the detection of cancerous tumors, delineating the efficacy of surgical excision of tumour growths, diagnostic assessment of human cutaneous melanoma, and ultrastructural detection of morphological changes in connective tissue [810 ]. compared to conventional diagnostic approaches, fluorescence spectroscopy is characterized by relatively high precision, efficacy, efficiency and cost - effectiveness. it was with this in mind that an alternative fluoroscopy - based modality for the detection of aa was developed and its applicability and generalizability within a clinical setting were investigated. based on prior diagnostic research experience and preliminary findings obtained within our own research group, a decision was made to conduct an extensive systematic analysis of fluorescence spectra of different tissue samples in which the presence or absence of amyloid deposits were previously confirmed. in so doing, patients affected with ra (n=99) between 39 and 65 years of age were examined (table 1). the first subgroup consisted of patients in whom the diagnosis of aa was confirmed by clinical observations, histopathological examinations and laboratory tests. the second cohort study subgroup consisted of study subjects in whom the intracorporeal presence of aa was excluded via the use of the same tissue specimens were collected from various sites, including adipose tissue from the abdominal fold, rectal mucosa or gingiva. following collection, each slice was subsequently transferred to a microscope slide, stained with congo red and observed under plane - polarized light with the use of a designated light microscope. in all cases, immunohistochemical examinations were performed on each tissue sample with the envision (dako) kit, which employed the following monoclonal antibodies : amyloid a component ; amyloid p component ; transthyretin ; and kappa and lambda light chains. with regards to specimen preparation for fluorescence spectroscopy, each paraffin - embedded biopsied tissue sample was characterized by strong autofluorescence due to the presence of paraffin. as a result, to this end, each collected tissue sample was transferred to a xylene - containing tissue bath and then dried by gradual heating. the treated tissue sample was then transferred to a microscope slide for observation via fluorescence spectroscopy. prior to performing the fluorescence spectroscopy, each collected tissue sample was independently coded such that the investigator was blinded without knowledge of the code - breaking procedures. in so doing, emitted fluorescence spectra were obtained between 260 nm and 450 nm with a scanning speed of 1500 nm / min and a spectral resolution of 2.5 nm. since the microscope used glass slides that were impermeable to uv light, specially constructed spectrofluorometer sample holders were used to facilitate detection of the fluorescence signal at the superior surface of the sample. in addition, the incident light beam was transmitted at a 60 angle to the surface of the tissue sample and the reflected fluorescence signal was detected perpendicularly to the sample tissue surface. this experimental configuration was found to optimize the fluorescence signal by maximizing the transmission of the generated fluorescence beam to the filter and photodiode array detector. since fluorescence intensity is influenced by the size of the examined sample, values obtained for the generation of spectral images were normalized for this parameter. fluorescence spectra were eventually generated by the collation of all spectra for the amyloidosis - affected and control groups by obtaining average and standard deviation values at each particular emitted fluorescence wavelength. in so doing, a direct comparison of the obtained spectra between these 2 separate subgroups was obtained. following fluorescence spectroscopy, tissue samples were subjected to histopathological examinations to determine whether sample preparation for fluorescence spectroscopy had the potential to damage the biopsied tissue in any way. figure 1 illustrates typical sample spectra obtained from the amyloid - containing and amyloid - free control groups. for this set of results, the influence of anatomical collection sites for the amyloid - containing tissue samples, the generated fluorescence spectra were characterized by a maximum emission peak at ~340 nm, with a shoulder region at ~280 nm. with regards to the amyloid - free control group tissue samples, the spectral image was typified by a single max value at approximately 340 nm, with no shoulder region. the presence of a shoulder emission region at ~280 nm at the quantum limit region of the fluorescence spectrum for amyloid - containing tissue samples was accepted as a preliminary diagnostic criterion for secondary amyloidosis. further investigations focused on analyzing the influence, if any, of anatomical collection site on the spectral images obtained from amyloid - containing and control group tissue samples. for this set of data analysis the following parameters were taken into account : presence or absence of secondary amyloidosis ; and anatomical site (abdominal fold, rectal mucosa, and gingiva). in so doing, for all amyloid - containing biopsy tissue samples, a single emission maxima at ~350 nm with a shoulder region at ~280 nm were observed. in contrast, all amyloid - free tissue samples were typified by only one max at approximately 340 nm. the anatomical tissue site location did not appear to influence the generated spectral image - the images within each amyloid subcategory (amyloid - containing and the amyloid - free groups) were similar. since the anatomical site did not appear to influence the generated spectral image, all the data was collated, averaged and graphed separately for the amyloid - containing and amyloid - free subgroups (figure 2). it can be seen that since the 2 resultant spectra do not superimpose at the initial wavelength values of the graph, the 2 spectra can be considered to be significantly different. table 1 depicts the sensitivity and specificity of the fluorescence spectroscopy technique used in the current study. the results indicate that the specimen collection site did not appear to influence these parameters. the minor differences observed between each collection site may be due to differences in the number of samples across each subcategory, as well as the inherent heterogenicity of each collected tissue. furthermore, the reported sensitivity values may be due to the limit of detection of the spectrofluorometer instrument. tissue samples were further processed for histopathological examinations and this indicated that the samples used for fluorescence spectroscopy were not damaged in any way and could potentially be used in further processing measures and observations. the results of the current research indicate that amyloid - containing and amyloid - free tissue samples are characterized by significantly different fluorescence spectra. while amyloid - containing tissue samples displayed fluorescent emission spectra characterized by 2 max values, the control - group biopsy specimens were typified by spectral images with 1 emittance peak. furthermore, when the fluorescence spectra of amyloid - containing tissue samples were analyzed, no detectable influence of collection site on generated spectra was apparent. such results are encouraging since it may thus be assumed that the inherent autofluorescence of the tissue collection site may be obviated from the final analysis. in so doing, the current fluorometric approach for the detection of aa may possibly be resistant to the variability in collection site types. however, it may be necessary to extend the current research by collecting tissue samples from other anatomical sites in order to delineate the diagnostic rigor of the current approach. in addition to these results, a further analysis indicated that both age and sex of the participating subjects did not appear to influence the generated fluorescence spectra of both amyloid - containing and control group tissue samples. with regards to the sensitivity of the current technique, amyloid - free tissue samples were always correctly diagnosed. however, while the sensitivity of amyloid - containing tissue samples was relatively high, some margin for error still existed. as a result, in some instances amyloid - containing tissue samples appeared to be amyloid - free, indicative of a false - negative result and a misdiagnosis in practice. this is most probably due to the limit of detection of the detection instrument used in the current study. the diagnostic error was probably due to trace quantities of amyloid deposits in the tissue samples that current conventional approaches could detect, while the resultant emitted fluorescence was below the limit of detection of the fluorometer used in the current study., tissue samples will be quantitatively spiked with known amounts of amyloid fibrous protein aggregates, and appropriate calibration curves will be constructed. this may also help assess the applicability of the current approach in the diagnosis of initial onset of aa. other types of amyloidosis (eg, primary, inherent) may also be investigated to gauge the applicability of the current spectroscopic approach. to the best of our knowledge, this study represents the first of its kind in which this type of fluorescence spectroscopic approach was used for the detection of amyloid deposits in these tissue types in a relatively large number of subjects. (1989) used the fluorescent dye thioflavin t1 to determine the presence of amyloid fibrils in mouse liver tissue in vitro. koh. (2006) utilized temporal resolution - based fluorescence microscopy to detect isolated -amyloid peptide deposits. while these reports describe novel approaches, the methodologies generally involve multiple staining steps, are relatively cumbersome and have not always been used for amyloid - containing human tissue samples. as a result, refinement of the current approach may allow fluorescence spectroscopy to be used as a fast, cost - effective preliminary diagnostic procedure prior to histopathological examination of the same tissue sample. this may help in the early detection of secondary amyloidosis, especially in less well - developed clinical settings. in addition, the current research could be extended to investigate amyloid aggregation processes via the use of time - resolved spectroscopy, since such amyloid structures have been implicated in the development of various pathologies. in conclusion, the results of the current study indicate that fluorescence spectroscopy may potentially be used in the diagnosis of aa. although current amyloid - detection approaches are characterized by a relatively larger sensitivity index, the relatively positive results of the current study are encouraging. efforts are currently underway to refine the spectroscopic approach used in this study to develop a fast, economical and user - friendly approach for the almost instantaneous detection of secondary amyloidosis in the general population in different clinical settings. it is also important that the conduct of such research does not harm the patient. the amount of material received for histopathological examination at time of biopsy is so large that the preparation of additional materials for the study autofluorescence does not present any difficulties, and performing an additional test may in some cases help in diagnosis. although histopathological examination is the gold standard in the case of amyloidosis, in some cases it is not clear. in such cases, this type of research is already taking place with the use of infrared spectroscopy in the study of alzheimer s disease and the study of skin autofluorescence in patients with cardiovascular disease. however, much work remains to be done, and at present we can not say whether this type of research will be possible when searching for deposits of amyloidosis. | summarybackgroundsecondary amyloidosis is a frequently reported complication of rheumatoid arthritis. currently, accepted diagnostic protocols for secondary amyloidosis involve histopathological and histochemical examinations of collected tissue specimens. the purpose of the current report was to evaluate the value of fluorescence spectroscopy as a supplementary tool in the diagnosis of secondary amyloidosis.material/methodstissue specimens were collected from abdominal folds, gingiva or rectal mucosa of 99 patients affected with rheumatoid arthritis. tissue samples were subjected to preliminary clinical observations, histopathological examinations and laboratory tests. these procedures were used to subdivide tissue samples into either amyloid - containing or amyloid - free control subgroups. all collected tissue samples were examined with the use of a designated spectrofluorometer and fluorescence spectral images were generated.resultsit was found that fluorescence spectra for amyloid - containing tissues were typically characterized by a double emittance peak. in contrast, amyloid - free samples were characterized by fluorescence spectra with a single max value. specimen collection site, age and sex did not appear to influence the morphology of electromagnetic spectra, which were generated for both amyloid - containing and amyloid - free tissue samples. the sensitivity of the fluorometric approach was ~78% and the specificity was 100%. possible shortcomings of the technique may be due to the limit of detection of the instrument used.conclusionsfluorescence spectroscopy may potentially be used as an effective, instantaneous and low - cost diagnostic tool for suspected secondary amyloidosis in patients affected with rheumatoid arthritis. |
it is a state of increased vulnerability to poor resolution of homoeostasis after a minor stressor event, which increases the risk of adverse outcomes, including falls, delirium, disability, long - term care, and death [1, 2 ]. between a quarter and half of people older than 85 years for older community residents, effective frailty prevention may potentially reduce serious frailty - related injuries. reducing frailty risk in older individuals is, therefore, an important public health objective. sarcopenia and osteoporosis are two distinct characteristics seen in older patients and are highly prevalent among elderly patients with frailty [4, 5 ]. sarcopenia, the age - related loss of skeletal muscle mass, is characterized by the deterioration of muscle quantity and quality leading to a gradual slowing of movement, a decline in strength and power, increased risk of fall - related injury, and often, frailty. osteoporosis is a progressive bone disease that is characterized by a decrease in bone mass and density which can lead to an increased risk of fracture. in osteoporosis, the bone mineral density (bmd) is reduced, bone microarchitecture deteriorates, and the amount and variety of proteins in bone are altered. osteoporosis is a common condition in elders and a powerful risk factor for adverse health outcomes such as fracture. intuitively, having a low muscle mass and strength with low bone mineral density (bmd) seems likely to lead to more physical functional limitations and frailty. however, we do not know the clinical characteristics of the individuals with both osteoporosis and sarcopenia, so - called sarco - osteoporosis. despite sharing common risk factors and biological pathways, the relationship between frailty and sarco - osteoporosis is not clear. the objective of our present study was to investigate the prevalence of sarco - osteoporosis among community - dwelling chinese elders and the relationship between sarco - osteoporosis and frailty. from august 2012 to august 2014, the patients who conducted comprehensive geriatric assessment (cga) from community - dwelling chinese elders (65 years) were recruited in changsha city and its surrounding area of china. individuals were originally excluded if unable to walk without the assistance of another person, or their renal function and liver function was abnormal, or their heart function classification was of grades iii and iv according to new york heart association (nyha) standard. a total of 360 subjects were screened and 316 of them had sufficient data for analysis, and their characteristics are presented in table 1. the study protocol was approved by the second xiangya hospital of central south university ethics committees in accordance with the declaration of helsinki and good clinical practices guidelines. participants completed a questionnaire and were interviewed by a cga nurse at the examination center and asked about health status, educational achievement, and smoking status. a selected medical history including a history of a physician diagnosis of diabetes mellitus, hypertension, coronary heart disease, dementia, parkinsonism, stroke, cancer, and chronic obstructive lung disease was obtained. body weight and height measurements were used to calculate a standard body mass index (bmi). participants were classified as frail, prefrail, and nonfrail according to a validated screening tool based on the presence or absence of five measurable characteristics by fried and colleagues : weakness, low physical activity, slowed walking speed, exhaustion, and weight loss. (1) weakness was defined as grip strength in the lowest quintile within groups defined by sex and bmi. participants reported their level of daily leisure physical activity in the past year ; (2) low physical activity was defined as either complete inactivity or performing low - intensity activities less than 1 h / wk ; (3) slowed walking speed was defined as usual walking speed in the slowest quintile within groups defined by sex and height. walking speed was measured on a 4 m course using photocell recordings at the course start and finish. the final measure averaged two walks ; (4) exhaustion was indicated by a response of occasionally or often / always to the statement, i felt that everything was an effort ; (5) weight loss was measured as self - reported unintentional weight loss more than 4.5 kg within the past year. individuals with three or more of the five components were defined as frail, those with one or two components were defined as prefrail, and those with none were defined as nonfrail. sarcopenia was defined as proposed by the asian working group for sarcopenia (awgs) with cutoff values for muscle mass measurements (7.0 kg / m for men and 5.7 kg / m for women by using bioimpedance analysis), handgrip strength (< 26 kg for men and < 18 kg for women), and usual gait speed (< 0.8 m / s). bone mineral density (bmd) of lumbar spine and femoral neck of all elderly adults were measured by dxa using qdr 4500a fan beam bone densitometer (hologic inc., bedford, ma, usa), according to the manufacturer 's recommended standard analysis procedures for the pa lumbar spine (vertebrae l2l4) and hip femoral neck. a long - term (exceeding 15 years) coefficient of variation (cv) for the bmd was not greater than 0.40%. with reference to the world health organization (who) definition, the diagnosis of osteoporosis was established when a bmd of 2.5 sd was lower than the peak mean of the same gender (t 2.5), and secondary osteoporosis was excluded. covariates were selected if they were considered to be related to frailty status, low bone mineral density, or sarcopenia. the covariates used were age, education (less than 9 years and more than 9 years), drinking and smoking (current drinking or current smoking or if stopped less than 4 years prior to the interview), supplemental vitamin d use or supplemental calcium use (less than twice every week and more than twice every week), physical activity (less than 30 minutes per day and more than 30 minutes per day), and the number of chronic diseases including diabetes mellitus, hypertension, coronary heart disease, dementia, parkinsonism, stroke, cancer, and chronic obstructive lung disease (less than 3 and more 3). data were analyzed including distribution of means and proportions of variables of interest across sex, musculoskeletal diseases, or frailty categories were compared using the student t - test and chi - square and trend tests. logistic regression using sarco - osteoporosis as the dependent variable for characteristics of the subjects, or using frailty / prefrailty (or nonfrailty) as the dependent variable for groups of musculoskeletal diseases classification, and adjusted for covariates was performed. no sarcopenia and no osteoporosis was set as the reference group. results were presented as odds ratio (or) with 95% confidence intervals (cis). all analyses were performed using the spss 19.0 package (spss, chicago, il). the mean age was 75.6 4.8 years for men and 74.9 5.2 years for women, and the average bmi value was 23.1 kg / m and 23.6 kg / m in men and women, respectively, with no significant difference between the two groups. in the total body bioimpedance analysis, women presented a higher percentage of total fat mass as compared to men. men had significantly higher values than did women for all bmd measurements, grip strength, and walking speed. the proportions of current drinker or current smoker and chronic obstructive lung disease were higher in men than in women. however, the percentage of physically active individuals was lower in men than in women. there was no significant difference in education, vitamin d and calcium supplementation, proportions of diabetes, hypertension coronary heart disease, dementia, parkinsonism, stroke, and cancer among the two groups. n = 51) were classified as having sarcopenia (having osteoporosis or no osteoporosis) ; 29.3% (n = 48) of men and 46.1% (n = 70) of women were classified as having osteoporosis (having sarcopenia or no sarcopenia). the mean age of men or women in the sarco - osteoporosis group was significantly higher than the mean age of those in the other groups. the percentage of drinkers, smokers, or parkinsonism was significantly higher in the sarco - osteoporosis group than in the other groups among men. in a logistic regression model, 80 years old or women had an increased likelihood of sarco - osteoporosis compared to < 80 years old or men (or 4.8 ; 95% ci, 3.0510.76 ; p = 0.027 ; or : 2.6 ; 95% ci, 1.182.76 ; p = 0.036, resp.). moreover, higher level of comorbidity (the number of chronic diseases was more than 3) was associated with sarco - osteoporosis (or 3.71 ; 95% ci, 1.6110.43 ; p = 0.021). subject characteristics for frailty / prefrailty stratified by sex are shown in tables 4 and 5. frailty status was detected in 11.6% (n = 19) of the elderly men, prefrailty in 49.4% (n = 81), and nonfrailty in 39.0% (n = 64) of the elderly men and frailty in 17.1% (n = 26) of the elderly women, prefrailty in 48.7% (n = 74), and nonfrailty in 34.2% (n = 52) of the elderly women. in the frail group, sarco - osteoporosis occurred in 26.3% of men (n = 5), 38.5% of women (n = 10), and in lower proportion in the prefrail (13.6% of men, n = 11 ; 16.2% of women, n = 12) and the nonfrailty groups (1.6% of men, n = 1 ; 1.9% of women, n = 1) (tables 4 and 5). the mean age of men or women in the frailty / prefrailty group was significantly higher than that of those in the nonfrailty group. the associations between sarco - osteoporosis and frailty / prefrailty in men and women, as assessed by logistic regression analysis, are shown in tables 6 and 7, respectively. after adjusting for subjects aged 80 years or more, drinking and smoking, education, body mass index and percentage of fat in the whole body scan, chronic medical history, sarcopenia (or 3.11 ; 95% ci, 1.656.63 ; p = 0.018 in men ; or 3.38 ; 95% ci, 1.417.62 ; p = 0.025 in women), and osteoporosis (or 2.07 ; 95% ci, 1.0913.12 ; p = 0.037 in men ; or 2.41 ; 95% ci, 1.2614.15 ; p = 0.024 in women) were independently associated with frailty. furthermore, the likelihood of being frail was substantially higher in the presence of sarco - osteoporosis (or 4.16 ; 95% ci, 2.1717.65 ; p = 0.019 in men ; and or 4.67 ; 95% ci, 2.4218.86 ; p = 0.007 in women.) (tables 6 and 7). this cross - sectional study examined the association between sarco - osteoporosis (the individuals with both sarcopenia and osteoporosis, as diagnosed by awgs / who criteria) and frailty in 316 community - dwelling elderly chinese men and women. we found that, in the frail group, sarco - osteoporosis occurred in 26.3% of men and 38.5% of women, but in lower proportion in the prefrail group (13.6% of men and 16.2% of women) or in the nonfrailty group (1.6% of men and 1.9% of women). in other words, the percentages of sarco - osteoporosis were higher in the frailty / prefrailty groups than in the nonfrailty group in both men and women. furthermore, the likelihood of being frail / prefrail was substantially higher in the presence of sarco - osteoporosis (or 4.16 ; 95% ci, 2.1717.65 ; p = 0.019 in men ; and or 4.67 ; 95% ci, 2.4218.86 ; p = 0.007 in women) than in the presence of sarcopenia or osteoporosis alone (or 3.11 ; 95% ci, 1.656.63 ; or 2.07, 95% ci, 1.0913.12 in men and or 3.38 ; 95% ci, 1.417.62 ; p = 0.025 ; or 2.41 ; 95% ci, 1.2614.15 ; p = 0.024 in women, resp.) frailty is a practical, unifying notion in the care of elderly adults that directs attention away from single - system illness towards a more holistic viewpoint of the patient. reduction of the occurrence or severity of frailty is likely to have large benefits for individuals, their families, and society. some clinical trials have confirmed that complex interventions including exercise, nutrient supplement, and pharmacological agents can increase the likelihood of continuing to live at home, mainly through a reduced need for care - home admission and fewer falls [8, 9 ]. to identify the subjects with high risk factors for frailty should be an essential part of further complex intervention. the immune system, skeletal muscle, brain, and endocrine system are intrinsically interrelated and are the organ systems that are best studied in the development of frailty. notably, frailty has also been associated with loss of physiological reserve in the cardiovascular, renal, respiratory, haemopoietic, and clotting systems [13, 14 ], and nutritional status can also be a mediating factor [1, 1517 ]. the results from our study implied that sarcopenia and osteoporosis were predictors of frailty. importantly, the results suggested that the joint predictive value of sarcopenia and osteoporosis was stronger than that of sarcopenia or osteoporosis alone. this finding supports the idea that, when physiological decline reaches an aggregate crucial level, frailty becomes evident. the results from the women 's health and aging study (whas) ii showed almost sixteen percent had sarcopenia concomitant to severe osteopenia / osteoporosis in community - dwelling older women according to baumgartner / who criteria. our present study for the first time showed the epidemiology of sarco - osteoporosis and its associative clinical characteristics in community - dwelling elderly chinese men and women. sarco - osteoporosis was prevalent in 10.4% of men and 15.1% of women among the study population. sarco - osteoporosis prevalence was lower than that of isolated sarcopenia (15.9% of men and 18.4% of women) or isolated osteoporosis alone (18.9% of men and 30.9% of women). we also found that 80 years old or women had an increased likelihood of sarco - osteoporosis compared to < 80 years old or men (or 4.8, 95% ci : 3.0510.76, p = 0.027 ; or 2.6, 95% ci : 1.182.76, p = 0.036, resp.). moreover, higher level of comorbidity (the number of chronic diseases was more than 3) was associated with sarco - osteoporosis (or 3.71, 95% ci : 1.6110.43, p = 0.021). muscle / bone relationships have recently been noted as a new research field related to the interactions among several organ systems. muscle / bone relationships include two factors : local control of muscle to bone and systemic humoral interactions between muscle and bone. further progress in understanding the common genetic etiology of osteoporosis and sarcopenia will provide valuable insight into important biological underpinnings for both conditions and may translate into new approaches to reduce the burdens of both conditions through improved diagnosis, prevention, and early targeted treatment.. osteoporosis and sarcopenia may be affected by genetic polymorphisms of several genes, such as androgen receptor, estrogen receptor, catechol - o - methyltransferase, igf - i, vitamin d receptor, and low - density - lipoprotein receptor - related protein 5. vitamin d, the growth hormone / insulin - like growth factor i axis, and testosterone are physiologically and pathologically important as endocrine factors. mechanical stress changes, such as immobilization and lack of gravity, greatly influence both muscle and bone. these findings suggest the presence of interactions between muscle and bone, which might be very important for understanding the physiology and pathophysiology of sarco - osteoporosis. the loss of muscle strength and mass during the aging process causes structural changes in the microarchitecture of the bones and decreases mineral density, resulting in bone quality decline. these factors of skeletal muscle and bone activate a vicious cycle leading to accelerated frailty and ultimately to physical disability. there are shared factors between sarcopenia and frailty such as slow walking speed and grip strength. if sarcopenia is integrated into the diagnosis of frailty, it would be positively identified and graded for severity. and it would help essential research to gain a deeper insight into the complex mechanisms of frailty and aid the development and evaluation of interventions to improve outcomes. first, the subjects were recruited from a community - based elderly population, represented a single chinese older adults. second, previous studies used ewgs criteria for the definition of sarcopenia to obtain a sufficient number of subjects within the group for statistical analysis. in contrast, we used the criterion of awgs for defining sarcopenia, which is known to be more suitable for chinese. first, the sample size of the subgroups in the analysis is relatively small and provides limited statistical power, and further investigation of the joint effects of sarcopenia and osteoporosis on frailty is needed. second, the individuals were originally excluded if unable to walk without the assistance of another person, or their renal function and liver function is abnormal, or their heart function classification is of grades iii and iv according to nyha standard ; this may have biased our results towards an underestimation of the risk of frailty associated with sarcoosteopenia. third, dxa scan presents some limitations in bmd evaluation in elderly people, like aortic calcifications and spine osteoarthritis that may produce an increase, up to 10%, in bmd of the lumbar spine, and this could underestimate the real prevalence of sarco - osteoporosis in this population and, consequently, the association with frailty status. therefore, findings from this study should be interpreted in the context of the complexity of skeletal muscle and bone as well as multifactorial nature of the frailty syndrome. despite these limitations, our findings are helpful for us to better understand sarco - osteoporosis and provide a basis for making an optimal prediction about frailty among community - dwelling chinese older people. in conclusion, sarco - osteoporosis defined by awgs / who criteria is present in 10.4% of men and 15.1% of women aged over 65 years, and its prevalence rate is higher in community - dwelling chinese people aged 80 and over. the joint effect of sarcopenia and osteoporosis may be tightly linked to the risk of frailty. assessment of both bone and muscle mass / function in older adults could potentially enhance frailty risk prediction. further prospective study is needed to clarify the roles of sarco - osteoporosis in the occurrence of frailty and frailty - related health outcomes. although no causal attribution is possible in this analysis, sarco - osteoporosis may explain some of the increases in frailty risk currently related to age. therefore, it is appropriate to consider sarcopenia together with osteoporosis in the elderly population. | the aim was to apply awgs criteria to estimate the prevalence of sarco - osteoporosis and investigate its relationship with frailty, in a sample of 316 community - dwelling chinese older people. regression analysis was performed using frailty as the dependent variable. the results showed that the prevalence rate of sarco - osteoporosis was 10.4% in older men and 15.1% in older women. 80 years old (or 4.8 ; 95% ci, 3.0510.76 ; p = 0.027), women (or 2.6 ; 95% ci, 1.182.76 ; p = 0.036), and higher level of comorbidity (or 3.71 ; 95% ci, 1.6110.43 ; p = 0.021) were independently associated with the likelihood of being sarco - osteoporosis. in the frail group, sarco - osteoporosis occurred in 26.3% of men, in 38.5% of women, and in lower proportion in the prefrail (13.6% of men ; 16.2% of women) and nonfrail group (1.6% of men ; 1.9% of women) (p < 0.05, resp.). furthermore, the likelihood of being frail / prefrail was substantially higher in the presence of sarco - osteoporosis (or 4.16 ; 95% ci, 2.1717.65 ; p = 0.019 in men ; and or 4.67 ; 95% ci, 2.4218.86 ; p = 0.007 in women). the results indicate that patients with sarco - osteoporosis are more likely to be 80 yrs with higher burden of comorbidities and to have frailty / prefrailty, especially for women. |
polycyclic aromatic hydrocarbons (pahs) are characterized by the presence of two or more fused benzene rings arranged in various configurations. pahs are ubiquitous airborne environmental pollutants that arise from the incomplete combustion of fossil fuels and are also present in automobile exhaust and first- and secondhand cigarette smoke. pahs are one of the major classes of carcinogens found in tobacco smoke and are suspect lung carcinogens. benzo[a]pyrene (b[a]p), the most studied pah, has been recently upgraded to a group 1 known human carcinogen by the international agency for research on cancer. pahs are not biological reactive, and the biotransformation of pahs to electrophilic metabolites is required to elicit their tumorigenic effects. there are three major routes for pah activation which include the formation of radical cations, diol epoxides, and electrophilic and redox - active o - quinones. in the o - quinone pathway, akrs catalyze the oxidation of proximate pah carcinogens, trans - dihydrodiols, to yield ketols which spontaneously rearrange to catechols. pah catechols are not stable and undergo autooxidation to form pah o - quinones with the concomitant production of reactive oxygen species (ros) (figure 1). pah o - quinones are electrophilic and highly reactive with endogenous nucleophiles and yield l - cysteine, n - acetyl - l - cysteine, and gsh conjugates. electrophilic pah o - quinones can also form both depurinating dna adducts in vitro(13) and stable covalent dna adducts in vitro and in human lung cells. apart from their electrophilicity, pah o - quinones are also redox active and undergo nonenzymatic or enzymatic reduction to reform catechols at the expense of consuming nadph. enzymes that contribute to this redox cycling include nad(p)(h):quinone oxidoreductase (nqo1), carbonyl reductases (cbr1 and cbr3), and akrs themselves. ros produced by redox cycling of the pah o - quinones mediates dna damage and can lead to 7,8-dihydro-8-oxo-2deoxyguanosine (8-oxo - dguo) lesions which contribute to g - to - t transversions in p53. it was found that even nanomolar concentrations of pah o - quinones were able to generate sufficient ros to cause a significant increase in 8-oxo - dguo. metabolic activation of b[a]p by akrs and detoxication of b[a]p-7,8-dione by ugts. using b[a]p as a representative pah and stable isotope dilution liquid chromatography tandem mass spectrometry, we found that all three pathways of pah activation were functional in human bronchoalveolar h358 cells. also, using a549 cells which show high constitutive expression of akrs, we found that b[a]p-7,8-trans - dihydrodiol (an akr substrate) was converted to b[a]p-7,8-dione and that the ros produced increased the level of 8-oxo - dguo in cellular dna as measured by stable isotope dilution liquid chromatography importantly, the level of 8-oxo - dguo produced from b[a]p-7,8-trans - dihydrodiol was elevated further in the presence of a comt inhibitor suggesting that a redox cycle was occurring. these observations led to a systematic study of the role of phase ii conjugating enzymes in intercepting pah - catechols to prevent redox cycling. human catechol - o - methyl transferase (comt) and sulfotransferases (sult) 1a1, 1a3, and 1e1 were able to detoxify b[a]p-7,8-dione by intercepting b[a]p-7,8-catechol through the formation of o - methylated - b[a]p-7,8-catechol and o - sulfated - b[a]p-7,8-catechol, respectively. another superfamily of phase ii metabolic enzymes, uridine diphosphate glucuronosyltransferases (ugts), are microsomal enzymes which catalyze the transfer of the glucuronosyl group from uridine 5-diphospho - glucuronic acid (udpga) to substrates that contain alcohols, amines, or carboxylic acids as functional groups. ugts are divided into two main subfamilies ugt1 and ugt2 based on amino acid sequence identity and are responsible for the glucuronidation of a variety of endogenous compounds and xenobiotics. ugts are widely distributed in a variety of tissues, including the liver, intestine, brain, and kidney, and the aerodigestive tract, etc. ugt1a7, ugt1a8, ugt1a9, and ugt1a10 and ugt2b7 are active against several pah metabolites, while ugt1a10 > ugt1a9 > ugt1a1 > ugt1a7 are the preferred isoforms for catalyzing the glucurondiation of b[a]p-7,8-trans - dihydrodiol. however, the glucuronidation of pah catechols by ugts has not been previously examined. studies of b[a]p-7,8-dione metabolism and disposition in three human lung cells hbec - kt, h358, and a549 cells showed rapid disappearance of the quinone accompanied by the formation of phase ii conjugates and a n1 or n3-adenine adduct originating from the nucleotide pool. in a549 cells, one of the phase ii conjugates was o - monoglucuronsyl - b[a]p-7,8-catechol indicating that ugts play a role in the interception / detoxication of pah catechols. in the present study, we investigated whether glucuronidation catalyzed by human ugts is a feasible detoxication pathway for b[a]p-7,8-dione and identified two major enzyme isoforms responsible for the glucuronidation of b[a]p-7,8-catechol. 7-hydroxy-4-(trifluoromethyl)coumarin(hfc), alamethicin, and uridine-5-diphosphoglucuronic acid (udpga) were purchased from sigma - aldrich co. (st. louis, mo). [c]-uridine-5diphosphoglucuronic acid (180 mci / mmol) was purchased from perkin elmer inc. all solvents were of hplc grade, and all other chemicals used were of the highest grade available. ugt1a1, 1a3, and 2b7 supersomes (microsomes from baculovirus infected insect cells expressing ugts) were obtained from bd biosciences (san jose, ca) and titered before use, using standard substrates. a549 cells (human lung adenocarcinoma cells) were from american type culture collection (atcc number ccl-185) and cultured in f-12k medium (kaighn s modification) with supplementation of 10% heat - inactivated fbs, 2 mm l - glutamine, 100 units / ml penicillin, and 100 g / ml streptomycin. hepg2 cells (hepatoma cells) from atcc were cultured in eagle s minimal essential medium supplemented with 10% heat - inactivated fetal bovine serum, 1% l - glutamine, and 100 units / ml penicillin / streptomycin solution. h358 cells (human bronchoalveolar cells) were purchased from american type culture collection (atcc number crl-5807) and cultured in rpmi 1640 medium containing 10% heat - inactivated fbs, 2 mm l - glutamine, 100 units / ml penicillin, and 100 g / ml streptomycin. hbec - kt cells (immortalized human bronchial epithelial cells) originating from a patient without lung cancer were kindly provided as a gift by dr. john minna at university of texas southwestern medical center and cultured in keratinocyte - serum - free medium with 0.10.2 ng / ml recombinant egf, 2030 g / ml bovine pituitary extract, and 2 mm l - glutamine. beas-2b cells (normal human bronchial epithelium cells) were obtained from american type culture collection (atcc number crl-9609) and cultured in begm bronchial epithelium medium (cambrex cc-3170). cells were maintained at 37 c in a humidified atmosphere containing 5% co2 and 95% o2. cultured cells used in the experiments were confined to passage numbers of 1020. extraction of total rna from each cell line was conducted using the rneasy kit (qiagen, valencia, ca). reverse transcription (rt) was conducted by using geneamp rna pcr core kit according to the manufacturer s protocol (applied biosystems, carlsbad, ca). an aliquot of 1 l of cdna synthesized in the above rt reaction was used for pcr. the pcr system (25 l) contained 1 pcr buffer (10 mm tris - hcl buffer, ph 8.3, and 50 mm kcl), 2.5 mm mgcl2, 250 m dntps, 0.2 m primers, and 0.5 u of taq dna polymerase (applied biosystems, carlsbad, ca). the sequences of the forward and reverse primer pairs for the amplification of ugt1a1, 1a3, 1a7, 1a8, 1a9, 1a10, and 2b7 and -actin transcripts are shown in table 1. pcr amplification was performed with a mycycler thermal cycler pcr system (bio - rad, ca) using the following protocol. after an initial denaturation step at 94 c for 3 min, amplification was conducted by denaturation at 95 c for 30 s, annealing at 61 c (for ugt1a1), 63 c (for ugt1a8), or 57 c (for other ugt isoforms) for 30 s, and extension at 72 c for 45 s for 35 cycles. the final extension reaction was performed at 72 c for 7 min. the control samples were amplified using the same conditions as those described above. an aliquot of 20 l of pcr products was analyzed by electrophoresis in 2% agarose gel with ethidium bromide and visualized under uv light. the reverse primer for the amplification of ugt2b7 is located between 931 - 912 nts. the same exon-3 derived antisense primer was used for all rt - pcr amplification of family 1 ugts. ugt enzyme assays were conducted as recommended by bd biosciences (san jose, ca). briefly, the reaction system was composed of 50 mm tris buffer of ph 7.4, 10 mm mgcl2, 1 mm udpga, 0.025 mg / ml alamethicin, 50 m 7-hydroxy-4-(trifluoromethyl)coumarin, and 10 g of ugt supersome in a final volume of 100 l. after incubation at 37 c for 15 min, the reactions were terminated by the addition of 50 l of 94% acetonitrile/6% glacial acetic acid and were centrifugated at 16,000 g for 10 min. an aliquot of 100 l of supernatant was carefully pipetted, and 50 l was injected into hplc / pda for the quantification of 7-hydroxy-4-(trifluoromethyl)coumarin glucuronide. the reversed - phase column (agilent zorbax - ods c18, 5 m, 4.6 250 mm, ca) was used for the separation of substrate from product. elution conditions used a flow rate of 1 ml / min with 80%:20% water / methanol (v / v) containing 0.1% formic acid. the methanol concentration was increased from 20% to 80% over 3 min and kept at 80% methanol for 4 min, then changed back to 20% methanol in 1 min followed by a re - equilibration phase of 7 min at 20% methanol. the experiments were conducted in 1.5 ml amber glass vials with polytetrafluoroethylene / silicone septa closures. the reaction system was composed of 10 mm kpo4 buffer at ph 7.4, 1.0 mm dithiothreitol, 5.0 mm mgcl2, 0.025 mg / ml alamethicin, 1 mm [c]-udpga (4 dpm / pmol), 020 m b[a]p-7,8-dione, and 1530 g of microsomes containing human recombinant ugts in a final volume of 0.2 ml. the reactions were initiated by the addition of udpga and incubated for 30 min (for ugt 1a1) or 60 min (for ugt 1a3 and 2b7) at 25 c. the incubation time and amount of enzyme used were always in the linear range as determined by plots of initial velocity versus incubation time or enzyme concentration. the reactions were terminated by the addition of 50 l of ice - cold 1% formic acid and were chilled on ice. the reaction mixtures were extracted with 0.5 ml aliquots of ethyl acetate twice by vortex mixing and centrifuged at 16,000 g to help phase separation. the combined ethyl acetate layer was backwashed with 0.2 ml of 1% formic acid by vigorous vortexing and centrifuged at 16,000 g. the ethyl acetate was then dried by a speedvac concentrator (thermo scientific). the residue was dissolved in 100 l of methanol and analyzed by scintillation counting or by hplc analysis. kinetic analyses using nonlinear regression were performed by fitting the michaelis menten equation to the data with the program grafit, where v is the initial velocity of the reaction, [s ] is the molar concentration of the substrate, and km is the michaelis because of the iterative fits of the equations to each data set, each fit provided estimates of the kinetic parameters as a mean se. because it is not possible to normalize vmax values to ugt protein expression, vmax values are apparent values only based on total protein. b[a]p-7,8-dione was prepared in hbss buffer containing 1 mm sodium pyruvate at final concentration of 2 m with 0.2% dmso and then was used to treat a549, hbec - kt, and h358 cells (5 10) at confluency. the media were collected at 0 and 24 h and acidified subsequently with 0.1% formic acid before extraction with 2 1.5-fold volume of cold h2o - saturated ethyl acetate. the organic phases of ethyl acetate were combined and dried by a speedvac concentrator (thermo scientific). the o - glucuronsyl - b[a]p-7,8-catechol conjugates formed in ugt reaction systems were analyzed by a waters alliance 2695 chromatographic system (waters corp., milford, ma) in tandem with a waters 996 photodiode array detector and a -ram inline radiometric detector (in / us systems inc., tampa, fl) or with a finnigan tsq quantum ultra spectrometer (thermo fisher, san jose, ca). chromatographic separation was conducted on a reverse - phase column (agilent zorbax - ods c18, 5 m, 4.6 250 mm, ca). the c18 reverse - phase column was eluted with the following linear gradient of h2o (0.1% formic acid ; solvent a)/meoh (solvent b) at a flow rate of 0.5 ml / min. solvent b was changed from 50 to 95% (v / v) over 15 min, kept at 95% over 10 min, changed from 95 to 50% over 1 min, and kept at 50% for equilibration for 4 min. the o - glucuronsyl - b[a]p-7,8-catechols formed in reactions with [c]-udpga were eluted from the c18 reverse - phase column and introduced into the inline radiometric detector using a mixture of the scintillant with the hplc effluent at a flow rate 1.5 ml / min. the o - glucuronsyl - b[a]p-7,8-catechols generated with unlabeled udpga were eluted from the c18 reverse - phase column and analyzed by electrospray ionization ms / ms. the mass spectrometer analysis was carried out in the positive or negative ion mode with the following parameters : spray voltage (4500 v at positive ion mode or 2000 v at negative ion mode), vaporizer temperature (400 c), sheath gas pressure (35 arbitrary units), auxiliary gas pressure (10 arbitrary units), capillary temperature (350 c), and collision energy (20 v). the molecular masses of the metabolites were acquired by detecting the molecular ion from q1 full scan, and the corresponding mass spectrum of each metabolite was obtained from a q3 full scan of the product ions of the molecular ions. we performed rt - pcr to identify the ugt isoforms expressed in hepg2 cells and four human lung cells. the ugts selected were those that have been previously characterized for the glucuronidation of b[a]p-7,8-trans - dihydrdodiol (ugt1a10 > ugt1a9 > ugt1a1 > ugt1a7) and the catechol estrogens (ugt1a8, ugt1a9, and ugt2b7). although rt - pcr is a semiquantitative approach to analyze gene expression, we found that ugt1a1, 1a3, and 2b7 were expressed at the mrna level in hepg2 and a549 cells among the seven different ugt isoforms examined (figure 2). the current studies focused on the glucuronidation of b[a]p-7,8-catechol by recombinant human ugt1a1, 1a3, and 2b7, which were major ugt isoforms in a549 cells. (hepg2, hepatoma cells ; a549, human lung adenocarcinoma cells ; h358, human bronchoalveolar cells ; hbec - kt, immortalized human bronchial epithelial cells ; beas-2b, normal human bronchial epithelial cells.) supersomes (bd - biosciences) containing overexpressed human recombinant ugts were tittered in standard assays. the standard assay for ugt activity using 7-hydroxy-4-(trifluoromethyl)coumarin as a substrate demonstrated that the specific activities of ugt1a1, 1a3, and 2b7 were 1.18 (0.8), 0.96 (0.7), and 1.74 (1.51) nmol of 7-hydroxy-4-(trifluoromethyl)coumarin glucuronide formed / min / mg, respectively, which are comparable to those values previously reported as indicated by the values in parentheses. ugt1a1, 1a3, and 2b7 were all able to catalyze the glucuronidation of b[a]p-7,8-catechol in the presence of udpga (figure 3a, b, c), while no conjugates were generated in the absence of udpga (figure 3d). ugt1a3 and 2b7 catalyzed the formation of one o - glucurosonyl - b[a]p-7,8-catechol (m2), while ugt1a1 catalyzed the formation of two o - glucuronsyl - b[a]p-7,8-catechols (m1, m2). the uv spectra of the two glucuronides of b[a]p-7,8-catechol (m1, m2) were quite different (figure 4). when they were compared with the uv spectrum of the o - glucuronsyl - b[a]p-7,8-catechol found in a549 cell culture media, the formation of glucuronides of b[a]p-7,8-catechol was confirmed by conducting the glucuronidation studies in the presence of [c]-udpga. [c]-o - glucuronsyl - b[a]p-7,8-catechol was detected with hplc - ram analysis (figure 3 e). hplc / uv / ram identification of b[a]p-7,8-catechol glucuronide metabolites (m1 and m2). b[a]p-7,8-catechol (20 m) was generated in situ under anaerobic conditions in an incubation buffer containing 1 mm dithiothreitol. b[a]p-7,8-catechol was converted to the o - glucuronide(s) by ugt supersomes supplemented with udpga (a, ugt1a1 ; b, ugt1a3 ; and c, ugt2b7) in the presence of udpga. (e) hplc - ram chromatogram of o - monoglucuronsyl - b[a]p-7,8-catechol formed by ugt1a3 in the presence of [c]-udpga. the delayed retention time of the m2 metabolite is due to the fact that the radiometeric detector is in - line and downstream from the absorbance (pda) detector. b[a]p-7,8-catechol (20 m) was generated in situ under anaerobic conditions in an incubation buffer containing 1 mm dithiothreitol. uv spectra of m1 and m2 were collected by an in - line waters 996 photodiode array detector. to determine whether b[a]-7,8-catechol formed mono- or bis - glucuronide conjugates, lc - ms / ms was used to further characterize the structure the m1 and m2 metabolites. the molecular ions of both metabolites (m1, m2) were the same and showed a ([m + h ]) m / z = 461 in the positive ion mode and a ([m h ]) m / z = 459 in the negative ion mode, respectively, indicating that both m1 and m2 are o - monoglucuronsyl - b[a]p-7,8-catechols where the position of glucuronidation is different. ms / ms analysis provided further evidence to the identity of the metabolites. in the positive ion mode, the product ions of the two metabolites were identical, and characteristic scission under collision - induced dissociation occurred at the c o glycosidic bond with the loss of 176 amu resulting from the loss of the monodehydrated glucuronic acid moiety to yield a fragment ion m / z = 285. oh bonds resulted in a daughter ion of m / z = 267 representing the loss of h2o. rearrangement resulting in a change of the remaining phenolic group from a c oh to c = o bond is followed by the loss of c = o group which resulted in a fragment ion at m / z = 239. in the negative ion mode, product ion spectra of two metabolites also demonstrated characteristic cleavage at the c o glycosidic bond with a loss of 176 amu resulting in daughter ions at m / z = 283. lc - ms / ms analyses in both the positive and negative ion modes confirmed that the metabolites (m1 and m2) formed in the reaction system were o - monoglucuronsyl - b[a]p-7,8-catechols. since the mass spectra of m1 and m2 were identical (figure 5), it was not possible to distinguish the position of glucuronic acid conjugation in the two isomers. however, our studies on the sulfonation of b[a]p-7,8-catechol catalyzed by sults also led to the formation of two regioisomeric o - monosulfated catechols, where the earlier eluting species was shown by 2d - nmr to be the o7-monosulfate. thus, in this study the later eluting m2 metabolite is tentatively assigned as the o8-monoglucuronsyl - b[a]p-7,8-catechol. lc - ms / ms in the selected reaction monitoring mode (srm) further confirmed that ugt1a1 catalyzed the formation of isomeric o - monoglucuronsyl - b[a]p-7,8-catechols (m1, m2) and ugt1a3 and that 2b7 catalyzed the formation of only one o - monoglucuronsyl - b[a]p-7,8-catechol (m2) (figure 6). lc / ms / ms identification of o - monoglucuronsyl - b[a]p-7,8-catechols (m1 and m2). b[a]p-7,8-catechol (20 m) was generated in situ under anaerobic conditions in the presence of 1 mm dithiothreitol. b[a]p-7,8-catechol was converted to o - glucuronide(s) in the presence of udpga and ugts in the incubation buffer. the organic phase was then dried and dissolved in meoh for lc - ms / ms analysis (positive ion mode, ms spectrum of isomer m1 and ms spectrum of isomer m2). lc - ms / ms detection of o - monoglucuronsyl - b[a]p-7,8-catechols (m1 and m2) formed by recombinant ugt1a1 (a), 1a3 (b), 2b7 (c), and by a549 (d) at 24 h. b[a]p-7,8-catechol (20 m) was generated in situ under anaerobic conditions in the presence of 1 mm dithiothreitol. b[a]p-7,8-catechol was converted to the o - glucuronide(s) in the presence of udpga and ugts in the incubation buffer (a, ugt1a1 ; b, ugt1a3 ; and c, ugt2b7). (d) b[a]p-7,8-dione (2 m, 0.2% dmso) in hbss medium was incubated with a549 cells and the culture media collected at 0 and 24 h, respectively. o - monoglucuronsyl - b[a]p-7,8-catechols were analyzed with lc - ms / ms in a negative ion mode by monitoring the mass transition m / z 459 283 ([m h ] [m h - glucuronic acid group ]). to elucidate the steady state kinetic properties of the recombinant ugts to glucuronidate b[a]p-7,8-catechol, we performed discontinuous assays to monitor the initial velocity of conjugate formation using [c]-udpga as the cofactor. because of substrate solubility, the substrate concentration was limited to 020 m. apparent utilization ratios (vmaxapp / km) showed that ugt1a1 supersomes were the most catalytically efficient for the glucuronidation of b[a]p-7,8-catechol, followed by ugt1a3 and 2b7 supersomes (table 2). the km values of ugt1a1 and 1a3 were 9.6 and 8.5 m, respectively, which were slightly lower than that of ugt2b7 at 16.3 m indicating that the substrate concentration at which half maximal velocity was observed was similar in each case. kinetic characterization of glucuronidation of b[a]p-7,8-catechol by ugts. kinetic analyses were performed by fitting the michaelis menten equation to the data. reactions contained 10 mm kpo4 buffer at ph 7.4, 1.0 mm dithiothreitol, 5.0 mm mgcl2, 1 mm [c]udpga, 020 m b[a]p-7,8-dione, and 1530 g of human recombinant ugt supersomes at 25 c. a, b, and c, velocity versus [s ] curve for the glucuronidation of b[a]p-7,8-catechol by ugt1a1, 1a3, and 2b7, respectively. only one o8-monoglucuronsyl - b[a]p-7,8-catechol (m2) was detected by lc - ms / ms in the srm mode in a549 cells after incubation of the cells with 2 m b[a]p-7,8-dione for 24 h. by contrast, o - monoglucuronsyl - b[a]p-7,8-catechol was not detected in h358 and hbec - kt cell lines, which was consistent with the low mrna level of ugts in these cell lines (figures 2 and 6d). we have been conducting a systematic study to elucidate the roles of human enzymes in catalyzing the redox cycling of pah o - quinones and the roles of phase ii enzymes in conjugating pah catechols that arise from the akr pathway of pah activation. previous studies have shown that akrs are efficient pah o - quinone reductases and that the pah catechols formed can be intercepted by comts and sults. we now show that ugt1a1, 1a3, and 2b7 are all expressed in a549 cells. of these, ugt1a3 and 2b7 are the enzymes most likely involved in b[a]p-7,8-catechol glucuronidation based on the glucuronide product profile. while recombinant ugt1a1 expressed in supersomes produces two regioisomeric monoglucuronides with b[a]p-7,8-catechol, only one of the isomers is produced in a549 cells and corresponds to the single isomer produced by ugt1a3 and ugt2b7. the monoglucuronide formed is tentatively assigned as o8-monoglucuronsyl - b[a]p-7,8-catechol based on differences in retention time observed for the regioisomeric o - monosulfated b[a]p-7,8-catechols. it is noteworthy that ugt2b7 is also the dominant isoform involved in the glucuronidation of the structurally related catechol estrogens. ugt2b7 catalyzes the glucuronidation of 4-hydroxycatecholestrogens (hydroxylated estrone / estradiol), which are structurally similar to b[a]p-7,8-catechol. the glucuronidation of 4-hydroxycatecholestrogens shows preference for the c4-hydroxyl group, which would be equivalent to the c7 position of b[a]p-7,8-catechol. although ugt1a1 was found in the kinetic analysis to have the highest utilization ratio (vmax / km) and catalyzed the formation of both regioisomeric b[a]p-7,8-catechol glucuronides, no significant m1 was found in a549 cells indicating that it did not contribute to the detoxication of this catechol in these cells. it is noteworthy that only a549 cells expressed the ugt isoforms studied to any extent. the low expression of ugts might be anticipated due to the low level of expression previously reported in human lung specimens. zheng at al. measured the expression of multiple ugts in 32 human lung tissues by duplex rt - pcr and found that ugt1a1, ugt1a3, ugt1a4, ugt1a6, ugt1a7, ugt1a8, ugt1a9, and ugt1a10 were not expressed. similarly, ugt2b4, ugt2b7, ugt2b15, and ugt2b17 were not detected. in later studies, dillinger. showed that ugt1a10 was found in low amounts in human lung specimens. the difference in ugt isoform expression observed in our study versus that observed in the earlier work could reflect differences in measuring ugt expression in human bronchial epithelial cells versus whole lung tissue. other ugts that could have been included in our study would be ugt2b10, but up until now, this has only been shown in glucuronidate tobacco specific nitrosamines. ugt1a1, ugt1a9, and ugt2b7, which are predominately expressed in the liver, were found to form the 7s - monoglucuronide from ()-b[a]p-7,8-trans - dihydrodiol, whereas the extrahepatic ugt1a7, ugt1a8, and ugt1a10 could form either the 7r - monoglucuronide or the 8s - monoglucuronide, i.e., they produce two different enantiomers. by contrast, ugt1a1 formed two different regioisomeric b[a]p-7,8-catechol monoglucuronides, whereas ugt1a3 and ugt2b7 produced a single regioisomeric o - monoglucuronsyl - b[a]p-7,8-catechol. the ability of ugts to form different enantiomers from b[a]p-7,8-trans - dihydrodiol may be related to the fact that the dihydrodiol is nonplanar and has both axial and equatorial alcohols. by contrast no bis - glucuronide has been observed with either b[a]p-7,8-trans - dihydrodiol or b[a]p-7,8-catechol possibly due to steric hindrance. we next compared the specific activities for the redox cycling of b[a]p-7,8-dione catalyzed by akrs, with the specific activities observed for o - methylation, o - sulfation, and o - glucuronidation of b[a]p-7,8-catechol catalyzed by comts, sults, and ugts in table 3. these data reveal that the rates of redox cycling are much greater than the rates of phase ii conjugating reactions. although some uncertainties exist in this comparison due to the differences in expression levels of these enzymes in specific cells, it is unlikely that these will account for the > 6,000-fold difference in the specific activities of nqo1 and ugt1a3 to use b[a]p-7,8-dione and b[a]p-7,8-catechol as substrates, respectively. thus, the ability to intercept the pah catechols and prevent redox cycling is not an efficient process and could be easily overwhelmed in a cellular environment. this is supported by earlier work in which we showed that b[a]p-7,8-trans - dihydrodiol (akr substrate) and b[a]p-7,8-dione (akr product) produced significant ros and 8-oxo - dguo formation in a549 cells. although ugts were not detected in hbec - kt, h358, and beas-2b under current culture conditions, the contribution of ugts in human lung cells to b[a]p-7,8-catechol conjugation may be affected by the induction of ugts. it has been proposed that the expression level of ugt1a4 in the human small airway epithelium was elevated as a result of nuclear factor erythroid 2 p45-related factors (nrf2) activation. however, ugt1a4 does not appear to be responsible for b[a]p-7,8-catechol glucuronidation in our experiments to date. it has also been demonstrated that ugt1a10 and ugt1a8 are coordinately regulated by the aryl hydrocarbon receptor (ahr) and nrf2 and that the nrf2 response requires the presence of ahr. both pahs and pah o - quinones such as b[a]p-7,8-dione are the ligands of the ahr, which is required for the induction of ugts by nrf2. with the induction of ugts, the akr1c genes involved in the formation and redox cycling of pah o - quinones are also highly induced by the nrf2-keap 1 system. inducers that activate nrf2 include electrophiles and ros and not surprisingly pah o - quinones. thus, the induction of akr1c genes with the products of pah - trans - dihydrodiol oxidation, namely, pah o - quinones, could lead to an exacerbation of ros formation which may not be easily countered by ugt induction. | polycyclic aromatic hydrocarbons (pahs) are environmental and tobacco carcinogens. proximate carcinogenic pah trans - dihydrodiols are activated by human aldo - keto reductases (akrs) to yield electrophilic and redox - active o - quinones. interconversion among benzo[a]pyrene (b[a]p)-7,8-dione, a representative pah o - quinone, and its corresponding catechol generates a futile redox - cycle with the concomitant production of reactive oxygen species (ros). we investigated whether glucuronidation of b[a]p-7,8-catechol by human udp glucuronosyltransferases (ugts) could intercept the catechol in three different human lung cells. rt - pcr showed that ugt1a1, 1a3, and 2b7 were only expressed in human lung adenocarcinoma a549 cells. the corresponding recombinant ugts were examined for their kinetic constants and product profile using b[a]p-7,8-catechol as a substrate. b[a]p-7,8-dione was reduced to b[a]p-7,8-catechol by dithiothreitol under anaerobic conditions and then further glucuronidated by the ugts in the presence of uridine-5-diphosphoglucuronic acid as a glucuronic acid group donor. ugt1a1 catalyzed the glucuronidation of b[a]p-7,8-catechol and generated two isomeric o - monoglucuronsyl - b[a]p-7,8-catechol products that were identified by rp - hplc and by lc - ms / ms. by contrast, ugt1a3 and 2b7 catalyzed the formation of only one monoglucuronide, which was identical to that formed in a549 cells. the kinetic profiles of three ugts followed michaelis menten kinetics. on the basis of the expression levels of ugt1a3 and ugt2b7 and the observation that a single monoglucuronide was produced in a549 cells, we suggest that the major ugt isoforms in a549 cells that can intercept b[a]p-7,8-catechol are ugt1a3 and 2b7. |
lysosomal cysteine proteases, the cathepsins, classified as clan c1,1 were long believed to be responsible for the terminal protein degradation in the lysosomes. this view has changed since they are involved in a number of important cellular processes, such as antigen presentation,2 bone resorption,3 apoptosis4 and protein processing,5 as well as several pathologies such as cancer progression,6 inflammation7 and neurodegeneration.8 so far, cathepsin d, an aspartic protease, and cysteine cathespins b, h, l, s and few others were associated with cancer progression. 9 - 12 cysteine cathepsins are single - gene products, but the protein products may be polymorphic, due to allelic variants of the gene, alternative rna splicing and/or post - translational modifications. similar to other proteases, cathepsins are regulated at every level of their biosynthesis, in particular, by their compartmentalization to lysosomes, activation of pro - enzyme forms and ultimately by their endogenous protein inhibitors. 4, 13, 14 among them the best characterized are cystatins, which comprise a superfamily of evolutionary related proteins, each consisting of at least one domain of 100 - 120 amino acid residues with conserved sequence motifs. 4, 13, 14 cystatins function as reversible, tight - binding inhibitors of cysteine proteases, and generally do not possess specific inhibitory activity to particular cathepsin. type i cystatins (the stefins), stefins a and b, are cytosolic proteins, lacking disulphide bridges. type ii cystatins are more numerous, comprising at least 14 members (for recent review, see keppler., besides well - known cystatins c, d, e / m, f, s, sa, sn this group contains the male reproductive tract cystatins 8 (cres, cystatin - related epididymal spermatogenic protein), 9 (testatin), 11 and 12 (cystatin t), the bone marrow - derived cystatin - like molecule clm (cystatin 13) and the secreted phosphoprotein ssp24 (cystatin 14). type iii cystatins, the kininogens, are large multifunctional plasma proteins, containing three type ii cystatin - like domains. another two types of cystatins, fetuins and latexins are constituted by two tandem cystatin domains, however, they do not exhibit inhibitory activity against cathepsins, and are not the subject of the present review. a broad spectrum of biological roles have been suggested for cystatins, including a role in protein catabolism, regulation of hormone processing and bone resorption, inflammation, antigen presentation and t - cell dependent immune response as well as resistance to various bacterial and viral infections.16, 17 cystatins have been suggested as modulators of the proteolytic system in several diseases, including immune disorders and cancer. 9, 16, 18 to highlight the function of cystatins in regulation of proteolysis as well as their functions other than protease inhibition, we review recent finding on the status of type i (stefins a, b) and type ii cystatins (cystatins c, f and e / m) in pathologies, including their suppressive vs. promotional function in the tumor immunology. stefin a (also named cystatin a, acid cysteine protease inhibitor, epidermal sh - protease inhibitor) and stefin b (also named cystatin b, neutral cysteine protease inhibitor) are representatives of family i cystatins. stefins a and b exibit 54% sequence identity, both are a 98-amino acid protein with a molecular mass of 11,175 da and 11,006 da, respectively. 19, 20 the first three - dimensional structure of a stefin was the crystal structure of the recombinant stefin b in complex with papain. the stefin molecule consists of a five stranded antiparallel -sheet wrapped around a five turn -helix with an additional carboxyl- terminal strand that runs along the convex side of the sheet. the n - terminus and the two -hairpins form the edge of the wedge shaped surface, which bind into the active site cleft of cysteine proteases. 21 the three - dimensional structure of stefin a has been determined in solution and in complex with cathepsin h, the latter being similar to stefin b - papain complex with a few distinct differences 19,22. both, stefin a and stefin b, have been shown to form protein structures known as amyloid fibrils, although stefin a under more hursh conditions than stefin b. 23 recently, the crystal structure of stefin b tetramer has been determined which involves pro at the position 74 in a cis isomeric state being essential in stefin b amyloid - fibril formation. 24,25 stefin a was purified from rat skin as a first identified mammalian cystatin 26 and has been found in other epithelial cells, 27 - 31 in neutrophils from the liver, 32 in dendritic reticulum cells of lymphoid tissue, 33 in hassall 's corpuscles and in thymic medullary cells. 34 the selective expression of the inhibitor correlates with the tissues participating in the first - line defence against pathogens. analysis of proteins uniquely involved in the development of the skin and skin immune system revealed strong expression of stefin a in neonatal mouse skin and decreasing with age suggesting an important role in the development of the epidermis 35. 20, 36 - 38 subcellularly, it was found mainly in the nucleus of proliferating cells and both in the nucleus and in the cytoplasm of differentiated cells. 39 it has been suggested that stefin b regulates the activity of cathepsin l in the nucleus. nuclear stefin b interacted with cathepsin l and with histones in the nucleus, but it did not bind to dna. increased expression of stefin b in the nucleus delayed cell cycle progression that was associated with the inhibition of cathepsin l in the nucleus. stefin b could thus play an important role in regulating the proteolytic activity of cathepsin l in the nucleus, protecting substrates such as transcription factors from its proteolytic processing. stefin a is involved in cellular proliferation and could be a useful target for diseases of abnormal proliferative conditions. its mrna level is increased in psoriatic plaques of the psoriasis vulgaris, a common inflammatory disease of the skin, characterized by hyperproliferation of skin cells that ultimately leads to red, scaly plaques. 41 polymorphysm in the gene for stefin a has been associated with atopic dermatitis, a chronic inflammatory skin disease often associated with a defective epidermal barrier. 42, 43 stefin a is able to protect skin barrier from allergic reactions, including atopic dermatitis. inhibition of proteolytic activity of major mite allergens, der f 1 and der p 1, by stefin a blocks the up - regulation of il-8 and gm - csf release from keratinocytes stimulated with the allergens. 44, 45 loss - of - function mutations in the gene for stefin a has been identified as the underlying genetic cause of another skin disease, exfoliative ichthyosis. 46 stefin b was found to form a multi - protein complex specific to the cerebellum with five other proteins and none of them is a protease : the protein kinase c receptor (rack-1), brain -spectrin, the neurofilament light chain (nf - l), one protein from the myotubularin family and one unknown protein. stefin b multiprotein complex is proposed to have a specific cerebellar function and the loss of this function might contribute to the disease in epm1 patients. 47 epm1 is a degenerative disease of the central nervous system also known as progressive myoclonus epilepsy of the unverricht - lundborg type. altogether 10 different mutations in stefin b gene underlying epm1 have been reported, of these the most common change an expansion of a normally polymorphic 12-nucleotide repeat in the promoter region is found that is associated with reduced protein levels. 48 five different mutations in the coding region of the stefin b gene were found causing protein truncation (r68x), frameshift (k73fsx2) and missense mutations (g4r, q71p and g50e). the k73fsx2-truncated mutant protein localizes to cytoplasm and nucleus, whereas r68x mutant is rapidly degraded. two missense mutations, g4r affecting the highly conserved glycine, critical for cathepsin binding, and q71p, fail to associate with lysosomes. these data imply an important lysosome - associated function for stefin b and suggest that loss of this association contributes to the molecular pathogenesis of epm1. 53 accumulation of protein agregates characterize many neurodegenerative diseases, including alzheimer 's disease (ad), parkinson 's disease, dementia, multiple system atrophy, huntington 's disease, and the transmissible " prion " dementias. 54, 55 amiloid- (a) is a soluble peptide, but can form aggregates, either oligomeric or fibrillar that are neurotoxic in ad. 56 stefin b has been found to be an a-binding protein thus it is likely to have a role in ad. it interacts with a in vitro and in cells and is supposed to have a " chaperone - like " function with binding the a and inhibiting its fibril formation. among type ii cystatins, the most prominent cystatins in immune cells are cystatins c and f, the former being the most abundant human cystatin. cystatin c was discovered first as a ' post--globulin ' or ' -trace ' and was the first cystatin determined for amino acid sequence. 58 later it was shown that its amino acid sequence was highly similar to cystatin, isolated from chicken egg white. 59 mature human cystatin c is composed of 120 amino acid residues and has a molecular mass of 13,343 da. the cystatin c cdna sequence revealed that cystatin c is synthesized as a preprotein with a 26 residue signal peptide.60 the gene, encoding cystatin c is typical house - keeping gene type, which is expressed in a variety of human tissues and cells. however, like the most of other type ii cystatins, cystatin c is secreted and can be found in high concentrations in body fluids, in particular high levels have been found in seminal plasma and cerebrospinal fluid. cystatin c is strong inhibitor of all papain - like proteases (clan c1) 61, 62 and asparaginyl endopeptidase / legumain (clan c13) 63 and could be seen as a major human extracellular cysteine protease inhibitor. cystatin c has been suggested as regulating cathepsin s activity and invariant chain (ii) processing in dendritic cells (dcs), 64 however, further studies excluded a role in controlling mhc ii - dependent antigen presentation in dcs. 65 additionally, the maturation process of dcs leads to reduced levels of cystatin c and colocalization studies do not support intracellular interactions among cystatin c and its potential target enzymes cathepsins h, l and s in immature or mature dcs. 66 a better candidate for regulating the proteolytic activity of cysteine proteases within the dcs was shown to be cystatin f. 67 cystatin f was discovered by three independent groups. two of them identified the new inhibitor by cdna cloning and named it leukocystatin and cystatin f 68,69. the third group found overexpressed mrna encoding cystatin f in liver metastatic tumors and identified it as cmap (cystatin - like metastasis associated protein). 70 human cystatin f is synthesized as a 145 amino acids pre - protein with a putative 19 residues signal peptide. 68, 69 although it is made with a signal sequence, only a small proportion is secreted 69 and importantly, it is secreted as a disulphide - linked dimer 71 which is inactive until it is reduced to its monomeric form. 72 it is glycosylated 68, 69 and mannose-6-phosphate modification of its n - linked saccharides is used for targeting to the endosomes and lysosomes. 73 glycosylation at asn62 is proposed to protect the intermolecular disulphide from reduction, explaining unusually strong reducing conditions needed to monomerize dimeric cystatin f in vitro. 72, 74 inactive dimer to active monomer conversion is also achieved with proteolitic cleavage by so far unidentified protease action on the extended n - terminal region of cystatin f. 75 being glycosylated, inacive cystatin f can be internalized through the mannose-6-phosphate receptor pathway and activated within different cells. 73 all these facts strongly imply on intracellular action of cystatin f as well as on in trans activity of its secreted inactive inhibitor which can be internalized and activated inside another cells. cystatin f tightly inhibits cathepsins f, k, v, whereas cathepsins s and h are inhibited with lower affinities and cathepsin x is not inhibited at all. 69, 76 c13 cysteine protease involved in antigen pocessing, mammalian legumain or asparaginyl endopeptidase (aep), is also inhibited by cystatin f although it showes reduced affinity for aep compared with cystatins c and e / m. 63 the inhibitor is expressed selectively in immune cells such as cytotoxic t cells, natural killer cells (nk cells), monocytes, dcs (figure 1). 67 - 69, 77, 78 its levels and localization are controlled according to the physiological state of the cells. it is strongly up - regulated in monocyte - derived dcs undergoing lps - induced maturation and downregulated in tpa- (causing monocytic differentiation towards a granulocytic pathway) or atra- (causing monocytic differentiation towards macrophages) stimulated u937 cells. 77, 79 the unique features of cystatin f suggests that this immune - cell specific inhibitor plays a role in immune response - related processes through inhibition of specific enzyme targets, even though the details of its role remain unexplained. it is likely that in dcs, cystatin f could regulate the activity of cathepsin l and thus controlling the processing of procathepsin x, which promotes cell adhesion via activation of mac-1 (cd11b / cd18) integrin receptor. 67 one of the protease targets of cystatin f is cathepsin c 75, the cysteine protease that activates the granzymes in cytotoxic t cells, nk cells and several of effector proteases of neutrophils and mast cells. 75, 80, 81 cystain f is a strong inhibitor of cathepsin c only as an n - terminally truncated form. 75 our recent work suggests potential regulation of split anergy in nk cells through inhibition of cathepsin c and consequently downstream regulation of granzymes (manuscript in prep). jewett and colleagues indicate that induction of split anergy in nk cells may be an important physiological step required for the conditioning of the nk cells to support differentiation of the stem cells. in this regard they proposed that conditioned or anergized nk cells may play a significant role in differentiation of the cells by providing critical signals via secreted cytokines as well as direct cell - cell contact. to be conditioned to drive differentiation, nk cells may have to first receive signals through their key surface receptors either from stem cells or other immune effectors or fibroblasts in the inflammatory microenvironment and lose cytotoxicity and gain cytokine producing phenotype (split anergy). these alterations in nk cell effector function will ultimately aid in driving differentiation of a population of surviving healthy as well as transformed stem cells. regulation of cystatin f and consequently cathepsin c and granzymes by nk cell surface receptors could be the mechanism which conditions nk cells to undergo split anergy and become regulatory nk cells. for cystatin m a downregulated mrna in metastatic breast tumor cells was identified by differential display when compared to normal and primary breast tumor cells. 82 independently, the same molecule was found by others in cdna libraries derived from epithelial cells, and was designated cystatin e. 83 cystatin e / m is a 14.5 kda secreted protein that has an overall structure similar to other family ii cystatins, such as a signal peptide and two intrachain disulfide bonds. like cystatin f 68, 69, it possesses the unusual characteristic of being a glycoprotein, carrying an n - linked carbohydrate chain at position 108. 82, 83 this protein is only distantly related to the other known family members as reflected by the genomic position of the cystatin e / m gene on chromosome 11q13 84, whereas all other family ii cystatin genes are clustered in a narrow region on chromosome 20p11.2. 85 cystatin e / m is synthesized as a pre - protein with a putative 28 residues while mature cystatin e / m contains 121 amino acids. 83 high expression levels are largely confined to skin epithelia, which emphasizes its prominent role in cutaneous biology. 86 its expression was also reported recently in breast tissue 87 and in oligodendrocyte- and astrocyte - like cells of human brain. 88 cystatin e / m is a high - affinity inhibitor of cathepsins v, l 89 and asparaginyl endopeptidase / legumain, 63 with lower affinity it binds also cathepsin b. 83 in vivo mouse models have revealed that cystatin e / m is a key molecule in a biochemical pathway that controls skin barrier formation. 90, 91 the terminal stage of the differentiation process of keratinocytes is desquamation, which involves degradation of lipids in the intercellular spaces and loss of residual intercellular desmosomal connections. cells reaching the skin surface are continuously sloughed and replaced by inner cells differentiating and moving outward. 92 cystatin e / m could have a role in desquamation by the regulation of cathepsin v protease activity or could regulate cathepsin l and legumain activities cosslinking of structural proteins by transglutaminase 3 in the keratinocyte differentiation process of the epidermis and the hair follicle. 93 in addition, it might also be involved in the regulation of cathepsin d activity, as it is known that cathepsin l can process and activate cathepsin d 94, 95 and mature cathepsin d is also important in the desquamation process of the skin. 96 - 98 the studies regarding implication of other type ii cystatins in immune system are not numerous. cystatins s, sn and sa were first isolated from human saliva and later identified as a cysteine protease inhibitors and their role in controlling cysteine proteases derived from bacteria has been suggested. 99 - 102 these three proteins with similar sequence (88% identity at the protein level) are distantly related to cystatin d (less then 60% identity at the protein level with cystatins s, sn and sa). 103 although they are possessing inhibitory activity, they are all poorer inhibitors of cysteine cathepsins than cystatin c. 104 inhibition of bacterial cysteine proteases have been tested by cystatins c, sn, s and chicken cystatin and they are generaly inactive against bacterial cysteine proteases. 105 - 107 in contrast to bacterial cysteine proteases, they are more efficient against cysteine proteases from parasits ; for example, cystatins sa and sn are inhibitors of trypanosome cruzain 108, chicken cystatin inhibits congopain from trypanosoma congolense 109 and cruzipain, the major cysteine proteinase of the protozoan parasite trypanosoma cruzi. 110 these results suggest a possible defensive role for the host 's cystatins after parasite infection. cystatin sn has been found also in dendritic cells exposed to toxoplasma gondii 111 and it could thus modulate antigen presentation. salivary cystatins may have a role in the host defense mechanism against virus infections as they can interfere with events in viral replication. cystatin d has been found to be a potent inhibitor of coronavirus replication 112 and cystatins s, sa and sn can suppress the infectivity of adenovirus 113 and herpes simplex virus 1. 114 human genom contains several related genes encoding gycoproteins that possess sequence similarity with other type ii cystatins. the shared characteristic of three subgroup members is an expression pattern limited primarily to the male reproductive tract. testatin 115 and cystatin t 116 are specifically expressed in the testis and cystatin - related epididymal spermatogenic protein (cres) exhibits highly tissue - specific expression in the reproductive tract. 117 although they possess a typical c - terminal pw motif, they lack the n - terminal glycine and motif qxvxg important for inhibition of cysteine proteases. changes in cystatin c expression and localization have been associated with various neurodegenerative pathologies. for example, a point mutation in the cystatin c gene, resulting in the substitution of leu to gln, is responsible for the dominantly inherited icelandic type of amyloidosis, hereditary cystatin c amyloid angiopathy (hccaa). patients with this disease suffer from successive brain hemorrhages concluding in death at the age of 30. in first line the formation of cystatin c aggregates are responsible for the outcome and progression of the disease, however, the loss of inhibitory function due to aggregation and lower concentration in brain can not be excluded. 118 the levels of cystatin c were highly reduced in cerebrospinal fluid of individuals suffering from multiple sclerosis (ms) compared with healthy individuals. its low concentration suggests that the inhibition of cysteine proteinases is impaired in this disease ; hence higher activity of cysteine proteinases could initiate or increase the breakdown of myelin. 119 cystatin c was shown to be implicated in alzheimer 's disease (ad). it co - deposits with amyloid - beta (a) in amyloid plaques of ad patients 120, associates with a and inhibits a oligomerization in vitro and in vivo. 121, 122 thus, cystatin c could protect the brain from amyloid - induced toxicity and may have therapetic implications for ad. 56 however, there are opposing results demonstrating neuronal cell death upon injecting cystatin c into the brain of ad mouse model, 123 showing that cystatin c triggers a accumulation by inhibiting cathepsin b - induced a degradation. 124 cystatin c may have an important role in brain injuries as its enhanced expression has been observed in response to different types of insults to the brain, such as ischemia and epilepsy. 125 - 127 regarding constant serum concentrations of cystatin c it is a suitable marker for glomerular filtration rate (gfr) and kidney function and thus has a possible application as a replacement for creatinine. 128, 129 studies on the sensitivity and specificity of cystatin c for detecting impaired gfr strongly suggest it to be superior diagnostic marker to creatinine for detecting impaired gfr. 130 however, caution is necessary in case of patients with malignancy or other diseases with increased activity of cyteine proteases which exhibit increased cystatin c levels irrelevant to kidney function. 131 it has been suggested that disturbance of the cystatin e / m - cathepsin pathway could contribute to dysregulated skin barrier function as observed in the inflammatory dermatoses. atopic dermatitis and psoriasis are two common chronic inflammatory skin diseases in which the expression of many genes and the formation of the epidermal barrier are altered. recent genetic studies have revealed that abnormalities in epithelium - expressed genes are an important etiological factor. 86 have shown decreased mrna and protein expression levels of cystatin e / m and cathepsin v in the inflamed skin during atopic dermatitis and psoriasis, which suggests their prominent role in these inflammatory diseases. it was found to be massively expressed in the microglia, monocyte / macrophage cells in the cental nervous system, only during acute demyelination and ceased in chronic phase, in which remyelinating ability is lost. expression of cystatin f thus indicates the occurrence of ongoing demyelination / remyelination during multiple sclerosis and might serve as a good indicator for this process, however, it is not clear whether changes in cystatin f levels result from or cause arrested remyelination. 135 the level of cystatin f mrna was increased in the granulocytes of the patients with polycythemia vera, a myeloproliferative disorder characterized by an increased proliferation of cells of myeloid lineages 136 and downregulated in peripheral blood mononuclear cells of patients with chronic fatigue syndrome. 138 given the high concentrations of cystatins in saliva and tears it is likely to have a more specialized role. 139 cystatin sn was found to inhibit the human cathepsins b, h and l and cystatin sa was found to inhibit human lysosomal cathepsin l, the proteases that are involved in periodontal tissue destruction suggesting the involvment of salivary cystatins sa and sn in the control of the proteolytic events. 108 however, weak inhibitory activity against cathepsins b, h, l and potent inhibition of cysteine proteases from parasitic organisms cleary indicate that salivary cystatins help to control the oral microorganisms rather than prevent periodotal diseases. there are an accumulating number of studies showing that cystatins are implicated in the progression of various types of cancer. in general, their localization and regulation of tumor associated proteolytic activity can be recognized by two distinct mechanisms. type i cystatins, stefins a and b are up - regulated in tumor tissue and, up to a certain level could counter - balance the over - expressed tumor - associated proteolytic activity. their function as tumor suppressors is supported by survival analysis, which associated their high levels with better outcome of cancer patients. for example, in brain tumors, increase of cathepsin b expression and lower levels of its inhibitor stefin a were associated with tumor malignancy 140. on the other hand, type ii cystatins, at least cystatins c and e / m are generally down - regulated in tumors. although their role remains protective, their lower levels could allow a surplus of harmful tumor associated proteolytic activity. outside the cells, higher levels of type ii cystatins may impair extracellular activity of cysteine proteases, associated directly or indirectly with the degradation of ecm and resulting in tumor cell invasion and metastasis. however, higher levels of cystatins in body fluids have been associated with poor prognosis in cancer patients supporting their role in regulation of proteases involved in the regression of tumors. in melanoma and colorectal cancer, increased extracellular levels of cystatin c as well as stefins correlated significantly with high risk of adverse outcome in cancer patients (figure 2). cathepsin b / cystatin c complex was also found to be less abundant in sera of patients with tumors suggesting an imbalance between the enzyme and its inhibitor in cancer patients. 141 animals with excluded expression of type i or type ii cystatins experience better outcome with regard to tumor growth and metastasis as the wild type ones. these contradictive results can be explained by the fact that type ii cystatins are involved also in processes resulting in tumor regression such as anti - tumor immune response, apoptosis, cell migration and seeding. 9 in particular, the role of cysteine proteases is very important for proper maturation of antigen presenting cells, antigen processing and the presentation to t cells, therefore, the enhanced inhibition may affect the activation of naive t cells by tumor associated antigens and impair t cell dependent anti - tumor immune response, known to be effective for eradication of tumor cells. higher levels of cystatins may affect also the innate immunity, as mentioned above, increased levels of cystatin f inactivate cathepsin c and thus impair activation of granzymes and cytotoxicity of nk cells. 75, 142 on the other hand, patients with higher local levels of stefins a and b in non - small cell lung tissue than in control lung tissue exhibited a better survival probability implying on their ability to counteract harmful tumor - associated proteolytic activity. 143 also, the overexpression of cystatin c in tumor cells inhibited melanoma metastasis formation. 144 thus, besides the concentration, cell and tissue localization of cystatins could make a critical switch between harmless and harmful and their application as anti - cancer agents has to be considered with caution and should be directed specifically to cysteine proteases which promote specific stage of tumor progression. for instance, the expression and the role of cystatin e / m in tumorigenesis may differes depending on the stage of the cancer. since it was initially identified as a downregulated mrna in metastatic breast tumor cells compared to normal and primary breast tumor cells 82, it is likely that the loss of the expression of cystatin e / m is associated with the progression of a primary tumor to a metastatic phenotype. furthermore, scid mice orthotopically implanted with cystatin e / m - expressing mda - mb-435s breast cancer cells show significantly delayed primary tumor growth compared to controls, the incidence of metastasis, however, appeared to be unaltered, confirming that cystatin e / m suppressed tumor cell proliferation at the secondary site. 145 higher level of cystatin f mrna in the colorectal cancer tissue correlated both with liver metastasis and with worse patient prognosis. 146 therefore, it is possible that a determination of the cystatin f mrna expression may help in the identification of patients at high risk for metastasis, and these patients could thereby benefit from careful examinations and extensive treatments. the pathways by which cystatins modulate immune response unrelated to their function of cysteine protease inhibitors, can not be excluded, as shown for cystatins of filarial nematodes 147, chicken cystatin 148, 149 and cystatin c. 150 the latter has been shown to antagonize tgf- binding to the cell surface receptors of normal and cancer cells by interacting physically with the tgf- type ii receptor and abrogating the binding of tgf-. tgf- is a multifunctional cytokine endowed with both tumor - suppressing and tumor - promoting activities. in normal cells tgf- inhibits proliferation and induce apoptosis, however during the transformation to cancer cell it becomes a trophic factor for the transformed cells promoting their proliferation. 151, 152 interestingly, down regulation of tumorogenesis through a similar mechanism has been shown also for a family-3 cystatin (ahsg) which inhibits colon carcinogenesis by suppressing tgf- signal transduction by blocking tgf-1 binding to cell surface receptors. 153 there is an evidence indicating that cystatin e / m is a tumor suppressor factor important in breast and brain malignancy reducing tumor cell proliferation, its expression is markedly decreased in carcinoma cells. 155, the effects of cystatin e / m expression on malignant properties were examined on human breast carcinoma mda - mb-435s cells, which are highly tumorigenic and metastatic and normally do not express cystatin e / m. the constitutive cystatin e / m expression in transfected cells resulted in a significant reduction of cell proliferation, migration, matrigel invasion, and adhesion to endothelial cells. cell migration and matrix invasion appeared to be controlled by the lysosomal cysteine proteases, as both recombinant cystatin e / m and e-64 were able to block these processes. in contrast, reduction of cell proliferation and adhesion to an endothelial cell monolayer were both independent of the inhibition of lysosomal cysteine proteases. cystatin e / m could thus act through various mehanisms, besides inhibition of cysteine proteases also by inhibition of legumain and modulation of gene transcription. the latter is confirmed by the fact, that cystatin e / m expression in mda - mb-435s cells significantly changed the expression of extracellular matrix components, cytokines, kinases, and phosphatases, as well as several key transcription factors, including downregulation of autotaxin, a signaling molecule that has been linked to breast cancer invasiveness. 156 furthermore, epigenetic studies reported dna methylation - dependent silencing of the cystatin e / m gene in breast cancer cell lines and in primary breast tumors. hypermethylation was significantly associated with gene silencing and loss of cystatin e / m protein expression, whereas enhanced gene expression was measured in breast cancer cells cultured on the dna demethylating agent. 154, 157, 158 collectively these data suggest that cystatin e / m could act as a tumor supressor through various mehanisms. they exert several immunomodulatory functions by controlling the activity of cysteine proteases or by other mechanisms not related to their inhibitory function. they may contribute to the proteolytic processing of progranzymes and other substrates, major histocompatibility complex class ii antigen presentation, maturation of dendritic cells, modulation of integrin function and formation of the skin barrier. it has become evident that disturbances in expression and localization of cystatins may be implicated in several pathological processes, such as inflammatory skin diseases and neurodegenerative disorders. in particular for stefin b and cystatin c, association with amyloid - beta in alzheimer 's disease has been demonstrated. in cancer, inhibitory effect of cystatins could beneficially counteract tumor - associated proteolytic activity or, could also be harmful when impairing the functions in cysteine cathepsins in anti - tumor immune response and is strongly depended on their cell and tissue localization. | cystatins comprise a large superfamily of related proteins with diverse biological activities. they were initially characterised as inhibitors of lysosomal cysteine proteases, however, in recent years some alternative functions for cystatins have been proposed. cystatins possessing inhibitory function are members of three families, family i (stefins), family ii (cystatins) and family iii (kininogens). stefin a is often linked to neoplastic changes in epithelium while another family i cystatin, stefin b is supposed to have a specific role in neuredegenerative diseases. cystatin c, a typical type ii cystatin, is expressed in a variety of human tissues and cells. on the other hand, expression of other type ii cystatins is more specific. cystatin f is an endo / lysosome targeted protease inhibitor, selectively expressed in immune cells, suggesting its role in processes related to immune response. our recent work points on its role in regulation of dendritic cell maturation and in natural killer cells functional inactivation that may enhance tumor survival. cystatin e / m expression is mainly restricted to the epithelia of the skin which emphasizes its prominent role in cutaneous biology. here, we review the current knowledge on type i (stefins a and b) and type ii cystatins (cystatins c, f and e / m) in pathologies, with particular emphasis on their suppressive vs. promotional function in the tumorigenesis and metastasis. we proposed that an imbalance between cathepsins and cystatins may attenuate immune cell functions and facilitate tumor cell invasion. |
the study sample consisted of 39 nw (bmi < 85th% for age and sex), 64 ob (bmi 95th% for age and sex), and 17 obese adolescents with type 2 diabetes, who were participants in our national institutes of health funded k24 grant of insulin resistance in childhood and had complete data relevant to this investigation. some of these participants were reported before (10). nw and ob participants were recruited through newspaper advertisements and fliers posted on bus routes, on the university campus, and in children s recreational areas. adolescents with type 2 diabetes were recruited from the diabetes center at children s hospital of pittsburgh and were negative for glutamic acid decarboxylase and insulinoma - associated protein-2 autoantibody as reported before (11). subjects with type 2 diabetes were treated with lifestyle intervention alone (n = 4), metformin with lifestyle intervention (n = 10), metformin + insulin (n = 1), or insulin alone (n = 2). in diabetic subjects, metformin and long - acting insulin none of the other participants, some of whom were reported previously (12), took any other medications that affect glucose, fat, or protein metabolism. studies took place at the children s hospital of pittsburgh national institutes of health funded pediatric clinical and translational research center (pctrc) after institutional review board approval. parental consent and all participants were documented to be in good health by history, physical examination, and routine hematological and biochemical tests. subject characteristics and metabolic profile the afternoon prior to testing, all subjects were admitted to the pctrc for testing the next morning after a 1012 h overnight fast. total - body lipolysis was measured at baseline for 2-h using a primed (1.2 mol / kg) constant rate (0.08 mol / kgmin) infusion of [h5]glycerol, as described (14,15). in vivo is was evaluated with a 3-h hyperinsulinemic - euglycemic clamp begun immediately after the baseline period. briefly, intravenous crystalline insulin (humulin ; lilly, indianapolis, in) was infused at a constant rate as reported (16,17), and plasma glucose was clamped at 100 mg / dl (5.5 mmol / l) with a variable - rate infusion of 20% dextrose based on arterialized plasma glucose determinations every 5 min. continuous indirect calorimetry by a ventilated hood system (deltatrac metabolic monitor ; sensormedics, anaheim, ca) was used to evaluate substrate oxidation during the last 30 min of the baseline period on two separate occasions within a 2- to 3-week period, and the last 30 min of the 3-h clamp on one occasion, as described (14,15). archived fasting plasma samples (80c) were analyzed for acylcn and amino acid profiles by ms / ms of butyl derivatives as described previously (7). acylcn species were quantified by calculating the concentrations of metabolites relative to the deuterated internal standard closest in mass (eight different masses ; cambridge isotope laboratories, andover, ma). each acylcn species presented in the tables and figures was detected in each plasma sample. body composition was assessed by dual - energy x - ray absorptiometry and subcutaneous and visceral adiposity by a single slice computed tomography scan at l4-l5 as reported previously (19,20). data are not available for subjects whose weight exceeded the limit for dual - energy x - ray absorptiometry scan (n = 13 ; 9 males/4 females ; 6 black/7 white ; 8 ob/5 type 2 diabetes), and for computed tomography imaging (n = 7 ; 2 males/5 females ; 3 black/4 white ; 2 nw, 4 ob, and 1 type 2 diabetes). including or excluding these 13 individuals did not influence the results, and therefore they were kept in the analyses. plasma glucose was determined with a glucose analyzer (yellow springs instrument co., yellow springs, oh), and insulin by radioimmunoassay as before (21). pancreatic autoantibodies were determined in the northwest lipid metabolism and diabetes research laboratories, university of washington (seattle, wa) using the national institute of diabetes and digestive and kidney diseases sponsored standardization assay (11). ffas were determined by an enzymatic colorimetric assay (wako nonesterified fatty acid c test kit ; wako, osaka, japan). deuterium enrichment of glycerol in plasma was determined on a 5971 mass spectrometer coupled to a hewlett - packard co. 5890 series ii gas chromatograph according to our previously described method (22). total - body lipolysis was calculated from the rate of appearance (ra) of glycerol in plasma according to steady - state tracer dilution equations (14,15). plasma ffa ra was calculated by multiplying glycerol ra by three, because when a triglyceride molecule is hydrolyzed, one glycerol molecule and three fatty acid molecules are produced (14). basal and insulin stimulated glucose oxidation and lipid oxidation rates were the calculated mean from indirect calorimetry measurements according to the equation developed by frayn (23). this equation (23) was developed using a triacylglycerol very similar to the average composition of human adipose tissue and is more reflective of human obesity. anova with bonferroni post hoc correction for quantitative variables and test for categorical variables were used to examine subject characteristics among the three groups. ancova models were used to assess differences in amino acid and acylcn values among the three groups, adjusting for age, tanner stage, and race. to assess the relationships between fatty acid and amino acid derived acylcn species, plasma amino acids, is, and fox, bivariate pearson or spearman correlations were applied according to data distribution. partial correlation analyses between is or fox and acylcn or amino acids were used to adjust for tanner stage because data suggest that increased lipid oxidation during puberty may contribute to the transient insulin resistance of puberty (14), adiposity measures (bmi, percent body fat [% bf ], and visceral adipose tissue [vat ]), and sex. the statistical analyses were performed using pasw statistics (version 18 ; spss inc., the afternoon prior to testing, all subjects were admitted to the pctrc for testing the next morning after a 1012 h overnight fast. total - body lipolysis was measured at baseline for 2-h using a primed (1.2 mol / kg) constant rate (0.08 mol / kgmin) infusion of [h5]glycerol, as described (14,15). in vivo is was evaluated with a 3-h hyperinsulinemic - euglycemic clamp begun immediately after the baseline period. briefly, intravenous crystalline insulin (humulin ; lilly, indianapolis, in) was infused at a constant rate as reported (16,17), and plasma glucose was clamped at 100 mg / dl (5.5 mmol / l) with a variable - rate infusion of 20% dextrose based on arterialized plasma glucose determinations every 5 min. continuous indirect calorimetry by a ventilated hood system (deltatrac metabolic monitor ; sensormedics, anaheim, ca) was used to evaluate substrate oxidation during the last 30 min of the baseline period on two separate occasions within a 2- to 3-week period, and the last 30 min of the 3-h clamp on one occasion, as described (14,15). archived fasting plasma samples (80c) were analyzed for acylcn and amino acid profiles by ms / ms of butyl derivatives as described previously (7). acylcn species were quantified by calculating the concentrations of metabolites relative to the deuterated internal standard closest in mass (eight different masses ; cambridge isotope laboratories, andover, ma). each acylcn species presented in the tables and figures was detected in each plasma sample. body composition was assessed by dual - energy x - ray absorptiometry and subcutaneous and visceral adiposity by a single slice computed tomography scan at l4-l5 as reported previously (19,20). data are not available for subjects whose weight exceeded the limit for dual - energy x - ray absorptiometry scan (n = 13 ; 9 males/4 females ; 6 black/7 white ; 8 ob/5 type 2 diabetes), and for computed tomography imaging (n = 7 ; 2 males/5 females ; 3 black/4 white ; 2 nw, 4 ob, and 1 type 2 diabetes). including or excluding these 13 individuals did not influence the results, and therefore they were kept in the analyses. plasma glucose was determined with a glucose analyzer (yellow springs instrument co., yellow springs, oh), and insulin by radioimmunoassay as before (21). pancreatic autoantibodies were determined in the northwest lipid metabolism and diabetes research laboratories, university of washington (seattle, wa) using the national institute of diabetes and digestive and kidney diseases sponsored standardization assay (11). ffas were determined by an enzymatic colorimetric assay (wako nonesterified fatty acid c test kit ; wako, osaka, japan). deuterium enrichment of glycerol in plasma was determined on a 5971 mass spectrometer coupled to a hewlett - packard co. 5890 series ii gas chromatograph according to our previously described method (22). total - body lipolysis was calculated from the rate of appearance (ra) of glycerol in plasma according to steady - state tracer dilution equations (14,15). plasma ffa ra was calculated by multiplying glycerol ra by three, because when a triglyceride molecule is hydrolyzed, one glycerol molecule and three fatty acid molecules are produced (14). basal and insulin stimulated glucose oxidation and lipid oxidation rates were the calculated mean from indirect calorimetry measurements according to the equation developed by frayn (23). this equation (23) was developed using a triacylglycerol very similar to the average composition of human adipose tissue and is more reflective of human obesity. anova with bonferroni post hoc correction for quantitative variables and test for categorical variables were used to examine subject characteristics among the three groups. ancova models were used to assess differences in amino acid and acylcn values among the three groups, adjusting for age, tanner stage, and race. to assess the relationships between fatty acid and amino acid derived acylcn species, plasma amino acids, is, and fox, bivariate pearson or spearman correlations were applied according to data distribution. partial correlation analyses between is or fox and acylcn or amino acids were used to adjust for tanner stage because data suggest that increased lipid oxidation during puberty may contribute to the transient insulin resistance of puberty (14), adiposity measures (bmi, percent body fat [% bf ], and visceral adipose tissue [vat ]), and sex. the statistical analyses were performed using pasw statistics (version 18 ; spss inc., as expected, ob and diabetic subjects had body composition indices that were all significantly higher (p < 0.001) than their nw peers but were not different from each other (table 1). adolescents with type 2 diabetes were older with more advanced pubertal stages than the other two groups. as expected, hba1c and fasting glucose were higher in the subjects with type 2 diabetes compared with the other two groups (table 1). fasting plasma insulin was higher in the ob and diabetic subjects compared with the nw subjects. fasting glycerol ra, ffa ra, and fox were significantly higher in ob and adolescents with type 2 diabetes compared with nw adolescents, but were not different from each other. the significant differences among the groups in glycerol ra and ffa ra disappeared after adjusting for bmi and tanner stage with no change in the other variables. the difference in fox between nw and ob subjects disappeared after adjusting for bmi and tanner stage. fasting ffa levels did not differ among the three groups despite rates of whole body lipolysis in ob and adolescents with type 2 diabetes that were double that of nw youth. during the hyperinsulinemic - euglycemic clamp, is was significantly lower in ob and diabetic youth compared with nw and in adolescents with type 2 diabetes compared with ob when expressed per fat - free mass. plasma very long- and long - chain acylcn (c18:2-cn to c14:0-cn), derived primarily from the entry of fatty acids into -oxidation, were similar in all three groups (supplementary table 1). plasma short- and medium - chain acylcn species were lower in youth with type 2 diabetes compared with nw, with a trend toward lower values in the ob subjects. specifically, c10:1-, c10-, and c6-cn, fatty acid derived intermediates, and c2-cn, the last product of fatty acid oxidation, were lower in youth with type 2 diabetes compared with nw and ob subjects (fig. 1). in addition, both c3-cn and c5-cn, intermediates derived from bcaa oxidation, were significantly lower in the diabetic group compared with nw. similarly, c4-cn, a short chain species that derives from both the latter stages of fatty acid oxidation and from the bcaa valine, was lower in the group with type 2 diabetes relative to nw. lastly, free carnitine (freecn) was lower in adolescents with type 2 diabetes compared with nw, with a similar tendency (p < 0.083) in ob. plasma free carnitine and acylcn concentrations in nw, ob, and type 2 diabetic (t2 dm) subjects. bonferroni post hoc analyses for significant (p < 0.05) differences between any two groups are indicated with the same letter. plasma amino acids were lowest in adolescents with type 2 diabetes compared with both ob and nw adolescents (supplementary table 2). fasting plasma concentrations of neutral bcaa including leucine and isoleucine (which in ms / ms appear together at the same mass) and valine were lower in adolescents with type 2 diabetes compared with both the nw and ob subjects (fig. the other neutrally transported amino acids, phenylalanine and methionine, exhibited similar patterns, with the last member, tyrosine, trending in that direction (this method does not report tryptophan values). similarly, the diabetic subjects had lower levels of the gluconeogenic amino acid alanine relative to the other two groups. in the ob and diabetic youth, the basic amino acids, histidine and arginine, were lower relative to the nw subjects, as was plasma serine (fig. plasma citrulline was also reduced in diabetic adolescents, paralleling the decrease in arginine and refuting any possibility that the arginine reduction results from enhanced nitric oxide synthesis. when data were analyzed for acylcn and amino acids for each sex separately, the trends were similar with variable statistical significance (data not shown) because of the limited number of subjects in each group. plasma amino acid concentrations in nw, ob, and type 2 diabetic (t2 dm) subjects. p anova among the three groups is shown above each species. bonferroni post hoc analyses for significant (p < 0.05) differences between any two groups are indicated with the same letter. however, these relationships disappeared after adjusting for bmi, tanner stage, and sex. amino acids valine, phenylalanine, methionine, tyrosine, alanine, histidine, and serine showed a negative association with rates of fox (table 2). however, after adjusting for bmi, tanner stage, and sex these relationships disappeared. similar results were observed upon adjustment for % bf or vat differences (data not shown). significant correlations of acylcn and amino acids with fasting fox, bmi, and is from the hyperinsulinemic - euglycemic clamp is was positively associated with freecn, c6-cn, and c10:1-cn (table 2). the relationships between is and acylcn species disappeared after adjustment for adiposity indices (bmi, % bf, or vat), tanner stage, and sex. all amino acids, except for phenylalanine, methionine, tyrosine, and alanine, were positively associated with is. the relationship between is and arginine, histidine, serine, and glycine remained significant after adjustment for adiposity measures, bmi or % bf, or vat, tanner stage, and sex. as expected, ob and diabetic subjects had body composition indices that were all significantly higher (p < 0.001) than their nw peers but were not different from each other (table 1). adolescents with type 2 diabetes were older with more advanced pubertal stages than the other two groups. as expected, hba1c and fasting glucose were higher in the subjects with type 2 diabetes compared with the other two groups (table 1). fasting plasma insulin was higher in the ob and diabetic subjects compared with the nw subjects. fasting glycerol ra, ffa ra, and fox were significantly higher in ob and adolescents with type 2 diabetes compared with nw adolescents, but were not different from each other. the significant differences among the groups in glycerol ra and ffa ra disappeared after adjusting for bmi and tanner stage with no change in the other variables. the difference in fox between nw and ob subjects disappeared after adjusting for bmi and tanner stage. fasting ffa levels did not differ among the three groups despite rates of whole body lipolysis in ob and adolescents with type 2 diabetes that were double that of nw youth. during the hyperinsulinemic - euglycemic clamp, is was significantly lower in ob and diabetic youth compared with nw and in adolescents with type 2 diabetes compared with ob when expressed per fat - free mass. plasma very long- and long - chain acylcn (c18:2-cn to c14:0-cn), derived primarily from the entry of fatty acids into -oxidation, were similar in all three groups (supplementary table 1). plasma short- and medium - chain acylcn species were lower in youth with type 2 diabetes compared with nw, with a trend toward lower values in the ob subjects. specifically, c10:1-, c10-, and c6-cn, fatty acid derived intermediates, and c2-cn, the last product of fatty acid oxidation, were lower in youth with type 2 diabetes compared with nw and ob subjects (fig. both c3-cn and c5-cn, intermediates derived from bcaa oxidation, were significantly lower in the diabetic group compared with nw. similarly, c4-cn, a short chain species that derives from both the latter stages of fatty acid oxidation and from the bcaa valine, was lower in the group with type 2 diabetes relative to nw. lastly, free carnitine (freecn) was lower in adolescents with type 2 diabetes compared with nw, with a similar tendency (p < 0.083) in ob. plasma free carnitine and acylcn concentrations in nw, ob, and type 2 diabetic (t2 dm) subjects. bonferroni post hoc analyses for significant (p < 0.05) differences between any two groups are indicated with the same letter. plasma amino acids were lowest in adolescents with type 2 diabetes compared with both ob and nw adolescents (supplementary table 2). fasting plasma concentrations of neutral bcaa including leucine and isoleucine (which in ms / ms appear together at the same mass) and valine were lower in adolescents with type 2 diabetes compared with both the nw and ob subjects (fig. the other neutrally transported amino acids, phenylalanine and methionine, exhibited similar patterns, with the last member, tyrosine, trending in that direction (this method does not report tryptophan values). similarly, the diabetic subjects had lower levels of the gluconeogenic amino acid alanine relative to the other two groups. in the ob and diabetic youth, the basic amino acids, histidine and arginine, were lower relative to the nw subjects, as was plasma serine (fig. plasma citrulline was also reduced in diabetic adolescents, paralleling the decrease in arginine and refuting any possibility that the arginine reduction results from enhanced nitric oxide synthesis. when data were analyzed for acylcn and amino acids for each sex separately, the trends were similar with variable statistical significance (data not shown) because of the limited number of subjects in each group. plasma amino acid concentrations in nw, ob, and type 2 diabetic (t2 dm) subjects. bonferroni post hoc analyses for significant (p < 0.05) differences between any two groups are indicated with the same letter. fasting fox was negatively associated with freecn, c5-cn, c6-cn, and c10:1-cn. however, these relationships disappeared after adjusting for bmi, tanner stage, and sex. amino acids valine, phenylalanine, methionine, tyrosine, alanine, histidine, and serine showed a negative association with rates of fox (table 2). however, after adjusting for bmi, tanner stage, and sex these relationships disappeared. similar results were observed upon adjustment for % bf or vat differences (data not shown). significant correlations of acylcn and amino acids with fasting fox, bmi, and is from the hyperinsulinemic - euglycemic clamp is was positively associated with freecn, c6-cn, and c10:1-cn (table 2). the relationships between is and acylcn species disappeared after adjustment for adiposity indices (bmi, % bf, or vat), tanner stage, and sex. all amino acids, except for phenylalanine, methionine, tyrosine, and alanine, were positively associated with is. the relationship between is and arginine, histidine, serine, and glycine remained significant after adjustment for adiposity measures, bmi or % bf, or vat, tanner stage, and sex. the primary focus of this study was to determine whether or not obese youth with or without type 2 diabetes show the fasting plasma metabolic signatures of elevated amino acid and medium- to short - chain acylcn species reported in adults. our findings demonstrate that the general trend, in both the ob and the diabetic youth, is for lower plasma short- and medium - chain acylcn with unchanged long - chain acylcn, in addition to lower plasma concentrations of most amino acids. thus, both the ob and the diabetic youth showed no evidence of defects in fatty acid or amino acid metabolism compared with their nw peers, despite being insulin resistant. obese adults with or without type 2 diabetes are characterized by dysregulated fatty acid and amino acid metabolism. recent investigations have applied comprehensive metabolomic profiling to gain a broader understanding of the metabolic differences between lean, obese, and diabetic adults. we and others recently demonstrated elevations in acylcn and amino acid concentrations in obese compared with lean (3,9), in adults with diabetes compared with nondiabetic individuals (68), and in lean insulin - resistant compared with lean insulin - sensitive adults (5). on the basis of these observations of altered fatty acid and amino acid metabolism in adults, we hypothesized that adolescents with type 2 diabetes would have higher concentrations of specific acylcn species and of specific plasma amino acids when compared with their ob and nw peers. in contrast to adults, our results show that adolescents with type 2 diabetes have lower concentrations of specific fatty acid and amino acid derived acylcn species along with lower plasma amino acid concentrations. the lower concentrations of fatty acid derived acylcn species observed in our diabetic adolescents are unlikely to be attributable to greater fatty acid reesterification because fasting ffas and the percentage of ffas reesterified were comparable among all three groups (data not shown). also, the concentrations of long - chain acylcn species (c18:2-cn to c14:0-cn) were comparable among groups, suggestive of similar inputs into -oxidation. additionally, the ratio between freecn and c16-cn, which is increased when carnitine palmitoyltransferase-1 function is blocked, did not differ among groups (nw, 761.0 48.9 ; ob, 820.0 64.2 ; and type 2 diabetes, 735.4 91.5 ; p = 0.69). yet, the presence of lower concentrations of the later -oxidation intermediates (c10:1-cn to c2-cn) in the diabetic subjects is consistent with enhanced rather than reduced utilization of these intermediates. these observations from metabolomics are consistent with the in vivo indirect calorimetry findings of increased fox and vo2 in youth with type 2 diabetes compared with nw, whether expressed in absolute terms or controlling for fat - free mass (data not shown). additionally, the lower amino acid concentration with the concomitant decreases in amino acid associated acylcn species (c3-cn, c4-cn, and c5-cn) derived from the catabolism of bcaa also suggests enhanced utilization through -oxidation. on the other hand, the lower plasma bcaa may be due to increased gluconeogenic drive typically seen in diabetes, with the bcaa being channeled to fuel gluconeogenesis through conversion to glutamate, pyruvate, and alanine. additionally, increased fat mass and higher rates of lipolysis in obesity and diabetes contribute increased glycerol which fuels gluconeogenesis (24) and may contribute to the lower bcaa concentrations being utilized as gluconeogenic substrates. the observed lower alanine concentrations in type 2 diabetic youth is in favor of increased gluconeogenic drive. lastly, a reduction in amino acids might also reflect decreased catabolism or increased anabolism in growing adolescents. we had shown that proteolysis and protein oxidation are lower during puberty compared with prepuberty in normal weight youth (25). even though data are lacking in obese youth, it is possible that obesity - associated hyperinsulinemia may further augment this together with their higher dietary intakes. on the other hand, there are data suggesting that bcaa uptake by adipose tissue is reduced in an adult transgenic moderately obese mouse model (26). it remains to be determined if such is the case in human obesity and whether or not there are differences in adipose tissue bcaa uptake between growing adolescents versus adults. although further studies are required to clarify the underlying mechanism(s) for these metabolic differences in youth, mitochondrial adaptation and metabolic plasticity early in the course of obesity in youth may be a likely explanation. similar adaptive mitochondrial responses were identified in a serial study of zucker diabetic fatty rats where the animals initially upregulated their oxidative capacity in the face of progressive insulin resistance (27). yet, the upregulated oxidative capacity was unable to stop the diabetic progression, and with age, the mitochondrial adaptive response was lost. consistent with our data, a recent investigation in young adults (mean age 22 years) demonstrated comparable long - chain acylcn species but lower medium - chain acylcn species in obese compared with lean individuals (28). these results support the hypothesis that adolescents and young adults with obesity and type 2 diabetes have not yet developed the mitochondrial defects that are documented in older adults (29,30), and they may even have enhanced mitochondrial activity as an adaptation. in support of the latter are previous reports of increased fox in obese children, proposed to be a metabolic defense against further weight gain (31,32). thus, with the present findings of enhanced rates of -oxidation in ob and diabetic youth, we propose that their mitochondrial function is not impaired. however, over time and with continued progressive obesity from childhood to adulthood, chronic exposure to excessive -oxidation results in mitochondrial overload (33) and oxidative stress, culminating in a reduced overall oxidative capacity, similar to the changes found in diet - induced insulin - resistant mice (34). our diabetic youth compared with nw were unable to suppress their fox during hyperinsulinemic conditions (30 vs. 70%), resulting in continued exposure and demand for excessive -oxidation. moreover, it remains to be determined if the progression to abnormal mitochondrial function could be prevented or aborted. we further propose, based on the inverse relationship between is and fox (r = 0.502, p < 0.001), that the increased rates of fox in youth with obesity and diabetes may play a role in their insulin resistance through the randle cycle of the competition between fatty acid and glucose oxidation (35). we previously demonstrated that the randle cycle is operative in pubertal adolescents, potentially explaining the physiology of pubertal insulin resistance (14,36). lastly, a cluster of obesity - related amino acid and acylcn metabolites have demonstrated an inverse relationship with is among adults. (3) observed associations between bcaa and insulin resistance. likewise, tai. (5) reported that insulin resistance was associated with leucine / isoleucine, phenylalanine, tyrosine, and alanine, and a cluster of bcaa and related amino acids identified by principal components analysis. thus, our second aim was to assess the relationship between is or fox with acylcn species or amino acid concentrations. we found that once we accounted for the level of adiposity (bmi, % bf, or vat), tanner stage, and sex, the relationship between acylcn species and is or fox disappeared. furthermore, the observed correlations between bmi and is and fox (table 2), suggest that the acylcn associations are mediated by obesity in our pediatric population. in contrast, the relationship between is and arginine, histidine, serine, and glycine concentrations remained significant after controlling for adiposity, tanner stage, and sex. the strengths of the current study are 1) the number of otherwise healthy nw and ob adolescents and youth with type 2 diabetes ; 2) the extensive investigations that included the state - of - the - art use of stable isotopes together with indirect calorimetry and the hyperinsulinemic - euglycemic clamp to assess in vivo lipolysis, lipid oxidation, and is ; 3) the comprehensive evaluation of whole body and abdominal adiposity ; and 4) a first - time metabolomics analysis in youth. however, ethically, patients can not be evaluated without the provision of proper therapy. moreover, the differing treatment modalities were driven by standard clinical recommendations and not by a research protocol. in summary, our results show that in contrast to adults, ob youth with or without type 2 diabetes do not demonstrate impaired fatty acid or amino acid metabolism. in fact, the metabolomics and the in vivo data favor enhanced mitochondrial function, as obese adolescents with type 2 diabetes demonstrate lower concentrations of the later -oxidation intermediates along with higher rates of fox. it is our theory that these contrasting results between adolescents and adults is a reflection of the duration of obesity and the consequent and gradual evolution of failure of mitochondrial adaptive mechanisms as the obese individual transitions from youth to adulthood and forward with continued obesity. future longitudinal studies of metabolomics will be required to test this theory and to determine if this early adaptive metabolic plasticity disintegrates over time and if intervention(s) could prevent such maladaptive progression into adulthood. | objectivewe compared acylcarnitine (acylcn) species, common amino acid and fat oxidation (fox) byproducts, and plasma amino acids in normal weight (nw ; n = 39), obese (ob ; n = 64), and type 2 diabetic (n = 17) adolescents.research design and methodsfasting plasma was analyzed by tandem mass spectrometry, body composition by dual energy x - ray absorptiometry and computed tomography, and total - body lipolysis and substrate oxidation by [2h5]glycerol and indirect calorimetry, respectively. in vivo insulin sensitivity (is) was assessed with a 3-h hyperinsulinemic - euglycemic clamp.resultslong-chain acylcns (c18:2-cn to c14:0-cn) were similar among the three groups. medium- to short - chain acylcns (except c8 and c10) were significantly lower in type 2 diabetes compared with nw, and when compared with ob, c2-, c6-, and c10-cn were lower. amino acid concentrations were lower in type 2 diabetes compared with nw. fasting lipolysis and fox were higher in ob and type 2 diabetes compared with nw, and the negative association of fox to c10:1 disappeared after controlling for adiposity, tanner stage, and sex. is was lower in ob and type 2 diabetes with positive associations between is and arginine, histidine, and serine after adjusting for adiposity, tanner stage, and sex.conclusionsthese metabolomics results, together with the increased rates of in vivo fox, are not supportive of defective fatty acid or amino acid metabolism in obesity and type 2 diabetes in youth. such observations are consistent with early adaptive metabolic plasticity in youth, which over time with continued obesity and aging may become dysfunctional, as observed in adults. |
idiopathic eruptive macular pigmentation (iemp) is a rare skin disorder characterized by the presence of asymptomatic, brown pigmented macules that involve the face, trunk and proximal extremities in children and adolescents. the first description of this condition was given by degos., these hyperpigmented macules gradually resolve over months or years without any residual pigmentation or scarring. we report a case of a 10-year - old girl who fulfilled all the criteria for this entity. a 10-year -old girl presented with asymptomatic brown macules over the trunk and proximal extremities, of three months duration. they progressively increased in number and size over a period of one month and became stable. the cutaneous examination showed multiple dark brown, discrete, round to oval macules and mildly elevated pigmented lesions over the anterior and posterior trunk and proximal extremities sparing the palms and soles [figures 1 and 2 ]. the individual lesion was 1 - 3 cm in diameter and the elevated lesions had a velvety appearance on the surface [figure 3 ]. the routine blood, urine and stool examinations, liver function, renal function and thyroid function tests revealed no abnormality. biopsy from elevated lesion on back showed acanthosis, moderate papillomatosis and uniformly prominent melanin in the basal layer of the epidermis with normal number of melanocytes [figure 4 ]. no new lesions or change in preexisting lesions was seen at six months of follow - up. dark brown discrete macules over the anterior trunk and proximal extremities characteristic macules on back velvety appearance of pigmented lesions acanthosis, papillomatosis and increased melanin in the basal layer (h and e, 45) iemp is a rare skin disorder characterized by asymptomatic, brown macules involving the neck, trunk and proximal extremities. though the first case was reported around 30 years back, the exact etiology and pathogenesis is still not known., in 1996 summarized the criteria for the diagnosis of this condition, namely (a) eruption of brownish - black, discrete, nonconfluent, asymptomatic macules involving the neck, trunk and proximal extremities in children and adolescents, (b) absence of any preceding inflammatory lesions, (c) no previous drug exposure, (d) basal layer hyperpigmentation of the epidermis with dermal melanophages without any basal cell damage or lichenoid infiltrate, and (e) normal mast cell counts. the youngest and oldest case reported in the literature is that of a one - year - old and a 50-year - old. the largest series of ten cases and nine cases have been described by jang. the differential diagnosis of iemp includes post - inflammatory hyperpigmentation, fixed drug eruption, urticaria pigmentosa, lichen planus pigmentosus and erythema dyschromicum perstans. iemp can be differentiated from these conditions by taking proper history and doing skin biopsy study. histopathologically, iemp shows acanthosis, basal layer hyperpigmentation of the epidermis with dermal melanophages without any basal cell damage or lichenoid infiltrate and normal mast cell count. some authors believe that iemp may be related nosologically to confluent and reticulate papillomatosis (crp) and eruptive acanthosis nigricans because both these conditions show histological findings of pigmented papillomatosis similar to our case. however, the clinical features and etiopathogenesis of these conditions are quite different. it is important to consider iemp in the differential diagnosis of pigmentary lesions as iemp is a self - resolving condition. the treatment of this condition is not required as spontaneous resolution of iemp is expected in a few weeks to few years. in our case, topical steroid was started for the first two weeks to speed up thinning of elevated velvety lesions but without any benefit. to include papillomatosis as one of the diagnostic criteria, more reports of iemp with papillomatosis | we present a case of an otherwise healthy 10-year - old girl who presented with asymptomatic brown macules over the trunk and proximal extremities, of three months duration. the cutaneous examination revealed multiple, dark brown, discrete, round to oval macules and a few mildly elevated lesions over the trunk and proximal limbs. the individual lesion was 1 - 3 cm in diameter and a few showed velvety appearance over the surface. darier 's sign was negative. the histopathological study from the velvety lesion showed acanthosis, papillomatosis and increased melanin in the basal layer. the upper dermis showed sparse perivascular infiltrate of lymphocytes without any dermal melanophages. it fulfilled the criteria for idiopathic eruptive macular pigmentation with additional histological finding of papillomatosis. |
methicillin resistant staphylococcus aureus (mrsa) is one of the most common pathogens responsible for hospital infections and, as recently discovered, also for community acquired infections. it can cause a broad spectrum of infections through local invasion, toxin mediated diseases to generalized infections. s. aureus is a bacterium commonly present in the human population and constant or part time carrier frequency in the nasal vestibule is estimated at 3060%,,,. therapeutic problems are mainly caused by infections with strains, which are resistant not only to methicillin (methicillin - resistant s. aureus, mrsa) and in consequence to all -lactam antibiotics but also to many other group of antimicrobial therapeutics. the presence of this pathogen enables its local distribution e.g. within a hospital ward and/or between hospital wards (hospital or healthcare acquired) ha - mrsa,,,,. when such a strain carries genes responsible for the resistance to many antibiotics, as in the in case of ha - mrsa strains,,,,, ; then it becomes a great problem both, therapeutically due to the limited number of antibiotics available, as well as economically due to the necessity of expensive drugs and the prolonged time spent in the hospital,,,,,,. the increase of costs is also a result of special procedure initiations needed for controlling the wide - spread of the pathogen, such as hygienic and isolation procedures, identifying the carrier and, next, its eradication,,. in some countries mrsa can constitute up to 80% of all s. aureus isolates in hospitals. in some polish hospitals the percentage of mrsa strains reached up to ~60%. the frequency of bacteremia of this etiology, according to research carried out in the years 19992000 in europe, depending on the country ranged from 44.4% (greece) to 0.6% (denmark and the netherlands). according to the research of sentry carried out in the years 19971999 in usa, canada, latin america, and western pacific, the percentage of infection in these areas was 25.3 ; 19.2 ; 20.6 and 21.6, respectively. in our study we have analyzed the impact of modified procedures on epidemiological situation of mrsa during the last three years comparing two similar hospitals in northern poland. gdansk is the city that lies on the southern edge of gdansk bay (of the baltic sea), in a conurbation with the city of gdynia, spa town of sopot, and suburban communities, which together form a metropolitan area called the tricity (trjmiasto), with a population of over 800,000. gdansk itself has a population of 455,830 (june 2010), making it the largest city in the pomerania region of northern poland. the 608-bed regional hospital contains three internal departments, cardiology, neurology, pediatric ward, surgery, orthopedic, icu (adult), icu neonatal, obstetrics and gynecology, neonatology, laryngology, ophthalmic ward and dialysis unit. the yearly admittance rate and average hospitalization time are presented in table 1 (tab. 1) and table 2 (tab. population of the citizens with access to this hospital is estimated on the level of 205,000. bacteriology lab has an access to the analytical software whonet (who) and vitek (biomerieux, france). the modified epidemiological procedure concerning mrsa carriers and patients (procedure 1) has been implemented within the hospital. procedure 1 is characterized by the following criteria : epidemiological procedure concerning initial mrsa, visa or vrsa isolation.patient suspected of being infected (colonized) with mrsa, should be placed in a separate room, may be provided with other patients who have had the presence of a strain.the patients prescribed to eradication treatment should not be cohorted. the epidemiological investigation is being performed to determine the origin of the strain.in the case of hospital - acquired infections, the high - risk patients are being screened with microbiological tests (nasal - throat, respiratory tract and rectal isolations).the employed medical staff is trained over the prophylaxis of mrsa, visa and vrsa infections.the verification of the established epidemiological procedures is performed. patient suspected of being infected (colonized) with mrsa, should be placed in a separate room, may be provided with other patients who have had the presence of a strain. the epidemiological investigation is being performed to determine the origin of the strain. in the case of hospital - acquired infections, the high - risk patients are being screened with microbiological tests (nasal - throat, respiratory tract and rectal isolations). the employed medical staff is trained over the prophylaxis of mrsa, visa and vrsa infections. koszalin is the largest city of middle pomerania in north - western poland, possess a county - status city and is a capital of koszalin county of west pomeranian voivodeship since 1999. population of the citizens with access to this hospital is estimated on the level of 650,000 and koszalin itself has a population of 107,217 (2009). the 609-bed regional hospital contains two internal wards, cardiology, neurology, oncology, infectious diseases ward, pediatric, children surgery, general surgery, orthopedic, icu (adult), icu neonatal, obstetrics and gynecology, neonatology, laryngology, ophthalmic ward, dermatology and dialysis unit. the modified epidemiological procedure concerning mrsa carriers and patients (procedure 2) has been employed implemented in the hospital. procedure 2 is characterized by the following criteria : procedure of treatment of patients suspected of mrsa infection, infected (colonized) with mrsa (methicillin resistant staphylococcus aureus), vrsa (vancomycin resistant staphylococcus aureus), vre (vancomycin resistant enterococci) refers to medical staff, supporting staff, staff of hospital hygienepatient suspected of being infected (colonized) with mrsa should be placed in a separate room, may be provided with other patients who have had the presence of a strainroom equipped with items and equipment as the standard isolation of infections spreading through direct contactindicated the use of single sheetsin the case of reusable application, one should proceed as in terms of dirty sheetsroom doors must be closedmedical and hospital hygiene staff are informed on the reason of isolationone entering the room should put ondisposable protective apron, shoe pads, cap on headdisposable non - sterile gloves for nursing activities, sterile for aseptic operationssurgical mask in case of carrying of mrsa / vrsa / vre in patient 's airwaybefore leaving the room take off personal protective equipment and place in red bagbefore performing the steps, in the course if necessary, and upon completion, the procedure for hygienic hand - washing appliesmandatory reporting by consultants (visiting family) to a designated nurse to obtain information about safety precautionsavoidance of unnecessary traffic in the hall of the isolation wardrestriction of movements of the patient in the ward and outside full information about the precautionary measureswasted disposable equipment, dressings, etc. are subject to proceedings according to the instruction of medical wastereusable equipment is subject to disinfection processes, cleaning and sterilizationdealing with surfaces contaminated with biological material in accordance with the procedure for the safety and handling of infectious materialhygienic treatment of patient : the entire body must be washed daily with an antiseptic for this purpose, hair shampoo 2x a weekthe patient 's bedding and linen change every day, following the steps on - bed bedside management of patientcleaning the room twice a day and depending on the needsin order to identify carriers and patients infected with mrsa / vrsa material for microbiological examination should be collected from the nasal vestibule or perineum, or groin, or areas of the affected skin ; material for a directed search towards vre identification should be sampled from the anal area (or a stool sample) or the perineum, lesions of the damaged skin or the cathetered patient 's urine samplepatients, who have had contact with microbiologically diagnosed mrsa / vrsa / vre infected patient or mrsa / vrsa / vre carrier, should be cohortedif a patient is diagnosed as mrsa / vrsa / vre carrier and if allowed by the clinical condition, the patient should be discharged with recommendations for further treatment aimed at eliminating the carrier state - control swabs should be taken for at least 5 days after the procedure eliminating carrier state ; it is advisable to obtain a 3-time negative resultsthe re - taking of the patient to the hospital or taking a patient previously hospitalized in other wards / hospitals, where endemic or epidemic presence of mrsa / vrsa / vre was recorded, it is advisable to carry out directed microbial diagnostics ; until the results are obtained, strict adherence to the principles of insulation of directly transmitted infections must be appliedstaff colonized with mrsa / vrsa / vre should be removed from contact with patients, until elimination of the carrier state ; before taking the job a microbiological control of the mrsa / vrsa / vre carrier state should be performed, especially in the personnel previously employed in hospitals where the mrsa / vrsa / vre were registered. procedure of treatment of patients suspected of mrsa infection, infected (colonized) with mrsa (methicillin resistant staphylococcus aureus), vrsa (vancomycin resistant staphylococcus aureus), vre (vancomycin resistant enterococci) refers to medical staff, supporting staff, staff of hospital hygiene patient suspected of being infected (colonized) with mrsa should be placed in a separate room, may be provided with other patients who have had the presence of a strain room equipped with items and equipment as the standard isolation of infections spreading through direct contact indicated the use of single sheets in the case of reusable application, one should proceed as in terms of dirty sheets room doors must be closed medical and hospital hygiene staff are informed on the reason of isolation one entering the room should put on disposable protective apron, shoe pads, cap on head disposable non - sterile gloves for nursing activities, sterile for aseptic operations surgical mask in case of carrying of mrsa / vrsa / vre in patient 's airway before leaving the room take off personal protective equipment and place in red bag before performing the steps, in the course if necessary, and upon completion, the procedure for hygienic hand - washing applies mandatory reporting by consultants (visiting family) to a designated nurse to obtain information about safety precautions avoidance of unnecessary traffic in the hall of the isolation ward restriction of movements of the patient in the ward and outside full information about the precautionary measures wasted disposable equipment, dressings, etc. are subject to proceedings according to the instruction of medical waste reusable equipment is subject to disinfection processes, cleaning and sterilization dealing with surfaces contaminated with biological material in accordance with the procedure for the safety and handling of infectious material hygienic treatment of patient : the entire body must be washed daily with an antiseptic for this purpose, hair shampoo 2x a week the patient 's bedding and linen change every day, following the steps on - bed bedside management of patient cleaning the room twice a day and depending on the needs in order to identify carriers and patients infected with mrsa / vrsa material for microbiological examination should be collected from the nasal vestibule or perineum, or groin, or areas of the affected skin ; material for a directed search towards vre identification should be sampled from the anal area (or a stool sample) or the perineum, lesions of the damaged skin or the cathetered patient 's urine sample patients, who have had contact with microbiologically diagnosed mrsa / vrsa / vre infected patient or mrsa / vrsa / vre carrier, should be cohorted if a patient is diagnosed as mrsa / vrsa / vre carrier and if allowed by the clinical condition, the patient should be discharged with recommendations for further treatment aimed at eliminating the carrier state - control swabs should be taken for at least 5 days after the procedure eliminating carrier state ; it is advisable to obtain a 3-time negative results the re - taking of the patient to the hospital or taking a patient previously hospitalized in other wards / hospitals, where endemic or epidemic presence of mrsa / vrsa / vre was recorded, it is advisable to carry out directed microbial diagnostics ; until the results are obtained, strict adherence to the principles of insulation of directly transmitted infections must be applied staff colonized with mrsa / vrsa / vre should be removed from contact with patients, until elimination of the carrier state ; before taking the job a microbiological control of the mrsa / vrsa / vre carrier state should be performed, especially in the personnel previously employed in hospitals where the mrsa / vrsa / vre were registered. gdansk is the city that lies on the southern edge of gdansk bay (of the baltic sea), in a conurbation with the city of gdynia, spa town of sopot, and suburban communities, which together form a metropolitan area called the tricity (trjmiasto), with a population of over 800,000. gdansk itself has a population of 455,830 (june 2010), making it the largest city in the pomerania region of northern poland. the 608-bed regional hospital contains three internal departments, cardiology, neurology, pediatric ward, surgery, orthopedic, icu (adult), icu neonatal, obstetrics and gynecology, neonatology, laryngology, ophthalmic ward and dialysis unit. the yearly admittance rate and average hospitalization time are presented in table 1 (tab. 1) and table 2 (tab. population of the citizens with access to this hospital is estimated on the level of 205,000. bacteriology lab has an access to the analytical software whonet (who) and vitek (biomerieux, france). the modified epidemiological procedure concerning mrsa carriers and patients (procedure 1) has been implemented within the hospital. procedure 1 is characterized by the following criteria : epidemiological procedure concerning initial mrsa, visa or vrsa isolation.patient suspected of being infected (colonized) with mrsa, should be placed in a separate room, may be provided with other patients who have had the presence of a strain.the patients prescribed to eradication treatment should not be cohorted. the epidemiological investigation is being performed to determine the origin of the strain.in the case of hospital - acquired infections, the high - risk patients are being screened with microbiological tests (nasal - throat, respiratory tract and rectal isolations).the employed medical staff is trained over the prophylaxis of mrsa, visa and vrsa infections.the verification of the established epidemiological procedures is performed. epidemiological procedure concerning initial mrsa, visa or vrsa isolation. patient suspected of being infected (colonized) with mrsa, should be placed in a separate room, may be provided with other patients who have had the presence of a strain. the epidemiological investigation is being performed to determine the origin of the strain. in the case of hospital - acquired infections, the high - risk patients are being screened with microbiological tests (nasal - throat, respiratory tract and rectal isolations). the employed medical staff is trained over the prophylaxis of mrsa, visa and vrsa infections. koszalin is the largest city of middle pomerania in north - western poland, possess a county - status city and is a capital of koszalin county of west pomeranian voivodeship since 1999. population of the citizens with access to this hospital is estimated on the level of 650,000 and koszalin itself has a population of 107,217 (2009). the 609-bed regional hospital contains two internal wards, cardiology, neurology, oncology, infectious diseases ward, pediatric, children surgery, general surgery, orthopedic, icu (adult), icu neonatal, obstetrics and gynecology, neonatology, laryngology, ophthalmic ward, dermatology and dialysis unit. the modified epidemiological procedure concerning mrsa carriers and patients (procedure 2) has been employed implemented in the hospital. procedure 2 is characterized by the following criteria : procedure of treatment of patients suspected of mrsa infection, infected (colonized) with mrsa (methicillin resistant staphylococcus aureus), vrsa (vancomycin resistant staphylococcus aureus), vre (vancomycin resistant enterococci) refers to medical staff, supporting staff, staff of hospital hygienepatient suspected of being infected (colonized) with mrsa should be placed in a separate room, may be provided with other patients who have had the presence of a strainroom equipped with items and equipment as the standard isolation of infections spreading through direct contactindicated the use of single sheetsin the case of reusable application, one should proceed as in terms of dirty sheetsroom doors must be closedmedical and hospital hygiene staff are informed on the reason of isolationone entering the room should put ondisposable protective apron, shoe pads, cap on headdisposable non - sterile gloves for nursing activities, sterile for aseptic operationssurgical mask in case of carrying of mrsa / vrsa / vre in patient 's airwaybefore leaving the room take off personal protective equipment and place in red bagbefore performing the steps, in the course if necessary, and upon completion, the procedure for hygienic hand - washing appliesmandatory reporting by consultants (visiting family) to a designated nurse to obtain information about safety precautionsavoidance of unnecessary traffic in the hall of the isolation wardrestriction of movements of the patient in the ward and outside full information about the precautionary measureswasted disposable equipment, dressings, etc. are subject to proceedings according to the instruction of medical wastereusable equipment is subject to disinfection processes, cleaning and sterilizationdealing with surfaces contaminated with biological material in accordance with the procedure for the safety and handling of infectious materialhygienic treatment of patient : the entire body must be washed daily with an antiseptic for this purpose, hair shampoo 2x a weekthe patient 's bedding and linen change every day, following the steps on - bed bedside management of patientcleaning the room twice a day and depending on the needsin order to identify carriers and patients infected with mrsa / vrsa material for microbiological examination should be collected from the nasal vestibule or perineum, or groin, or areas of the affected skin ; material for a directed search towards vre identification should be sampled from the anal area (or a stool sample) or the perineum, lesions of the damaged skin or the cathetered patient 's urine samplepatients, who have had contact with microbiologically diagnosed mrsa / vrsa / vre infected patient or mrsa / vrsa / vre carrier, should be cohortedif a patient is diagnosed as mrsa / vrsa / vre carrier and if allowed by the clinical condition, the patient should be discharged with recommendations for further treatment aimed at eliminating the carrier state - control swabs should be taken for at least 5 days after the procedure eliminating carrier state ; it is advisable to obtain a 3-time negative resultsthe re - taking of the patient to the hospital or taking a patient previously hospitalized in other wards / hospitals, where endemic or epidemic presence of mrsa / vrsa / vre was recorded, it is advisable to carry out directed microbial diagnostics ; until the results are obtained, strict adherence to the principles of insulation of directly transmitted infections must be appliedstaff colonized with mrsa / vrsa / vre should be removed from contact with patients, until elimination of the carrier state ; before taking the job a microbiological control of the mrsa / vrsa / vre carrier state should be performed, especially in the personnel previously employed in hospitals where the mrsa / vrsa / vre were registered. procedure of treatment of patients suspected of mrsa infection, infected (colonized) with mrsa (methicillin resistant staphylococcus aureus), vrsa (vancomycin resistant staphylococcus aureus), vre (vancomycin resistant enterococci) refers to medical staff, supporting staff, staff of hospital hygiene patient suspected of being infected (colonized) with mrsa should be placed in a separate room, may be provided with other patients who have had the presence of a strain room equipped with items and equipment as the standard isolation of infections spreading through direct contact indicated the use of single sheets in the case of reusable application, one should proceed as in terms of dirty sheets room doors must be closed medical and hospital hygiene staff are informed on the reason of isolation one entering the room should put on disposable protective apron, shoe pads, cap on head disposable non - sterile gloves for nursing activities, sterile for aseptic operations surgical mask in case of carrying of mrsa / vrsa / vre in patient 's airway before leaving the room take off personal protective equipment and place in red bag before performing the steps, in the course if necessary, and upon completion, the procedure for hygienic hand - washing applies mandatory reporting by consultants (visiting family) to a designated nurse to obtain information about safety precautions avoidance of unnecessary traffic in the hall of the isolation ward restriction of movements of the patient in the ward and outside full information about the precautionary measures wasted disposable equipment, dressings, etc. are subject to proceedings according to the instruction of medical waste reusable equipment is subject to disinfection processes, cleaning and sterilization dealing with surfaces contaminated with biological material in accordance with the procedure for the safety and handling of infectious material hygienic treatment of patient : the entire body must be washed daily with an antiseptic for this purpose, hair shampoo 2x a week the patient 's bedding and linen change every day, following the steps on - bed bedside management of patient cleaning the room twice a day and depending on the needs in order to identify carriers and patients infected with mrsa / vrsa material for microbiological examination should be collected from the nasal vestibule or perineum, or groin, or areas of the affected skin ; material for a directed search towards vre identification should be sampled from the anal area (or a stool sample) or the perineum, lesions of the damaged skin or the cathetered patient 's urine sample patients, who have had contact with microbiologically diagnosed mrsa / vrsa / vre infected patient or mrsa / vrsa / vre carrier, should be cohorted if a patient is diagnosed as mrsa / vrsa / vre carrier and if allowed by the clinical condition, the patient should be discharged with recommendations for further treatment aimed at eliminating the carrier state - control swabs should be taken for at least 5 days after the procedure eliminating carrier state ; it is advisable to obtain a 3-time negative results the re - taking of the patient to the hospital or taking a patient previously hospitalized in other wards / hospitals, where endemic or epidemic presence of mrsa / vrsa / vre was recorded, it is advisable to carry out directed microbial diagnostics ; until the results are obtained, strict adherence to the principles of insulation of directly transmitted infections must be applied staff colonized with mrsa / vrsa / vre should be removed from contact with patients, until elimination of the carrier state ; before taking the job a microbiological control of the mrsa / vrsa / vre carrier state should be performed, especially in the personnel previously employed in hospitals where the mrsa / vrsa / vre were registered. according to comparative analysis performed for two regional hospitals of northern poland with similar bed numbers (608 vs. 609) and similar ward profiles, the percentage of mrsa distribution was different. in 2008 at hospital no. 1 the rate of mrsa to all s. aureus isolates was 14.4% and at hospital no. however, in 2010, the rate was similar for the two described hospitals (table 2 (tab. the differences concern also the number of hospitalized patients, hospitalization time and number of microbiological tests performed (table 1 (tab. 1) and table 3 (tab. 25,000, the hospitalization time was 6.66.7 days with percentage of patients beds occupation of approx. 75%. at hospital 2 the number of hospitalized patients was higher ca. 31,000, nevertheless the average hospitalization time was shorter and was calculated as 4.64.9 days with lower percentage of patients beds occupation (62.368.2%). 1, the number of specimens/100 patients was as follows : in 2008 101.9, in 2009 110.7 and in 2010 122.7. at hospital 2 much smaller number of specimens/100 patients was performed in each year : 29.51, 32.1 and 45.6, respectively. in the last decade in poland, the monitoring of mrsa infections and carriers has been improved,. previously some health - associated centers introduced the survey, and epidemiological procedures concerning mrsa carriers and patients,,. currently, many hospitals in poland are involved within the european project ears - net (http://ecdc.europa.eu/en/activities/surveillance/ears-net/), what enables in future the unification of procedures and surveillance methods. nowadays, each medical center forms its own procedures on the basis of obtained results from epidemiological investigations. it is clearly visible that the comparison of even very similar hospitals is quite difficult. 2 in the same year in spite of a lower number of specimens send for bacteriological analysis, there were more isolations of s. aureus (533 vs. 478) and smaller number of isolation of mrsa with one outbreak in neurology ward (4 infected patients and 9 carriers). though the percentage of mrsa in 2010 year was slightly higher (6.5%), we have observed the decrease of mrsa hospital - acquired infections from 3 and 4 in 20082009 to 0 in 2010. it is interesting that in both hospitals the number of specimens send to bacteriology lab consequently increased whereas the frequency of mrsa isolations decreased. in our opinion this opinion could be supported by results from ears - net report where overall in poland mrsa isolation frequency in 2009 is 20% (http://ecdc.europa.eu/en/activities/surveillance/ears-net/). | in the present study we have analyzed the impact of modified mrsa screening of carriers and patients on epidemiological situation of mrsa during 20082010, comparing two regional hospitals with similar bed numbers and similar ward profiles in northern poland. in 2008 the proportion of mrsa to all s. aureus isolates was 14.4% resp. 6.0%, in 2009 8.3% resp. 4.7% and in 2010 6.5% in both hospitals. independent of the different prevention and intervention strategy in both hospitals the different mrsa incidence seems to be due to regional epidemic settings |
the history of psychiatry describes the medicalization processes and the mechanisms by which society has emarginated diversity and has always been imbued with the attitudes which mental health institutions and services have had towards the patients families. the advent of psychotherapy and psychoanalysis was the turning - point when theoretical and clinical medicine was first concerned with etiology of psychiatric disorders. this prompted a redefinition of the role of family and their importance in mental health care. as discussed by castel spirals of guilt are no longer constructed, the aim nowadays is rather to construct dialogues which support and promote the family as an entity capable of replicating and optimizing modern welfare systems. the role of family members has changed over the centuries, especially since the closing down of lunatic asylums, and this has led to responsibility for the patient being shared between the health service and the family network. this new scenario questioned the old concept of delegation to the custody of..., allegedly the only concrete answer to the family s perpetual oscillation between the need for assistance, the impossibility of a cure and the desire to get rid of a problem. now that the possibility of totally delegating the task of care to the health services is decreasing, the family begins to be defined as a resource for community psychiatric services. this different mindset, which views the family as an alternative to institutionalization, leads to it being seen as an essential entity in the setting up of community service dynamics. it is a new approach which is bound up with new care models and with the changes in the subjects involved. as discussed by saraceno there is a change in perspective, which the interviewees in our study, both the heads of the services and the workers, highlighted repeatedly ; the family is considered not only as an entity to be instructed and assisted, but also as an essential and necessary resource for the rehabilitation of the psychiatric patient. indeed, it is identified as one of the principal actors in the process and organization of care. the family s role is therefore redefined and it has gained importance to become the main focus for dialogue for mental health services. the new interactions between the mental health services and the family have led to different requests and, thus, different expectations, tools and resources. in the comparative literature on the welfare state, psychiatry assistance have represented a marginal theme. social policy scholars have in fact concentrated mainly on the core sectors of the welfare state. yet, the closure of the large mental hospitals in much of the western world has created the paradigmatic shift. it born a great deal of discourse about the experience between family members and services. in this field, moreover, research has tended to a large extent to focus on microsectoral aspects : family member needs and burden ; participation in decision - making and relationships with psychiatric workers. current research tends to focus on barriers between families and mental health services, such as staff attitudes and inadequate communication with family members. for example, slade. reported that family members were seen as pushy and demanding, prejudged if they tried to share information, and had to persevere in contacting services. alternatively, gray. found that family member were often discouraged from approaching services, and that they were seen as troublemakers and as part of the problem. gray interviewed sixty - five participants (directors, managers and senior staff from social, voluntary and healthcare organizations) who were encouraged to talk in detail about their understanding of family emotions. what emerged was a rich understanding of the broad spectrum of family members negative emotions (such as fear, anger and denial). participants noted a clear lack of emotional support for family carers, with accompanying feelings of marginalization, particularly during transitions and especially involving young family members as well as ethnic minorities. a year later, gray proposed drawing up a covenant between mental health services and family carers, one based on mutual obligations. he claimed that much of the relationship between families and services puts the interests of service users and providers ahead of family members and suggested that family members were hidden and invisible, trapped and isolated in narrow roles. therefore, a covenant would help clarify what professionals expect of carers ; and would ultimately improve engagement and relationships. in italy, the closing of lunatic asylums and the nationwide development of local services to treat people with psychiatric disorders and return them to the community have put these people back in their original social context and, especially, in their family. currently, the assumption is that much of psychiatry is oriented to the involvement of family members in the rehabilitation therapy of the patient. but, when family members considered to be collaborative ? currently, in italy lacks a literature that examines, in particular, the construction of the relationship between operators in psychiatric services, family and territory. the main objective of my research is to observe the intertwining of issues involving people with mental suffering, their families and services. the starting point is the concept of the family as a resource for the entire mental health service but : i) the sense of failure and denial of illness which often creates an obstacle to a collaborative approach ; ii) another obstacle to collaboration lies in the latent sense of guilt in family members ; iii) obstacle to collaboration could derive from some staff behavior and not only from the family with its problems. from this final point of view, collaboration is something that mental health services and family should approach from a position of parity and symmetry, with both sides reacting to the other s behavior and attitudes. hence, it becomes evident that those in charge should attribute positive worth to every sector of activity carried out within it, so as to make the unit work in function of the patients needs and the workers job satisfaction. it should not be overlooked here that the building up of relationships with the family, or with family associations, could, first of all, make it easier to listen to people who, though they may lack specific technical or scientific knowledge, are still people, to cite creer with a wealth of experience which can be of use. this experience should be exploited, since no system of treatment can work adequately and efficiently without complete collaboration from family members, without their first - hand knowledge and without a mutual sharing of the task. the interviews showed that the problems to be investigated were predominantly in the interpersonal sphere, that people s subjective impressions carry great weight, that society s interpretations of mental illness are significant and that everyone has their own personal cultural viewpoint. structured interviews were conducted among 26 health professionals that included psychiatrists, psychologists, nurses, rehabilitation professionals, social workers and head nurses. the study was carried out at the public mental health services in rome, also because there were links there with another, wider - ranging research project (it is an interdisciplinary research project drawn up by dr. maone, head of the via sabrata community, romea, with the participation of prof. cammarota of the university of messina and antonella torre, an operative in the mental health services). the starting point is the concept of the family as a resource for the entire mental health service ; a concept expressed across the whole range of interviews. these parents who have to deal with their son or daughter s schizophrenia are usually no longer young and may have some physical problems of their own, since onset of schizophrenia usually occurs between the ages of 20 and 30. these mothers and fathers will all tend to have different ways of facing up to the difficulties and of expressing their pain. in some cases, in the absence of parents, brothers and sisters will come. given that nowadays the majority of these services recognize the importance of family involvement by drawing up formal intervention plans, when family members considered to be collaborative ? but we are always looking for collaboration... sometimes you find the family in agreement and willing to collaborate. but the important thing is to build up a relationship with the family and proceed. (galia, psychiatrist, csm romed) well... yeah... at first there is some reluctance []. i must say that... mainly it s reluctance connected with the fact that, when parents send a son or daughter to us, they are unconsciously in competition with the community,... because, in a way, the separation, entering the community is nt something they view... it s seen as a problem, there s a sense of failure. (leopold, psychiatrist, community romeb) galia is psychiatrist in the a mental health centres, while leopold is a psychiatrist in a community which frequently works with family members, also using multi - family groups. galia thinks that the essential starting point for a collaborative approach with the family is for them to express agreement with therapy and treatment. non - acceptance by the family of the specific nature of their relative s disorder becomes an obstacle. galia makes it clear immediately that, even when this expression of agreement is not there at first and the family has an attitude of opposition or ambivalence, the possibility of building up a relationship with the family is still a priority for the mental health service. galia thinks that a collaborative approach is connected to how and whether family members accept the patient s disorder. the need to obtain the family s agreement to therapy and treatment is seen as something in which the service must invest energy and resources. leopold too talks about reluctance on the part of some family members which often becomes an obstacle to collaboration with the service and which is usually a consequence of a sense of failure or impotence. yet, taking a longer view, leopold makes it clear that, even when there are difficulties at first, the results can be amazing if pathways to mutual understanding and collaboration are created with the family. making the effort to work with the family brings gratification and excellent results, once the initial difficulties are overcome. an essential ingredient for a collaborative approach is acceptance on the part of the family of their relative s situation. these roots spread back to the process of stigmatization which society has applied to mental illness, deriving from the centuries - old view of madness as something frightening. only a few studies take into account this stigmatization process, which involves people close to psychiatric patients such as family members. they too become victims of the social exclusion strategies which lead to a sense of shame and isolation in addition to their suffering and despair. as discussed by hatfield, unlike other unfortunate events which can occur in the private family sphere, such as death or a physical illness, in the case of psychiatric illness sometimes the structures of socially formalized assistance and sympathy seem to be lacking. the sense of failure and denial of illness which often creates an obstacle to a collaborative approach, here are the stories of some events experienced by other staff : a personal memory of mine was [] a widowed mother with a schizophrenic son, the schizophrenia was nt responding to medication [] this boy caused a lot of problems, in his neighbourhood and in his family. [... ] when you set a sectioning process in motion, with signed documents, the police, two doctors and all the rest, well that s a big thing. when they got to the house, the mother would say : no, he s all right now []. this is an example of how a parent can be passive and aggressive at the same time, first they ask for help and then they refuse the help. then you get the parents who ca nt be collaborative because they are psychotic themselves. (mustaf, psycologist, community romeb) mustaf here illustrates the ambivalent attitude, present not only in this specific case, which causes parents to swing perpetually between fear and love, between despair and a search for protection. this attitude can be seen as a consequence of the fear of the son or daughter s reaction when the sectioning procedure is set in motion, but there is probably also fear of being abandoned by the institution which assists them. in the light of this, the road to information and awareness that the mental health services constructs with the family is an essential prerequisite for a relationship of trust. it is prevalently the chief nurses who express a concept of collaborative care which implies practical and concrete participation, to assist the mental health services. if we ask them to..... well, say.... to act in a certain way, like do nt come too often or please phone before you come, you know, not turning up unexpectedly without saying they re here..... they must nt go into the rooms..... and they do as we ask [... ] everybody has stuff to do, sure... but the patients are nt just parked here, we do require basic co - operation. [... ] it s true that we all have things that take up our time, but if you really want to, you can make time for something [] (ellen, head nurse, community romeb) we do believe that parents and family are of great value sometimes they can reach places we do nt, for example... if a patient is reluctant to take medication, a parent or other family member who has been well instructed can be a great help to us. also, if we give health education and explain what the early symptoms of a crisis can be, then sometimes we can avoid hospitalization... that means they also have an actual financial value, in terms of costs to society. (crisanta, head nurse, csm romed) in these cases yes, having contacts with the family that helps in giving medication ; i have the prescriptions written out by the doctor and then i give them to the family..... sometimes a mother will telephone to say look, he did nt take his medication today. (isabel, nurse, csm romea) ellen, who works in a residential community, says that the kind of collaboration that the mental health services are often obliged to ask the family for, because they lack either the staff or the time, is everyday tasks such as health checks with the doctor. when the family refuses to do this, ellen interprets it as an offloading of the patient by the family onto the mental health services. ellen s worry is that a residential community could become, in the eyes of some people, a sort of alternative to the old lunatic asylums. for ellen, non - abandonment and this kind of participation is a yardstick for measuring family collaboration. the community where ellen works does in - depth work with the patients families, both to have immediate support during treatment and to recreate relationships with an eye on the future. crisanta sees the collaborative assistance which is requested from the family as the services need for an extra eye, which checks and monitors the situation outside the structures of the service itself. the family thus takes on an active role in the therapeutic process and its management, as well as being an external support for the service. for this to be successful, however, it is necessary to work with the family, by giving them instruction and information about the warning signal symptoms which precede a crisis and about the importance of the patient following correct methods of medication. isabel, like ellen and crisanta, helps to clarify the difficult points where the services can not be effective on their own. in her view, family participation and collaboration is essential as an extension of the services efforts, as something which enables them to work better. what isabel actually makes clear is that, in spite of limited contact with family members and the difficulties encountered with them, the services real need and proper attention towards the patient are elements which can create a scenario where collaborative work with the family is a real possibility. it is clear so far that the majority of the workers interviewed in this study, even those who technically have less contact with the family, see collaboration as something which has to be constructed together. this attitude is also found among the psychologists : i mean, family collaboration can become something more than just..... i ll try to check that my son or my wife takes the medication... and you get the family noticing behavior which is completely different from usual and telling us about it []. very, very often there s denial ; some families say oh no, you say he s ill, but he s not.... (magda, psychologist, csm romed) a collaborative parent is one who says : doctor, i ve noticed that my son is doing this or doing that, maybe it has significance for you. then maybe i ll call them back to say : you were right, it was a good thing you pointed that out to me because it let me work on that aspect. intrusive is when they say : look here, you have to see that things are just not going right here. [] they criticize our work, the way we go about it ; they say you do nt understand the kid, i d better explain him to you. [they do it ] as a defense strategy [] (beverly, psychologist, csm romed) the family may be non - collaborative for two reasons : first when they are disturbed themselves ; and second, when the medical staff do nt create an alliance because, in their own minds, they believe that the parents are actually responsible for the disorder. (mustaf, psychologist, community romeb) magda and beverly are both psychologists in the same mental health centre, yet here are two people, in the same structure with the same professional role, who define collaboration differently. magda is one of the few interviewees who emphasizes the importance of collaboration viewed first and foremost as a journey to be made with the family. its basis is the ability and the need, not just of the service as a whole, but of each individual worker, to create a relationship with the family, through meetings and continuous dialogue. she believes that is the only way to obtain a type of collaboration which gives support to both the patient and the service. magda also thinks that non - acceptance of the patient s disorder is an obstacle to construction of collaborative relations with the family. in her view, it is necessary to make every effort to surmount this obstacle as soon as it appears. beverly makes it clear that she only considers the family collaborative if there is an a priori respect for the workers professional training and knowledge, with no intrusiveness ; in other words, the family should have implicit faith in the professionals opinion. here a detailed outline of the actions and the practical interventions necessary for a definition of collaborative is not given, just an indication of the attitude required by the worker of the family to facilitate its involvement in the care program drawn up for the patient : the attitude consists in respect for the healthcare professional. while beverly too sees family collaboration as a support for treatment, in her opinion an obstacle to collaboration lies in the latent sense of guilt in family members. thus, magda and beverly both consider a successful collaborative process with the family to be an effective tool for the worker in treatment of the patient, but have different opinions as to what constitutes an obstacle to its creation. on the one hand there is a belief in the possibility of creating collaborative pathways, also by overcoming human failings, but on the other the building of bridges seems to be compromised by the massive sense of guilt that families have. the constraints on collaboration created, in beverly s opinion, by the sense of guilt, which leopold also spoke of using the word failure, are viewed from an opposite point of view by mustaf. he observes what could be a mistaken attitude on the part of the mental health workers ; the apportioning of blame. mustaf is the only interviewee who, on identifying an obstacle to collaboration, suggests that it could derive from some staff behavior and not only from the family with its problems. from this point of view, collaboration is something that mental health services and family should approach from a position of parity and symmetry, with both sides reacting to the other s behavior and attitudes. the study did not reveal a common way of interpreting collaboration between mental health services and patients families. indeed, the interviews contained various points of view which, in most cases, tended to neglect the way workers should be and concentrated on what the family should do. even though the various healthcare professionals have different ideas about collaboration, practically all the mental health services in rome included in the study intended to continue to attempt collaboration with the family. they were aware, however, that many obstacles can be encountered : i) the sense of failure and denial of illness which often creates an obstacle to a collaborative approach ; ii) another obstacle to collaboration lies in the latent sense of guilt in family members ; iii) obstacle to collaboration could derive from some staff behavior and not only from the family with its problems. the study also shows that, in spite of all the structures included in the study having specific guidelines for ways of meeting with families, the actual relationships vary not only from one structure to another, but also between the staff members in the same structure. one of the main contradictions that emerges is the fact that in some structures, despite there being, in theory, a common code for collaborative action with family members, a grey area appears, within which individual workers have their own interpretations of what this actually involves. in the light of this, the tools and resources that the structure actually employs in practice are just as important as the theory in mediation of the relationship with the family. it was also observed that, very frequently, when the mental health service gives added value to all types of worker by actively involving them in work with family members, then each individual worker has a higher opinion of the worth of their job. this leads to an improved capacity for empathizing with the family, which the worker sees as needing support and assistance, as well as being able to give these things. seeing things from this perspective would mean that the mental health services must create places where workers and family can meet and establish a dialogue, so as to deepen the understanding of the daily difficulties that the family has to face. in conclusion services should create moments, such as multi - family groups or groups of information, managed by nurses and not only by doctors. | the family s role in patient care was greatly altered by law 180. this law, introduced in italy in 1978, led to a gradual phasing out of custodial treatment for psychiatric patients. this different mindset, which views the family as an alternative to institutionalization, leads to it being seen as an essential entity in the setting up of community service dynamics. we interviewed health professionals in order to understand obstacles of collaboration between family members and mental health care workers. the goal was to uncover actions that promote collaboration and help build alliances between families and psychiatric workers. results showed that health professionals view the family as a therapeutic resource. despite this view, family members were rarely included in patient treatment. the reasons is : the structures have a theoretical orientation of collaboration with the family but, for nurses not are organized a few meeting spaces with family members. services should create moments, such as multi - family groups or groups of information, managed by nurses and not only by doctors. these occasions it might facilitate the knowledge between professionals and family members. |
we followed the prisma (preferred reporting items for systematic reviews and meta - analyses) checklist for reporting systematic reviews and meta - analyses (6). we systematically searched medline, embase, central, cinahl, and web of science through october 2011 for studies in humans of the association between metabolic syndrome and cancer. our core search consisted of the terms metabolic syndrome, insulin resistance syndrome, and syndrome x, combined with specific terms for each cancer site : colorectal (colon and rectum), gastric, esophageal, hepatobiliary (liver and gallbladder), pancreas, lung, bladder, thyroid, renal, leukemia, malignant melanoma, multiple myeloma, and non - hodgkin lymphoma for both sexes and prostate, breast, ovary, and endometrium for single sex. relevant journals, bibliographies, reviews, and personal files were hand searched for additional articles. we included studies if 1) their aim was to assess the effect of metabolic syndrome on risk of cancer or association with cancer, 2) they reported the definition of metabolic syndrome according to criteria of national or international scientific associations, federations, or organizations (traditional definitions) or if they used proxy indicators in the absence of the original data (nontraditional definitions), and 3) they included at least three factors, even in the absence of others. we included cohort studies, nested case - control studies, control arms from clinical trials, case - control studies, patient series, and mortality studies. we specified that every study must either report risk estimates (relative risks [rrs ], odds ratios, hazard ratios, and standardized incidence ratio) with 95% cis separately for men, women, or both or must report sufficient data to estimate these. if a site - specific dataset had been published more than once, we used the most recent publication. we included a specific cancer site in the analysis if there were at least two cohort datasets. we excluded studies that were not published as full reports, such as conference abstracts and letters to editors, and studies of cancer precursors (e.g., colorectal adenoma). from each retrieved article, we extracted the following data : name of the first author, year of publication, country where the study was performed, specific outcomes, follow - up time, proportion of men and women, total number of individuals, number of cases, and risk estimates and their 95% cis (presence versus absence of metabolic syndrome)., k.e., and p.c.) until interrater reliability (0.60) was established. methodological quality of each study was assessed according to three study components that might affect the strength of the association between metabolic syndrome and cancer risk : length of follow - up for cohort studies, whether metabolic syndrome definition was traditional or nontraditional, and the extent of adjustments for potential confounding factors. we also collected, where available, risk estimates of the association with cancer for each single factor of the syndrome taken at its highest level. the primary end point was to assess the association between metabolic syndrome and cancer risk in cohort studies. for the main outcome at each cancer site, we graded the evidence for study quality and for the risk of bias : study quality was based on the number of datasets, number of events, width of cis, and heterogeneity ; risk of bias was mainly based on type of study and adjustment for confounders. heterogeneity of the effect across studies was assessed by q statistics, which is distributed as statistics (7). a value of p 0.1 ; otherwise, we used a random - effect model. we did subgroup analyses for each site to identify study - level factors that modify the association between the presence of metabolic syndrome and cancer risk : these factors include sex, subsite (e.g., colon and rectum), definition of metabolic syndrome (traditional versus nontraditional), and design ; for incidence of cancer, we considered cohort studies, nested case - control studies, and control arms of clinical trials. sensitivity analyses evaluated whether the results could have been affected markedly by a single study and were repeated using a fixed - effects model. publication bias was examined in funnel plots and with a regression asymmetry test : the egger test is best for cancer sites with 10 or more datasets. we used stata, version 9.0 (stata, college station, tx), to analyze data. we followed the prisma (preferred reporting items for systematic reviews and meta - analyses) checklist for reporting systematic reviews and meta - analyses (6). we systematically searched medline, embase, central, cinahl, and web of science through october 2011 for studies in humans of the association between metabolic syndrome and cancer. our core search consisted of the terms metabolic syndrome, insulin resistance syndrome, and syndrome x, combined with specific terms for each cancer site : colorectal (colon and rectum), gastric, esophageal, hepatobiliary (liver and gallbladder), pancreas, lung, bladder, thyroid, renal, leukemia, malignant melanoma, multiple myeloma, and non - hodgkin lymphoma for both sexes and prostate, breast, ovary, and endometrium for single sex. relevant journals, bibliographies, reviews, and personal files were hand searched for additional articles. we included studies if 1) their aim was to assess the effect of metabolic syndrome on risk of cancer or association with cancer, 2) they reported the definition of metabolic syndrome according to criteria of national or international scientific associations, federations, or organizations (traditional definitions) or if they used proxy indicators in the absence of the original data (nontraditional definitions), and 3) they included at least three factors, even in the absence of others. we included cohort studies, nested case - control studies, control arms from clinical trials, case - control studies, patient series, and mortality studies. we specified that every study must either report risk estimates (relative risks [rrs ], odds ratios, hazard ratios, and standardized incidence ratio) with 95% cis separately for men, women, or both or must report sufficient data to estimate these. if a site - specific dataset had been published more than once, we used the most recent publication. we included a specific cancer site in the analysis if there were at least two cohort datasets. we excluded studies that were not published as full reports, such as conference abstracts and letters to editors, and studies of cancer precursors (e.g., colorectal adenoma). from each retrieved article, we extracted the following data : name of the first author, year of publication, country where the study was performed, specific outcomes, follow - up time, proportion of men and women, total number of individuals, number of cases, and risk estimates and their 95% cis (presence versus absence of metabolic syndrome)., k.e., and p.c.) until interrater reliability (0.60) was established. methodological quality of each study was assessed according to three study components that might affect the strength of the association between metabolic syndrome and cancer risk : length of follow - up for cohort studies, whether metabolic syndrome definition was traditional or nontraditional, and the extent of adjustments for potential confounding factors. we also collected, where available, risk estimates of the association with cancer for each single factor of the syndrome taken at its highest level. the primary end point was to assess the association between metabolic syndrome and cancer risk in cohort studies. for the main outcome at each cancer site, we graded the evidence for study quality and for the risk of bias : study quality was based on the number of datasets, number of events, width of cis, and heterogeneity ; risk of bias was mainly based on type of study and adjustment for confounders. unless otherwise stated, we used the most adjusted risk estimate from each study. heterogeneity of the effect across studies was assessed by q statistics, which is distributed as statistics (7). a value of p 0.1 ; otherwise, we used a random - effect model. we did subgroup analyses for each site to identify study - level factors that modify the association between the presence of metabolic syndrome and cancer risk : these factors include sex, subsite (e.g., colon and rectum), definition of metabolic syndrome (traditional versus nontraditional), and design ; for incidence of cancer, we considered cohort studies, nested case - control studies, and control arms of clinical trials. sensitivity analyses evaluated whether the results could have been affected markedly by a single study and were repeated using a fixed - effects model. publication bias was examined in funnel plots and with a regression asymmetry test : the egger test is best for cancer sites with 10 or more datasets. we used stata, version 9.0 (stata, college station, tx), to analyze data. we screened 2,628 potentially relevant, nonduplicate articles. the score for concordance between reviewers rating the articles was 0.620.77 the final number of articles (850) included in the meta - analysis was 43 (supplementary fig. 1), which reported on 116 datasets (supplementary table 2). all articles were published in english. the analysis included 38,940 cancer cases (18,180 men and 20,201 women, plus 559 cases for gallbladder cancer not divided by sex). the median follow - up per cohort studies and per cancer site varied from 3 years (endometrium) to 12.2 years (prostate). notably, no north american population data contributed to the summaries for gallbladder, ovary, thyroid, and bladder cancers. the proportion of studies in which the definition of metabolic syndrome was traditional varied by cancer sites : higher for colorectal (14 vs. 8 nontraditional) ; approximately equal for breast, hepatobiliary and prostate ; and lower for pancreas. no further differentiation was made when the number of datasets for cancer site was four or fewer. the number of potential confounding factors (cancer - site specific risk factors) included in the adjusted analyses also varied (supplementary table 3). 1a and b shows the results of meta - analyses of rr (for presence of metabolic syndrome) in men and in women, respectively, for cohort studies only. separate meta - analyses for some relevant sites and for sex are given in supplementary figs. 2a ; 1.25, p 150 mg / dl (15) ; other single or combined factors explained part (from 30 to 50%) of the increased risk conveyed by metabolic syndrome : bmi (11,21), waist (16,19), bmi and lipid (17), bmi and dysglycemia (14), and hypertension (21). influence analysis showed that no single study affected the sex - specific summary estimates for most sites. moreover, we did not note funnel plot asymmetry for cancer sites where a sufficient number of datasets exited to run the egger test (colorectal cohorts men, p = 0.912 ; colorectal cohorts women, p = 0.201 ; and prostate cancer cohorts, p = 0.085). our results from meta - analyses of prospective cohort studies indicate that metabolic syndrome is consistently associated with an increased risk of several cancers in adults. however, many of the reported associations are small (rr between 1.1 and 1.6) and might differ between sexes for some sites and also across populations. in particular, the associations were stronger in women for some cancers (pancreas and rectal), and the magnitude of the associations was highest for sex - specific cancers (endometrial and breast postmenopausal). moreover, from analyses in which sufficient datasets existed, the association was stronger for colorectal cancer in female european populations (rr 1.64 [95% ci 1.172.28 ] ; five datasets with 2,665 incident cancers) and became protective for prostate cancer in the white u.s. populations, which needs confirmation from future studies. given the widespread diffusion of metabolic syndrome (1) and the increased cancer mortality associated with metabolic syndrome (2), the findings of the present meta - analysis may have a clinical significance. at least for some common cancer sites (colorectal cancer in both sexes, liver cancer in men, and pancreas cancer in women), we are confident that the results are real, as the grading for study quality was moderate to high and overall risk of bias was low. moreover, the inclusion of the few case - control studies did not change the overall estimates significantly. in general, the most robust association seems to be with colorectal cancer in both sexes and liver cancer in men. however, part of the association may be explained by the presence of obesity and overt hyperglycemia. metabolic syndrome may be a surrogate marker for other cancer risk factors, such as decreased physical activity, consumption of high calorie dense foods, high dietary fat intake, low fiber intake, and oxidative stress (1). excess adiposity, in particular visceral obesity, results in a state of chronic systemic low - grade inflammation, attributed to production of inflammatory cytokines by both adipocytes and infiltrating immune cells creating a protumorigenic environment (51). by contrast, adiponectin levels are inversely associated with risk of some cancer, and some polymorphisms of adiponectin and its receptor genes are associated with multiple cancer risk (52). the altered balance between proinflammatory and antiinflammatory cytokines driven by central obesity might contribute to insulin resistance, a core component of the metabolic syndrome. the igf-1 axis has also been implicated in the progression of breast, pancreatic, and esophageal cancer (53) : levels of igf are influenced by circulating insulin levels, with increasing insulin leading to decreased levels of igf - binding proteins 1 and 2, thus increasing the bioavailability of igf. although not suggested by the formal statistical tests that we undertook, there is still a possibility of publication bias considering that the tests were likely to be underpowered. moreover, we can not exclude the possibility of residual confounding and bias because of misclassification. although the included studies attempted to control for various known risk factors, the possibility of residual or unmeasured confounding can not be ruled out. single - point measurement increases the chance of random measurement error, which may underestimate the reported associations. studies on the association between metabolic syndrome and cancer risk used different factors and cutoff points, which complicate comparisons between studies. additionally, metabolic factors were not directly measured in some cohorts but replaced either by proxy indicators of the factor (i.e., hypercholesterolemia as a proxy indicator of high triglyceride and/or low hdl cholesterol levels), self - reported diagnosis of diseases (i.e., diabetes, hypertension), or specific drug use (antidiabetic, antihypertensive, and antidyslipidemic). however, there was a consistent positive association between studies, despite the use of different definitions. our pooled estimates for the primary end point were based on prospective analyses with detailed adjustment for a wide range of variables. we used uniform methods and subgroup analyses to better define associations across cancer types between sexes, populations, cancer subsites, and definitions of metabolic syndrome. moreover, this is the first meta - analysis that entailed a comprehensive search for all studies that assessed association between metabolic syndrome and cancer risk. findings from this meta - analysis, which includes many recently published studies, suggest that metabolic syndrome is associated with increased risk of common cancers. the excess risk of cancer conferred by metabolic syndrome is low to moderate and in part explained by accompanying obesity of hyperglycemia. nevertheless, the increasing prevalence of metabolic syndrome worldwide and the high incidence of some malignancies, particularly colorectal and breast cancers, imply that every year many cases of cancer are attributable to metabolic syndrome. preventive strategies (primary prevention and early detection of cancer) are urgently needed, as has been suggested for patients affected by fully developed diseases, such as diabetes (54). moreover, patients with the metabolic syndrome, even in the absence of obesity or diabetes, should be encouraged to undergo appropriate cancer screenings, at least for some more frequently involved sites, as recommended for all people of their age and sex. more importantly, we need evidence of whether effective interventions to reduce the prevalence of metabolic syndrome in adult populations (55) will reduce cancer risk. the formulation of public health strategies based on sustained and bearable lifestyle changes can hopefully obtain significant results in the fight against cancer at the population level. | objectiveavailable evidence supports the emerging hypothesis that metabolic syndrome may be associated with the risk of some common cancers. we did a systematic review and meta - analysis to assess the association between metabolic syndrome and risk of cancer at different sites.research design and methodswe conducted an electronic search for articles published through october 2011 without restrictions and by reviewing reference lists from retrieved articles. every included study was to report risk estimates with 95% cis for the association between metabolic syndrome and cancer.resultswe analyzed 116 datasets from 43 articles, including 38,940 cases of cancer. in cohort studies in men, the presence of metabolic syndrome was associated with liver (relative risk 1.43, p < 0.0001), colorectal (1.25, p < 0.001), and bladder cancer (1.10, p = 0.013). in cohort studies in women, the presence of metabolic syndrome was associated with endometrial (1.61, p = 0.001), pancreatic (1.58, p < 0.0001), breast postmenopausal (1.56, p = 0.017), rectal (1.52, p = 0.005), and colorectal (1.34, p = 0.006) cancers. associations with metabolic syndrome were stronger in women than in men for pancreatic (p = 0.01) and rectal (p = 0.01) cancers. associations were different between ethnic groups : we recorded stronger associations in asia populations for liver cancer (p = 0.002), in european populations for colorectal cancer in women (p = 0.004), and in u.s. populations (whites) for prostate cancer (p = 0.001).conclusionsmetabolic syndrome is associated with increased risk of common cancers ; for some cancers, the risk differs betweens sexes, populations, and definitions of metabolic syndrome. |
it is now widely accepted that colorectal cancer (crc) arises through the accumulation of genetic and epigenetic changes. the order as well as the number of events are important in the process that transform normal cells into neoplastic precursors and subsequently into malignant tumors, which may further metastasize. inactivation of tumorsupressor genes, apc and tp53, as well as components of mismatch repair system, is commonly found in colorectal tumors. a recent study suggest alternative molecular pathways for colorectal carcinomas based on the observation that apc, kras2, and tp53 are all frequently mutated but rarely in the same tumor. twelve to fifteen percent of all primary colorectal carcinomas display microsatellite instability [5 - 8 ], a result of defect mismatch repair. the majority of colorectal carcinomas, however, harbor numerous aberrations at the chromosome level, and chromosomal instability seems to be pronounced in these tumors. this type of instability may be caused by varying mechanisms, including telomer dysfunction, defect dna double - strand break repair and disturbances during chromosome segregation. the genomes of hundreds of primary colorectal carcinomas have now been studied by conventional chromosomal banding technique as well as by comparative genomic hybridization (cgh) [11 - 28 ]. these studies have revealed a nonrandom pattern of genomic abnormalities in primary carcinomas, including frequent gain of material from chromosomes and chromosome arms 7, 8q, 13, and 20, and losses from 4, 8p, 14, 17p, and 18q. however, in spite of the fact that metastases are usually the ultimate cause of death in crc patients, the cytogenetic changes that characterize and presumably drive the advanced stages of this disease have been poorly described [reviewed in ]. we have recently analyzed 17 crc liver metastases, combining chromosome banding with cgh, and found that the former technique, in contrast to cgh profiles, did not always detect the cytogenetically abnormal clones. the use of cgh have provided profiles of various series of liver metastases [reviewed in ]. to our knowledge the genome aberrations in local recurrences and peritoneal carcinomatoses from crc patient have not previously been described. in order to identify genetic changes underlying the development of local and distant metastases, we have compared the genomes of primary crc with those of local recurrences, peritoneal carcinomatoses, and liver metastases using a molecular cytogenetic approach. the complete cgh profiles are presented in the supplementary table 1 (additional data file 1). all tumors, except one primary carcinoma, three local recurrences and one liver metastasis, exhibited dna copy number changes, and the overall copy number profiles for each tumor stage are illustrated in fig. 1. the number of imbalances per case ranged from 0 to 28 (median, 11). although all chromosomes were involved, the distribution of the imbalances was clearly nonrandom. the most common copy number changes, found in more than 20% of each tumor group, were gains of 7, 8q, 13q, and 20, and losses of 4q, 8p, 17p, and 18, but however, frequency variations were observed among primary tumors, local recurrences, and liver metastases (fig. twenty tumors showed amplifications (table 1, supplementary data, additional data file 1) in one to nine discrete regions : chromosome arms 13q (ten cases), 20q (nine cases), 8q (eight cases), 20p (six cases), 5p (one case), and chromosome x (two cases) and 7 (one case). comparison between carcinomatoses and the other stages of the crc were performed using two - sided fisher exact test. graphic comparison of the overall genomic gains (a) and losses (b) detected by cgh in different crc stages. for each case, the presence or absence of imbalance in every chromosome band (from 1p36 to xq28) was computed in a spreadsheet. the total number of imbalances detected in every band was then used to prepare the graphic comparison. (a) loss of 18q was one of the most frequent aberrations seen in all groups. in addition to 18q loss, primary carcinomas and liver metastases often showed loss of 8p. additionally, liver metastases and carcinomatoses frequently harbored loss of 4q. in addition, carcinomatoses often contained gains of 5p, 7p, 9q, 12, and 13q, whereas liver metastases often showed gains of 7p, and 13q. multiple samples from a single tumor (case no. 53r1 and 53r2 ; 64c1 and 64c2 ; 21l1 and 21l2 ; 42l1 and 42l2 ; 52l1 and 52l2 ; 76l1, 76l2, and 76l3) were counted as one sample. the number of imbalances per case for primary carcinomas, local recurrences, carcinomatoses, and liver metastases ranged from 0 to 16 (median, 10), 0 to 17 (median, 6), 1 to 20 (median, 13), and 0 to 28 (median, 14), respectively. increased copy numbers of chromosome arms 5p and 12p were significantly more frequent in the carcinomatoses than in the other lesions, except the association for 5p between carcinomatoses and primary carcinomas (table 1). a hierarchical cluster analysis was performed for the colorectal tumors based on the gains and losses from all chromosome arms (fig. furthermore, a separate cluster analysis for the 37 liver metastases is illustrated in fig. the majority of the liver metastases harbored changes at 7, 8p, 13q, 17p, 18, and 20 (pink tree). the top dendogram indicates that the liver metastases can be subdivided into two main groups (red and brown trees) according to the chromosome changes, one of these subgroup is characterized by additional changes at 4q, 5q, 6p, 8q, 16 (blue tree). chromosome arms 13p, 14p, 15p, 21p, 22p, and chromosome y, due to high content of heterochromatin, were excluded from the cluster analysis. the chromosome arms are given in the right dendogram (gains in green and losses in blue). each tumor sample is depicted and coded (white primary carcinomas ; yellow local recurrence ; green peritoneal carcinomatoses ; red liver metastases) at the top of the dendogram. each row represents the alterations from a separate chromosome arm over all tumor samples, and each column represents all changes in each tumor. chromosome arms 13p, 14p, 15p, 21p, 22p, and chromosome y, due to high content of heterochromatin, were excluded from the cluster analysis. the chromosome arms are given in the right dendogram (gains in green and losses in blue). each row represents alterations from a separate chromosome arm over all tumor samples, and each column represents all changes in each tumor. in four of the five patients from whom both primary carcinomas and liver metastases were analyzed, the liver metastases had more aberrations than the corresponding primary carcinomas (table 1, supplementary data, additional data file 1). gains of 8q (four cases) and 20q (three cases), and losses of 8p (three cases) and 18q (three cases) were seen in both the primary carcinomas and metastatic samples, whereas gain of 7p and loss of 15q were more common in the liver metastases than in the primary carcinomas (7p : in 5 metastases versus 2 primary carcinomas ; 15q : in 4 metastases versus 1 primary carcinoma). the present study is the first cgh study of peritoneal carcinomatoses and local recurrences from crc patients and includes one of the largest series of liver metastases analyzed. our data provide clues to the understanding of the genetic basis of the advanced disease stages. local recurrences are re - growth of tumor cells at the site of the primary carcinomas. we found that the median number of alterations in these tumors was lower than in the primary carcinomas. in a heterogeneous primary tumor, regrowth to a local recurrency might originate from genetically less complex cells in the primary tumor. this observation should be interpreted with caution since the analyzed primary carcinomas and local recurrences were not from the same patients, and in fact the ranges of aberrations were large for both tumor groups. although carcinomatosis is not always associated with widespread visceral or extra - peritoneal disease, it almost always reflects an incurable disease. the present comparisons of the distribution and frequency of specific chromosomal imbalances among the different tumor stages revealed significant differences between carcinomatoses and each of the other stages of the disease regarding gains at 5p and 12p (table 1). the frequencies of 5p gains were not significantly different between primary carcinomas and carcinomatoses (table 1), is best explained by small sample sets. however, these chromosome aberrations are indeed quite rare in primary colorectal carcinomas as evaluated from previous studies, and among 670 cases the average frequencies for 5p and 12p gains were less than 10% each [11 - 23,28,33 - 40 ]. summarized, the present study and the previous data, lead us to speculate that genes located at these chromosome arms are involved in development of peritoneal carcinomatoses. although gain of 5p12-p14 was confirmed in both samples from the same patient (fig. 4), a smallest region of overlapping gain can not determined from one case only. gains of 5p and 12p in carcinomatoses. gains of 5p (samples 17c, 64c1, and 64c2) and 12p (samples 36c, 64c1, and 62c2) were clearly detected by cgh. the central line (red) in the cgh profile shows the average fluorescence ratio along the chromosome, and the flanking curves (brown) represent the 95% confidence interval. the red and the green lines represent the cut - off values, 0.83 and 1.17, respectively. our findings agree well with previous cgh studies of primary colorectal carcinomas and liver metastases, as the type and the frequency of copy number changes are comparable [11 - 23,28,30,33 - 42 ]. the increasing number of aberrations found in distant metastases compared to primary carcinomas is also in line with a previous allelotype study in which we found a higher fractional allelic imbalance in liver metastases than in primary carcinomas and local recurrences. some observation from the results obtained from the five primary carcinomas and their corresponding liver metastases gain of 7p and loss of 15q were more frequent in the latter, and interestingly the three primary carcinomas that each showed one of these aberrations had synchronous liver metastases. when the present cases and those of previous reports were combined [11 - 23,28,41,42 ], statistical significant frequency differences between these two stages were found (7p : 216/670 versus 74/147, p 2 signals in at least 1/3 of the adenomas. this and the present results reveal a major cluster of changes in all advanced stages (containing gains of 7, 13q, 20q, and losses of 8p, 17p and 18q), one may therefore speculate that gains of 7, 13q, and 20q precede the losses of 8p, 17p, 18q, and all aberrations seem to be, in most cases, part of the clone within the primary tumor with the ability to metastasize, locally and peripheral. a number of genetic imbalances common to all tumor groups were demonstrated, as well as consistent genetic differences among the different stages. whereas local recurrences were genetically less complex than primary colorectal carcinomas, liver metastases and peritoneal carcinomatoses usually have more dna copy number changes than the lesions from which they originated. gains of 5p and 12p seem to be particularly important for the spread of crc cells within the peritoneal cavity. ten primary carcinomas, 14 local recurrences, 7 peritoneal carcinomatoses, and 42 liver metastases from 61 crc patients admitted to the norwegian radium hospital (oslo, norway) and to the lund university hospital (lund, sweden), were included in the present study. both primary carcinomas and liver metastases were obtained from each of five patients. among these, three (76, 93, 136) occurred synchronously and two (3, 112) metachronously. from each frozen tumor sample, a five m section was stained with hematoxylin and eosin and evaluated by a pathologist (j. m. n.). the tumor area was identified, and dissected to enrich the fraction of tumor cells before dna extraction, which was performed using standard phenol and chloroform extraction followed by ethanol precipitation (nucleic acid extractor, model 340a, applied biosystems, foster city, ca). the cgh method initially described by kallioniemi. was used with modifications previously described by kraggerud.. briefly, test (tumor) and reference (peripheral blood lymphocytes from 4 healthy females or 4 healthy males) dna were labeled in nick - translation reactions with a mixture of two fluorochrome - conjugated nucleotides (fitc-12-dctp and fitc-12-dutp for tumor dna, and texas red-6-dctp and texas red-6-dutp for reference dna, new england nuclear, boston, ma). the same amounts of labeled tumor and reference dna (1 g each) were mixed with 20 g of unlabeled cot-1 dna (life technologies, rockville, md), ethanol - precipitated, dried, and dissolved in hybridization buffer (vysis, downers grove, il). after denaturing, the dna was hybridized to normal, denatured metaphase spreads and incubated in a humidified chamber for 23 days at 37c. finally, the slides were washed and mounted in an antifade solution with dapi (vector laboratories, burlingame, ca). three images fitc (green) and texas red (red) hybridization signals and dapi counterstain were sequentially captured with a cohu 4900 ccd (12 bits gray scale) camera, using an automated filter wheel coupled to a zeiss axioplan fluorescence microscope (zeiss, oberkochen, germany), and a cytovision system (applied imaging, newcastle, uk). chromosomes were identified based on their inverted dapi banding, and fluorescence ratio profiles (green to red fluorescence) were calculated for each chromosome using data from at least eight representative copies of each chromosome (range 8 22). the y chromosome was not evaluated. due to the high quality dna extracted from frozen tissue, the threshold values 1.17 and 0.83 were used to score gain and loss of dna sequences, respectively. this threshold allows the detection of one chromosome copy number change present in at least 50% of the cells in a sample from a triploid tumor. amplification was defined as a ratio equal to or above 2.0, corresponding to the detection of at least 6 and 9 chromosomes in 50% of the cells in a diploid and triploid tumor, respectively. all scorings were performed independently by two of the authors (c. b. d. and r. a. l.) with few interobserver differences ; these were resolved after joint reevaluation. a negative (normal versus normal) and a positive (the colon cell line lovo with known copy number changes) control the description of the cgh copy number changes followed the guidelines suggested in the international system for human cytogenetic nomenclature. in order to distinguish between a cgh profile that was abnormal throughout the chromosome from one that returns to normal within the terminal band, we used " ter " in the former and the respective band number in the latter when writing the copy number changes. cgh profiles for 17 of the liver metastases have previously been published. for hierarchical clustering the average - linkage method the cluster analyses and the drawing of the dendogram were performed with j - express pro. for comparisons of different groups, the two - sided fisher exact test was used, and all the statistical analyzes were performed with the spsssoftware (spss, chicago, il). ten primary carcinomas, 14 local recurrences, 7 peritoneal carcinomatoses, and 42 liver metastases from 61 crc patients admitted to the norwegian radium hospital (oslo, norway) and to the lund university hospital (lund, sweden), were included in the present study. both primary carcinomas and liver metastases were obtained from each of five patients. among these, three (76, 93, 136) occurred synchronously and two (3, 112) metachronously. from each frozen tumor sample, a five m section was stained with hematoxylin and eosin and evaluated by a pathologist (j. m. n.). the tumor area was identified, and dissected to enrich the fraction of tumor cells before dna extraction, which was performed using standard phenol and chloroform extraction followed by ethanol precipitation (nucleic acid extractor, model 340a, applied biosystems, foster city, ca). the cgh method initially described by kallioniemi. was used with modifications previously described by kraggerud.. briefly, test (tumor) and reference (peripheral blood lymphocytes from 4 healthy females or 4 healthy males) dna were labeled in nick - translation reactions with a mixture of two fluorochrome - conjugated nucleotides (fitc-12-dctp and fitc-12-dutp for tumor dna, and texas red-6-dctp and texas red-6-dutp for reference dna, new england nuclear, boston, ma). the same amounts of labeled tumor and reference dna (1 g each) were mixed with 20 g of unlabeled cot-1 dna (life technologies, rockville, md), ethanol - precipitated, dried, and dissolved in hybridization buffer (vysis, downers grove, il). after denaturing, the dna was hybridized to normal, denatured metaphase spreads and incubated in a humidified chamber for 23 days at 37c. finally, the slides were washed and mounted in an antifade solution with dapi (vector laboratories, burlingame, ca). three images fitc (green) and texas red (red) hybridization signals and dapi counterstain were sequentially captured with a cohu 4900 ccd (12 bits gray scale) camera, using an automated filter wheel coupled to a zeiss axioplan fluorescence microscope (zeiss, oberkochen, germany), and a cytovision system (applied imaging, newcastle, uk). chromosomes were identified based on their inverted dapi banding, and fluorescence ratio profiles (green to red fluorescence) were calculated for each chromosome using data from at least eight representative copies of each chromosome (range 8 22). the y chromosome was not evaluated. due to the high quality dna extracted from frozen tissue, the threshold values 1.17 and 0.83 were used to score gain and loss of dna sequences, respectively. this threshold allows the detection of one chromosome copy number change present in at least 50% of the cells in a sample from a triploid tumor. amplification was defined as a ratio equal to or above 2.0, corresponding to the detection of at least 6 and 9 chromosomes in 50% of the cells in a diploid and triploid tumor, respectively. all scorings were performed independently by two of the authors (c. b. d. and r. a. l.) with few interobserver differences ; these were resolved after joint reevaluation. a negative (normal versus normal) and a positive (the colon cell line lovo with known copy number changes) control was included in every set of experiments. the description of the cgh copy number changes followed the guidelines suggested in the international system for human cytogenetic nomenclature. in order to distinguish between a cgh profile that was abnormal throughout the chromosome from one that returns to normal within the terminal band, we used " ter " in the former and the respective band number in the latter when writing the copy number changes. for hierarchical clustering the average - linkage method was used with pearson 's correlation similarity measure. the cluster analyses and the drawing of the dendogram were performed with j - express pro. for comparisons of different groups, the two - sided fisher exact test was used, and all the statistical analyzes were performed with the spsssoftware (spss, chicago, il). jnw, k - eg, and bj were responsible for referring the patients, collecting tissue specimens and clinical information. ral conceived the study, was responsible for its design and coordination, participated in the evaluation of the data and in the manuscript preparation. complete cgh profiles of 10 primary carcinomas, 14 local recurrences, 7 peritoneal carcinomatoses, and 42 liver metastases from colorectal cancer patients. this work was supported by a phd grant (cbd) from the norwegian foundation for health and rehabilitation (cbd), as well as grants from the norwegian cancer society (lt, ral), and the swedish cancer society (bj). | backgroundcolorectal cancer (crc) is one of the most common causes of cancer - related deaths in the western world, and despite the fact that metastases are usually the ultimate cause of deaths, the knowledge of the genetics of advanced stages of this disease is limited. in order to identify potential genetic abnormalities underlying the development of local and distant metastases in crc patients, we have, by comparative genomic hybridization, compared the dna copy number profiles of 10 primary carcinomas, 14 local recurrences, 7 peritoneal carcinomatoses, and 42 liver metastases from 61 crc patients.resultsthe median number of aberrations among the primary carcinomas, local recurrences, carcinomatoses, and liver metastases was 10, 6, 13, and 14, respectively. several genetic imbalances, such as gains of 7, 8q, 13q, and 20, and losses of 4q, 8p, 17p, and 18, were common in all groups. in contrast, gains of 5p and 12p were more common in the carcinomatoses than in other stages of the disease. with hierarchical cluster analysis, liver metastases could be divided into two main subgroups according to clusters of chromosome changes.conclusionseach stage of crc progression is characterized by a particular genetic profile, and both carcinomatoses and liver metastases are more genetically complex than local recurrences and primary carcinomas. this is the first genome profiling of local recurrences and carcinomatoses, and gains of 5p and 12p seem to be particularly important for the spread of the crc cells within the peritoneal cavity. |
study participants were from the chingford study, a well - described prospective population - based longitudinal study of osteoarthritis and osteoporosis, comprising 1,003 women aged 43 years or above at entry, derived from the age / sex register of a large general practice in chingford, north london. all the women lived within 5 miles of the general practice, 98% of the women were white, and they were predominantly middle class. participants were similar to women in the uk general population in terms of weight, height, and smoking characteristics. the study was approved by the local ethics committee, and written consent was obtained from each woman and has been described in detail previously [16, 17 ]. bmd was measured at the fn and ls at l1l4 by dual - energy x - ray absorptiometry (dxa ; hologic qdr 1000 for year 1 to year 3 ; hologic qdr 2000 for year 4 to year 6 ; hologic delphi w for year 8 to year 10, and hologic discovery for year 15 ; hologic, waltham, ma). a cross - calibration was performed each time the machine was upgraded. the cross - calibration involved scanning 30 patients on the old scanner and then again on the new one on the same day. intrascanner reproducibility, expressed as a coefficient of variation from duplicate measurements in healthy volunteers 1 week apart, was 1.6% at the fn and 0.8% at the ls. quality control was performed regularly using a phantom to ensure the reliability of the densitometer. serum samples were collected without any special patient preparation requirements at the first visit and stored at 45c in a freezer until assayed. serum dheas levels were measured by radioimmunoassay (ria) from the same batch a nonextraction, nonchromatographic method using i ligand as described previously. at each biannual visit, height and weight were measured and body mass index (bmi, weight [kg]/height [m ]) was calculated. in addition, information on smoking (never - smokers, ex - smokers, and current smokers), physical activity (inactive, moderately inactive, moderately active, and active), diet (milk pints, ounce of cheese, yoghurt pots) per week, estradiol, polypharmacy (current medication for blood pressure, diabetes, diuretics, and thyroid), family history (arthritis at hand and knee of child, mother, father, brother, sister, aunt, and grandmother), and rheumatoid arthritis (ra) was collected at the first visit. all women completed a standardized, nurse - administered questionnaire on medical history for a number of known risk factors for osteoporosis. medication information was detailed, particularly for those drugs that influence bone loss such as bisphosphonates and steroids. use of hormone - replacement therapy (hrt) was assessed, and women were classified as current and no hrt use for each visit year except year 5. since a series of bmd measurements during the 15-year follow - up period were available and the number of measurements and their time interval differed across individuals, a multilevel regression model for longitudinal data was chosen for the analysis. repeated measures of bmd at the fn and ls, sufficiently normally distributed, taken on a maximum of eight occasions per participant between baseline and the most recent clinical visit were used as outcomes in the analyses. a time variable, measured in years since baseline, indicated when each measurement was taken and represented follow - up time. the model allowed the investigation of changes in the influence of dheas on bmd over time by including dheas - by - time interaction terms. the coefficient for the dheas variable then indicated its influence on bmd at time zero (thus, baseline bmd), while the interaction term indicated the influence of dheas on bmd change per year from baseline (slope). these models account for within - subject correlation between repeated measures and allow for incomplete outcome data as long as a missing - at - random process can be assumed. therefore, all participants with at least two bmd measurements make a contribution to the estimates of association. the repeated measures of bmd were modeled to represent a linear change of bmd over time (slope). nonlinear (time squared) changes of bmd were also considered ; this was significant and, therefore, included in the model. the basic model was therefore reduced to include baseline dheas, time, time squared, and the dheas - by - time interaction. wald tests were performed to assess levels of significance for the fixed - effect parameters. based on the lrt, a random intercept model fitted the data well compared to other possible models. separate models were produced for the fn and ls bmd. to investigate whether the associations between baseline dheas and bmd at both sites were confounded, age, estradiol at baseline, and time - varying hrt and bmi as covariates were added to the model. since dheas was not associated with smoking, physical activity, diet, polypharmacy, family history, and ra, these variables were not included in the final model. from the final adjusted model, predicted average bmd trajectories over the follow - up time were also graphically presented by baseline dheas interquartile ranges. the model fit was as follows:\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{bmd}}_{\text{ij } } = \left ({ \beta_{00 } + { \text { u}}_{{0{\text{j } } } } } \right) _ { + } \beta _ { 10 } { \text{dheas}}_{\text{j } } + \beta _ { 20 } { \text{time}}_{\text{ij } } + \, \beta _ { 30 } \left ({ { \text{dheas}}_{\text{j } } { \text{time}}_{\text{ij } } } \right) + \, \beta _ { 40 } { \text{time}}_{\text{ij}}^{2 } + { \text{e}}_{\text{ij } } $ $ \end{document}where bmdij denotes bmd at the fn or ls on measurement occasion i (i = 18) of subject j (j = 1 n), dheasj was dheas at the baseline, and timeij and timeij2 were defined as the linear and quadratic forms of the time in years after baseline at which each measurement was taken, respectively. the terms u0j and eij, denoting the random intercept and error components, were assumed to be normally, independently, and identically distributed with mean zero and variance u02, e2, respectively. the intraclass coefficient of the bmd that attributed to subject effect (level 2) was estimated using the formula \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \rho ({ \text{y}_{\text{ij}}}/\text{x}_{\text{ij } }) = \frac{{\sigma^{2 } _ { { \text{u}}0}}}{{\sigma^{2 } _ { { \text{u}}0 } + \sigma^{2 } _ { \text{e } } } } $ $ \end{document}. a coefficient estimate was considered statistically significant if its p value (two - sided) was 0.05). however, those who had only one dxa scan, < 6% of the total study subjects, were excluded from the analysis and were significantly older and heavier and had higher dheas levels than those who had at least two dxa scans and were included in the analysis (all p < 0.02). the results of the unadjusted and adjusted associations between dheas and bmd change at fn and ls are presented in tables 3 and 4, respectively. bmd at the fn decreased 0.45% (95% ci 0.300.60%) per year, and the rate of decline was slowed down by 0.025% per squared year (table 3, fig. 1). the serum level of dheas at baseline was strongly and positively associated with the fn bmd at baseline. increase of dheas (each micromole per liter) was associated with 0.92% less bone loss at the fn (95% ci 0.561.27%, p < 0.0001). however, this strong association became slightly weaker over time by 0.017% (95% ci 0.00360.031%) per year as indicated by a significant dheas - by - time interaction (p = 0.013) (table 3). the negative and significant interaction between dheas and time reflects a small change in the degree to which baseline dheas predicts bone loss at the fn during the follow - up. the significance remained after adjustment for age and estradiol at baseline and time - varying bmi and hrt (fully adjusted beta = 0.49%, 95% ci 0.210.71%, p = 0.001), and all covariates were significant in the model, as expected (table 3). the results remained the same when the analyses were restricted to those women who were never on hrt during the 15-year follow - up. figure 1 illustrates the average fn bmd trajectories over time by baseline dheas interquartile ranges, which were predicted from the final adjusted multilevel model.table 3multilevel linear regression of bmd at the fn with regard to the study factorsunadjusted analysismultiple adjusted analysisfully adjusted analysisfncoeff. (95% ci)pdheas (mol / l)0.92 (0.561.27)<0.00010.35 (0.110.57)0.0070.49 (0.210.71)0.001time (year)0.45 (0.60 to 0.30)<0.00010.48 (0.65 to 0.34)<0.00010.49 (0.71 to 0.31)<0.0001time (year)0.025 (0.0130.036)<0.00010.027 (0.0180.035)<0.00010.028 (0.0180.036)<0.0001dheastime0.017 (0.031 to 0.0036)0.0130.017 (0.030 to 0.0057)0.0020.020 (0.037 to 0.0063)0.003baseline age (year)0.68 (0.87 to 0.53)<0.00010.61(0.86 to 0.41) < 0.0001bmi (kg / m)0.53 (0.480.56)<0.00010.61 (0.570.63)<0.0001hrt0.81 (0.501.07)<0.00010.92 (0.5561.21)<0.0001estradiol (pmol / l)0.018 (0.010.025)<0.0001the s are interpreted as percentage change in bmd at femoral neck per unit increase in the study factors listed in the table. for example, 0.45 for the time variable represents a 0.45% decrease in bmd at the femoral neck per year during the follow - up periodadjusted for baseline age, time - varying (bmi and hrt)further adjusted for estradioldheas, dehydroepiandrosterone sulfate ; fn, femoral neck bmd, bmi, body mass index ; hrt, hormone - replacement therapy ; dheas time, baseline dheas and time interaction ; time, quadratic form of time ; bmd, bone mineral densitytable 4multilevel linear regression of bmd at ls with regard to the study factorsunadjusted analysismultiple adjusted analysisfully adjusted analysislscoeff. (95% ci)pdheas (mol / l)0.72 (0.351.06)<0.00010.25 (0.015 to 0.48)0.0630.29 (0.038 to 0.56)0.078time (year)0.46 (0.60 to 0.31)<0.00010.45 (0.62 to 0.31)<0.00010.47 (0.69 to 0.29)<0.0001time (year)0.038 (0.0260.049)<0.00010.035 (0.0260.042)<0.00010.039 (0.0280.047)<0.0001dheas time0.037 (0.052 to 0.023)<0.00010.035 (0.050 to 0.022)<0.00010.033 (0.053 to 0.017)<0.0001baseline age (year)0.63 (0.82 to 0.47)<0.00010.51 (0.77 to 0.30)<0.0001bmi (kg / m)0.44 (0.380.49)<0.00010.55 (0.500.60)<0.0001hrt1.52 (1.251.76)<0.00011.69 (1.361.95)<0.0001estradiol (pmol / l)0.022 (0.0130.029)<0.0001the s are interpreted as percentage change in bmd at lumbar spine per unit increase in the study factors listed in the table. for example : 0.46 for time variable represents a 0.46% decrease in bmd at the lumbar spine per year during the follow - up periodadjusted for baseline age, time - varying (bmi and hrt)further adjusted for estradioldheas dehydroepiandrosterone sulfate, ls lumbar spine bmd, bmi body mass index, hrt hormone - replacement therapy, dheas time baseline dheas and time interaction, time quadratic form of time, bmd bone mineral densityfig. 1average predicted fn bmd trajectories over follow - up time by baseline dheas interquartile ranges, from the fully adjusted model, for the postmenopausal women multilevel linear regression of bmd at the fn with regard to the study factors the s are interpreted as percentage change in bmd at femoral neck per unit increase in the study factors listed in the table. for example, 0.45 for the time variable represents a 0.45% decrease in bmd at the femoral neck per year during the follow - up period adjusted for baseline age, time - varying (bmi and hrt) further adjusted for estradiol dheas, dehydroepiandrosterone sulfate ; fn, femoral neck bmd, bmi, body mass index ; hrt, hormone - replacement therapy ; dheas time, baseline dheas and time interaction ; time, quadratic form of time ; bmd, bone mineral density multilevel linear regression of bmd at ls with regard to the study factors the s are interpreted as percentage change in bmd at lumbar spine per unit increase in the study factors listed in the table. for example : 0.46 for time variable represents a 0.46% decrease in bmd at the lumbar spine per year during the follow - up period adjusted for baseline age, time - varying (bmi and hrt) further adjusted for estradiol dheas dehydroepiandrosterone sulfate, ls lumbar spine bmd, bmi body mass index, hrt hormone - replacement therapy, dheas time baseline dheas and time interaction, time quadratic form of time, bmd bone mineral density average predicted fn bmd trajectories over follow - up time by baseline dheas interquartile ranges, from the fully adjusted model, for the postmenopausal women the variance component corresponding to the random intercept is 0.014. since this estimate is substantially larger than its standard error (0.00068), there appears to be significant variation in the between - subject means. the two - variance components can be used to partition the variance across levels. the intraclass correlation coefficient or the proportion variance at the person level is estimated as = 0.94, meaning that roughly 94% of the variance of the fn bmd measures were attributable to the subject level ; about 6% is variance within individuals across time. this suggested that the variance within an individual across time does not account for much of the additional variance of the fn bmd. similarly, bmd at the ls decreased 0.46% (95% ci 0.310.60%) per year, and the rate of decline was slowed down by 0.038% per squared year (table 4, fig. 2). the serum level of dheas at baseline was strongly and positively associated with the ls bmd at baseline. increase of dheas (each micromole per liter) was associated with 0.72% less bone loss at the ls (95% ci 0.351.06%, p < 0.0001). however, this strong association became slightly weaker over time by 0.037% (95% ci 0.0230.052%) per year as indicated by a significant dheas - by - time interaction (p < 0.0001) (table 4). the association became weaker after adjustment for other covariates including age and estradiol at baseline and time - varying bmi and hrt (fully adjusted beta = 0.29%, 95% ci 0.038% to 0.56%, p = 0.078) (table 4). however, the association became stronger (p = 0.02) when the analyses were restricted to those women who were never on hrt during the 15-year follow - up. figure 2 shows the average ls bmd trajectories over time by baseline dheas interquartile ranges, which were predicted from the final adjusted multilevel model. the graphs, at different quartiles, start to converge slowly over the follow - up time, implying that the effect of baseline dheas on ls bmd loss diminishes over time.fig. 2average predicted ls bmd trajectories over follow - up time by baseline dheas interquartile ranges, from the fully adjusted model, for the postmenopausal women average predicted ls bmd trajectories over follow - up time by baseline dheas interquartile ranges, from the fully adjusted model, for the postmenopausal women the fixed - effect model estimates the repeated - measures variance as 0.0018 (within - subject variability) and the person level (between - subject variability) as 0.023. the intraclass correlation at the person level is estimated as = 0.93. this implies that about 93% of the variance in the ls bmd measures was variance between individuals (subjects) and that about 7% was variance within individuals across time. this large prospective population - based study examined the association between dheas level at baseline and bmd change at the fn and ls in postmenopausal women over a 15-year follow - up period. the data suggest that serum level of dheas at baseline is strongly associated with bmd but the association became slightly weaker over time. although the relationship between dheas and bmd at the fn and ls was reduced after adjusting for age and estradiol at baseline and time - varying bmi and hrt, an independent effect on bmd was still apparent at both sites. further, women who had higher dheas levels at baseline also had higher bmd at both fn and ls at the end of the 15-year follow - up. the results from previous cross - sectional studies on the effect of dheas on bmd in postmenopausal women with dheas in the normal range are inconsistent. our findings concur with studies that report a positive association between serum dheas and bmd [1921 ]. szathmari. found a positive relationship between dheas and bmd at both fn and ls sites in 105 women aged 4569 years, 76 postmenopausal and 29 premenopausal. in contrast, other studies found no association of circulating dheas either with absolute value of bmd [22, 23 ] or with rate of bone loss.. found no association between dheas and bmd at both fn and ls in 147 healthy or osteoporotic but otherwise normal premenopausal (n = 26 and n = 13, respectively) or postmenopausal (n = 40 and n = 68, respectively) women aged 40.1 9.9 and 61.9 8.9 years, respectively. likewise, murphy. found no significant association in a cross - sectional study of 90 community - based women, all at least 1 year since their last menstrual period, who were on average 9.6 years postmenopausal (mean 9.6 4.9 years, range 122). in a prospective study of 256 men (aged 5074 years) and 162 women (aged 5574 years), barrett - connor. found no association between dheas levels with bmd at any site in either sex, both before and after adjusting for age, obesity, cigarette smoking, and use of antihypertensive medications. a similar study found an insignificant association between dheas and bmd at the fn and ls in a population - based cohort of 159, non - hrt user, australian - born women who at baseline had a mean age of 50.0 years (sd = 2.4) with reported menstruation within the previous 3 months. the reason for the discrepancy is unclear ; possible explanations include small sample size, methodological differences [22, 23 ], and relatively short duration [10, 24 ]. studies have suggested the complexity of the potential mechanisms of the effects of dhea on bmd loss. there was evidence for a transient increase in serum estrogen for a few hours after oral dhea administration, and the correlations linking serum dheas levels to bone turnover markers, bmd, and osteoporotic fractures are strongest in elderly women, in whom peripheral dhea conversion is the only source of estrogens. however, the effect size of dheas on bone loss was even increased after further adjustment for estradiol in the current study, suggesting that dhea may have a direct effect on bone loss that is independent of estradiol. there is also a possibility that the effect of dhea on bone was liaised by the increase in serum testosterone concentration that occurs in response to dhea. testosterone replacement has been shown to inhibit bone resorption and stimulate formation in hypogonadal men. dhea replacement resulted in a marked increase in circulating igf - i levels, which may exert anabolic effects on bone. furthermore, treatment of oophorectomized rats with dhea increased bmd above that of intact animals in parallel with the increase in alkaline phosphatase, which may indicate a direct positive effect of dhea on osteoblasts. dhea administration reduced the loss of cancellous bone volume in rats in a dose - dependent manner by reducing bone remodeling. however, findings in rodents are of limited significance for humans as dhea physiology in humans is distinctly different from that in nonprimate mammals. the study population was all female ; thus, the results may not be generalizable. however, a similar significant association in males has been reported. multilevel analysis was used to take account of the correlation between repeated measures of fn and ls bmd, bmi, and hrt for the same individual. although missing outcome data can be comprised in this method of analysis, the missing - at - random assumption might be violated if missing occurs systematically with regard to dheas levels. however, the majority of loss to follow - up in the study was due to censoring, and there was no difference in bmd, age, bmi, and hrt use between women with and without dheas measurements, suggesting that this is not an issue. although the models fit the data properly, there was inconsiderable variability of bmd between and within subjects that remained unexplained ; this could be due to unknown genetic and environmental factors which were not taken into account. in addition, since dheas was measured only at baseline in this study, we could not exclude the possibility that changes of dheas accompanying bmd changes may be significant for the regulation of bmd. in conclusion, our data suggest that a high serum dheas level at baseline is associated with reduced bone loss at both fn and ls and this association diminishes over time. the nature of the association is unclear, but such an association implies that women might benefit from maintaining a high level of dheas for their bmd. | our aim was to examine the association between serum dehydroepiandrosterone sulfate (dheas) at baseline and bmd change at the femoral neck (fn) and lumbar spine (ls) in postmenopausal women during a 15-year follow - up. all participants were from the chingford study. bmd at the fn and ls were measured eight times during the 15-year follow - up by dual - energy x - ray absorptiometry. dheas at baseline was measured using radioimmunoassay. data on height, weight, and hormone - replacement therapy (hrt) status were obtained at each visit. multilevel linear regression modeling was used to examine the association between longitudinal bmd change at the fn and ls and dheas at baseline. postmenopausal women (n = 1,003) aged 4568 years (mean 54.7) at baseline were included in the study. after adjustment for baseline age, estradiol, hrt, and bmi, bmd at the fn decreased on average 0.49% (95% ci 0.310.71%) per year ; and the decline was slowed down by 0.028% per squared year. increase of dheas (each micromole per liter) was associated with 0.49% less bone loss at the fn (95% ci 0.210.71%, p = 0.001). however, this strong association became slightly weaker over time. similar but weaker results were obtained for ls bmd. our data suggest that high serum dheas at baseline is associated with less bone loss at both fn and ls and this association diminishes over time. the nature of the association is unclear, but such an association implies that, in managing bmd loss, women might benefit from maintaining a high level of dheas. |
four - month - old whole - body ampk2 mice (29) backcrossed to c57bl/6j mice for nine generations, and wild - type littermate controls were fed on either chow, chf, or chf+f diet for nine weeks. body weight and food consumption were recorded, and edta - plasma and tissues were collected for various analyses as described in the online appendix, available at http://diabetes.diabetesjournals.org/cgi/content/full/db09-1716/dc1. male mice were used for all the experiments, except for the measurements of hepatic ampk activity, which were performed on female mice. the experiments were conducted under the guidelines for the use and care of laboratory animals of the institute of physiology and followed the principles of laboratory animal care (national institutes of health publication no. 85 - 23, revised 1985). nonesterified fatty acids (nefas), triglycerides, and total cholesterol were determined in plasma using the following enzymatic photometric tests : nefa - c (wako chemicals, neuss, germany), triacylglycerols liquid, and cholesterol liquid (pliva - lachema diagnostika, brno, czech republic), respectively. plasma insulin was measured using the sensitive rat insulin ria kit (linco research, st. total adiponectin levels and adiponectin multimeric complexes were determined using western blotting (31). the tissue content of triglycerides was estimated in ethanolic koh tissue lysates as described before (8). the content and fatty acid composition of the phospholipid, diacylglycerol, triglyceride, and ceramide fractions were assessed in tissue lipid extracts ; gene expression was evaluated using real - time rt - pcr (see online appendix). livers were collected by freeze - clamping, ampk was immunoprecipitated from tissue extracts, and the activity was assayed using a peptide substrate (32) ; see the online appendix. five days before the experiment, an indwelling catheter was placed into the left femoral vein under anesthesia (33). mice were allowed to recover for 57 days, followed by a 6-h fast (8:00 a.m.2:00 p.m.) prior to the experiment. the whole - body glucose turnover was determined under basal (nonstimulated) and insulin - stimulated conditions (hyperinsulinemic - euglycemic clamp), using separate groups of mice. insulin (actrapid, novo nordisk pharma, denmark) was infused at a constant rate of 4.8 mu / kgmin for 3 h, while d-[3-h]glucose (perkin elmer, boston, ma) was infused at a rate of 15.9 kbq / min. throughout the infusion, glucose concentration and d-[3-h]glucose specific activity (during the last hour of infusion) mmol / l) was maintained by periodically adjusting a variable infusion of 33% glucose (33). at the end of a 3-h infusion period, mice were first anesthetized by diethylether, exsanguinated through the cervical incision, and then killed by cervical dislocation, and tissues (liver and quadriceps muscle) and edta - plasma were collected for biochemical analyses (see supplementary table 1 and research design and methods of the online appendix for details on basic clamp parameters and methodology). hepatocytes were isolated from livers of fed mice by a modification of the collagenase method (34) and seeded at a density of 0.5 10 cells per each 35-mm petri dish. rates of basal and insulin - stimulated de novo lipogenesis and aicar - stimulated fatty acid oxidation were measured using [1-c ] acetate and [1-c ] palmitate, respectively (see online appendix). nonesterified fatty acids (nefas), triglycerides, and total cholesterol were determined in plasma using the following enzymatic photometric tests : nefa - c (wako chemicals, neuss, germany), triacylglycerols liquid, and cholesterol liquid (pliva - lachema diagnostika, brno, czech republic), respectively. plasma insulin was measured using the sensitive rat insulin ria kit (linco research, st. total adiponectin levels and adiponectin multimeric complexes were determined using western blotting (31). the tissue content of triglycerides was estimated in ethanolic koh tissue lysates as described before (8). the content and fatty acid composition of the phospholipid, diacylglycerol, triglyceride, and ceramide fractions were assessed in tissue lipid extracts ; gene expression was evaluated using real - time rt - pcr (see online appendix). livers were collected by freeze - clamping, ampk was immunoprecipitated from tissue extracts, and the activity was assayed using a peptide substrate (32) ; see the online appendix. five days before the experiment, an indwelling catheter was placed into the left femoral vein under anesthesia (33). mice were allowed to recover for 57 days, followed by a 6-h fast (8:00 a.m.2:00 p.m.) prior to the experiment. the whole - body glucose turnover was determined under basal (nonstimulated) and insulin - stimulated conditions (hyperinsulinemic - euglycemic clamp), using separate groups of mice. insulin (actrapid, novo nordisk pharma, denmark) was infused at a constant rate of 4.8 mu / kgmin for 3 h, while d-[3-h]glucose (perkin elmer, boston, ma) was infused at a rate of 15.9 kbq / min. throughout the infusion, glucose concentration and d-[3-h]glucose specific activity (during the last hour of infusion) mmol / l) was maintained by periodically adjusting a variable infusion of 33% glucose (33). at the end of a 3-h infusion period, mice were first anesthetized by diethylether, exsanguinated through the cervical incision, and then killed by cervical dislocation, and tissues (liver and quadriceps muscle) and edta - plasma were collected for biochemical analyses (see supplementary table 1 and research design and methods of the online appendix for details on basic clamp parameters and methodology). hepatocytes were isolated from livers of fed mice by a modification of the collagenase method (34) and seeded at a density of 0.5 10 cells per each 35-mm petri dish. rates of basal and insulin - stimulated de novo lipogenesis and aicar - stimulated fatty acid oxidation were measured using [1-c ] acetate and [1-c ] palmitate, respectively (see online appendix). specific activities of ampk1 and ampk2 were evaluated in the liver of ad libitum - fed mice after nine weeks of the differential dietary treatment (fig. no significant effect of either diet (chow, chf, and chf+f) or genotype (wild - type versus ampk2) on ampk1-specific activity was observed, although the ampk1 activity tended to be higher in the ampk2 mice (fig. in contrast, ampk2 activity was stimulated by n-3 lc - pufas (chf+f diet ; fig. no changes were detected in the activity of ampk1 and ampk2 in the quadriceps muscle in response to n-3 lc - pufas (not shown). liver ampk1 (a) and ampk2 (b) activity in wild - type and ampk2 mice fed either a chow diet, chf, or chf+f for 9 weeks. the data are the means se (n = 58). in the ampk2 mice, p < 0.05 versus genotype chow ; p < 0.05 versus genotype chf. at four months of age, at the beginning of dietary treatments, wild - type and ampk2 mice fed the chow diet exhibited similar body weights (table 1). in mice of both genotypes, chf - feeding for nine weeks resulted in greater body weight gain compared with the chow - fed mice. however, this effect was less pronounced in ampk2 mice (table 1). in both wild - type and ampk2 mice, the chf+f diet induced smaller body weight gain than the chf diet (table 1 and supplementary figure 1). none of the differences in body weight gain could be explained by caloric intake, which was similar in all experimental groups (table 1). the weight of fat depots increased in response to chf feeding, while the chf+f diet partially prevented this increase (table 1). triglycerides and nefa levels in plasma of ad libitum - fed mice were similar in the chow- and chf - fed mice, while cholesterol levels were markedly and significantly elevated by the chf diet. triglycerides and nefa levels were strongly reduced, even below the levels observed in the chow - fed mice (table 1). metabolic and plasma parameters in wild - type and ampk2 mice data are means se of 2730 mice for metabolic parameters and 1315 mice for other measures. ampk2 and wild - type mice were fed either a chow diet, chf, or chf+f for nine weeks. body weight gain (see supplementary fig. 1) and plasma parameters were assessed in ad libitum - fed mice after nine weeks. plasma insulin levels were also assessed in fasted mice after eight weeks. at, adipose tissue ; a.u., arbitary units ; hmw, total adiponectin, ratio of high molecular weight to total adiponectin (for levels of all molecular weight forms of adiponectin ; nd, no data ; see supplementary fig. 2) ; p < 0.05 vs. genotype chow ; p < 0.05 vs. genotype chf ; p < 0.05 vs. wild - type on respective diet. after nine weeks of dietary treatment, no change was observed in blood glucose, but elevations were observed in plasma insulin levels in response to the chf diet in ad libitum - fed mice of both genotypes. however, the increase in plasma insulin levels was less pronounced in ampk2 mice, closely reflecting the genotype - dependent differences in body weight gain (table 1). a similar pattern of changes in insulin levels was also observed in fasted mice, in which n-3 lc - pufas significantly reduced insulin levels only in wild - type animals (table 1). as expected, plasma levels of total as well as high molecular weight form of adiponectin, an adipokine associated with increased insulin sensitivity (35), were increased 1.4- and 1.2-fold, respectively, in wild - type mice in response to n-3 lc - pufa supplementation (table 1 and supplementary fig. 2, available in an online appendix) ; however, no significant increase of plasma adiponectin by n-3 lc - pufas was observed in ampk2 mice. in further experiments, under basal conditions, glucose turnover rate (gto ; i.e., glucose uptake in peripheral tissues) was similar in all groups of mice (supplementary table 1). under insulin - stimulated conditions (fig. f), the amount of exogenous glucose required to maintain euglycemia during the clamp, i.e., the glucose infusion rate (gir), was 1.3-fold lower in ampk2 than in wild - type mice fed the chow diet (fig. gir was decreased by the chf diet to a similar level in mice of both genotypes, manifesting diet - induced insulin resistance. this was attributed to a decreased gto and, in particular, to an impaired suppression of hepatic glucose production (hgp) by insulin, with hgp being 8.5-fold higher in the chf - fed compared with the chow - fed wild - type mice (fig. 2c). in wild - type mice, chf+f diet feeding increased gir and gto (1.9- and 1.2-fold increase, respectively) as compared with chf - fed mice, while hgp was lowered to a similar level as in the chow - fed mice. these results document the protective effects of n-3 lc - pufas from high - fat diet - induced insulin resistance in wild - type mice, namely, at the level of hgp. in contrast, none of these beneficial effects of n-3 lc - pufas was observed in ampk2 mice, in which neither the gir (fig. 2b) differed between the chf+f - fed and the chf - fed mice, whereas the rate of hgp was even higher in the chf+f - fed than in the chf - fed ampk2 mice (fig. 2c). 2d), the rate of whole - body glycogen synthesis, which reflects insulin sensitivity of muscle glucose metabolism, was dependent on both diet and genotype (fig. 2e). in the chow - fed mice, the rate of whole - body glycogen synthesis tended to be higher in wild - type mice than in ampk2 mice. only in the former mice thus, in wild - type mice, glycogen synthesis was decreased 2.4-fold in response to the chf diet, while n-3 lc - pufas provided a partial protection from this decrease (fig. a similar pattern of changes in the glycogen synthesis rate in response to n-3 lc - pufas was observed when measured directly in the skeletal muscle (fig. thus, in accordance with the previous study (26), the results of clamp studies suggested impairment of insulin sensitivity in response to whole - body ablation of ampk2 in chow - fed mice. however, ampk2 mice seemed to be partially protected against chf - induced insulin resistance, while ampk2 was required for preservation of insulin sensitivity in the skeletal muscle and especially in the liver in response to n-3 lc - pufa feeding. gir (a), gto (b), hgp (c), whole - body glycolysis (gl - wb ; d), whole - body glycogen synthesis (gs - wb ; e) ; and glycogen synthesis in quadriceps muscle (gs - qm ; f) were measured in wild - type and ampk2 mice fed either a chow diet, chf, or chf+f for 9 weeks. the data are the means se (n = 58). p < 0.05 versus genotype chow ; p < 0.05 versus genotype chf ; p < 0.05 versus wild - type on respective diet. in addition to the ad libitum - fed mice (table 1), plasma lipid levels were also measured in fasted mice, as well as in mice subjected to hyperinsulinemic - euglycemic clamp (supplementary table 2). in contrast to the ad libitum - fed state, chf+f diet did not affect either triglyceride or nefa levels under fasting conditions. under the hyperinsulinemic - euglycemic conditions, both triglyceride and nefa levels were lower in the chf+f - fed than in the chf - fed wild - type mice (1.6-fold and 1.4-fold difference, respectively), but no such difference between the diets was observed in ampk2 mice. cholesterol levels were consistently decreased by n-3 lc - pufas independently of both the metabolic state and genotype (supplementary table 2). in ad libitum - fed mice of both genotypes, the hepatic triglyceride content was increased twofold by chf compared with the chow diet, while triglyceride accumulation was increased only 1.3-fold by chf+f diet in both genotypes, documenting a protection against hepatic triglyceride accumulation by n-3 lc - pufas (fig. 3a). under hyperinsulinemic - euglycemic conditions, n-3 lc - pufas also protected livers of wild - type mice against the chf - induced accumulation of triglycerides. however, this effect was absent in ampk2 mice (fig. 3b). moreover, a strong correlation was found between plasma nefa levels and hepatic triglyceride content assessed under the clamp conditions in the chf+f - fed ampk2 mice (r = 0.43, p < 0.05) but not in wild - type mice (r = 0.08, p = 0.40). triglyceride concentration in the livers of ad libitum - fed mice (a) and mice killed at the end of a 3-h infusion period of the hyperinsulinemic - euglycemic clamp (b). wild - type and ampk2 mice were fed either a chow diet, chf, or chf+f for 9 weeks. the data are the means se (a, n = 1315 ; b, n = 814). p < 0.05 versus genotype chow ; p < 0.05 versus genotype chf ; p < 0.05 versus wild - type on respective diet. for the detailed fatty acid composition of triglyceride fractions in the livers of ad libitum - fed mice, see supplementary table 4. we sought to determine whether the differential effect of n-3 lc - pufas on accumulation of liver triglycerides in wild - type and ampk2 mice under the clamp conditions could be explained by hepatic lipid metabolism. in cultured hepatocytes isolated from mice following the different dietary treatments, activities of both fatty acid oxidation and de novo fatty acid synthesis were evaluated. the stimulatory effects of 5-aminoimidazole-4-carboxamide-1--d - ribofuranoside (aicar), an ampk activator, and insulin on fatty acid oxidation and synthesis are shown in fig. 4a and b, respectively (for corresponding basal metabolic activities, see supplementary table 3, available in an online appendix). in hepatocytes from both chow and chf diet - fed mice, the absence of ampk2 was associated with a trend for lower aicar - stimulated fatty acid oxidation. although hepatocytes from chf - fed mice showed reduced stimulatory effect of aicar irrespective of the genotype, chf+f feeding normalized this defect in wild - type but not in ampk2 hepatocytes (fig. 5a), suggesting ampk - dependent induction of capacity for fatty acid oxidation by n-3 lc - pufas in the liver. the stimulatory effect of insulin on de novo fatty acid synthesis was reduced in hepatocytes from chf - fed wild - type mice, whereas it was retained in the hepatocytes from chf - fed ampk2 mice (fig. chf+f feeding tended to restore the stimulatory effect of insulin only in wild - type hepatocytes (fig. the effect of differential dietary treatment on the regulation of metabolic fluxes in the liver. aicar - stimulated fatty acid oxidation (a) and insulin - stimulated de novo fatty acid synthesis (b) in cultured hepatocytes isolated from wild - type and ampk2 mice fed for 9 weeks either a chow diet, chf, or chf+f. for basal the expression of scd-1 (c) and srebp-1c (d) genes was quantified in total rna isolated from the livers of mice subjected to hyperinsulinemic - euglycemic clamp following the differential dietary treatment for 9 weeks. the data are means se (isolated hepatocytes, n = 3 in triplets ; hepatic gene expression, n = 58). p < 0.05 versus genotype chow ; p < 0.05 versus genotype chf ; p < 0.05 versus wild - type on respective diet., arbitrary units. the composition of fatty acids in hepatic diacylglycerol fraction in ad libitum - fed wild - type and ampk2 mice : total fatty acids (tfas ; a), pufas (b), monounsaturated fatty acids (mufas ; c), and saturated fatty acids (sfas ; d). animals were fed either a chow diet, chf, or chf+f for 9 weeks. p < 0.05 versus genotype chow ; p < 0.05 versus genotype chf ; p < 0.05 versus wild - type on respective diet. for the detailed fatty acid composition of diacylglycerol fractions in the livers of ad libitum fed mice, see supplementary table 4. to further characterize hepatic effects of differential dietary treatment, the expression of selected genes was quantified in total rna isolated from the livers of mice subjected to hyperinsulinemic - euglycemic clamp (fig. 4c and d). feeding chf diet suppressed expression of lipogenic genes stearoyl - coa desaturase (scd-1) and srebp-1c in all groups (except for srebp-1c in ampk2 mice). this suppression was partially counteracted by chf+f diet in wild - type but not ampk2 mice (fig. 4c and d). together with the de novo fatty acid synthesis data, these results further support the ampk2-dependent improvement of liver insulin sensitivity by n-3 lc - pufas. to identify factors predisposing animals to insulin resistance in an ampk2-dependent manner, no major genotype - dependent differences in the contents of either ceramides or phospholipids were observed (supplementary table 4). in contrast, hepatic content of diacylglycerols was affected in a genotype- and diet - dependent manner (fig. wild - type mice fed the chf+f diet had lower diacylglycerol content than genotype - matched chf diet - fed mice, while this effect of the chf+f diet was not observed in ampk2 mice. moreover, the analysis of fatty acid composition of the diacylglycerol fraction in the liver revealed that wild - type as well as ampk2 mice fed chf diet were characterized by marked increase in the level of pufa but not monounsaturated or saturated fatty acids (fig. the increase in the pufa content tended to be smaller in the wild - type compared with ampk2 mice (1.7-fold and 2.2-fold, respectively). administration of n-3 lc - pufas completely prevented accumulation of hepatic polyunsaturated diacylglycerols in wild - type mice, whereas their level in the ampk2 animals, although decreased, was still significantly higher compared with genotype - matched chow - fed mice (fig. -linolenic acid (18:3n-3) appeared to be by far the most differentially regulated pufa in the diacylglycerol fraction in the two genotypes (supplementary table 4). in addition, chf+f diet markedly reduced hepatic content of monounsaturated diacylglycerols in wild - type but not in knockout animals (fig. hepatic diacylglycerol levels and their fatty acid composition were also analyzed in mice subjected to hyperinsulinemic - euglycemic clamp (supplementary table 5 and supplementary fig. no significant differences among the groups were observed in total diacylglycerols content or in their saturated or monounsaturated fatty acid fractions (supplementary fig. specific activities of ampk1 and ampk2 were evaluated in the liver of ad libitum - fed mice after nine weeks of the differential dietary treatment (fig. no significant effect of either diet (chow, chf, and chf+f) or genotype (wild - type versus ampk2) on ampk1-specific activity was observed, although the ampk1 activity tended to be higher in the ampk2 mice (fig. in contrast, ampk2 activity was stimulated by n-3 lc - pufas (chf+f diet ; fig. no changes were detected in the activity of ampk1 and ampk2 in the quadriceps muscle in response to n-3 lc - pufas (not shown). liver ampk1 (a) and ampk2 (b) activity in wild - type and ampk2 mice fed either a chow diet, chf, or chf+f for 9 weeks. the data are the means se (n = 58). in the ampk2 mice, at four months of age, at the beginning of dietary treatments, wild - type and ampk2 mice fed the chow diet exhibited similar body weights (table 1). in mice of both genotypes, chf - feeding for nine weeks resulted in greater body weight gain compared with the chow - fed mice. however, this effect was less pronounced in ampk2 mice (table 1). in both wild - type and ampk2 mice, the chf+f diet induced smaller body weight gain than the chf diet (table 1 and supplementary figure 1). none of the differences in body weight gain could be explained by caloric intake, which was similar in all experimental groups (table 1). the weight of fat depots increased in response to chf feeding, while the chf+f diet partially prevented this increase (table 1). triglycerides and nefa levels in plasma of ad libitum - fed mice were similar in the chow- and chf - fed mice, while cholesterol levels were markedly and significantly elevated by the chf diet. triglycerides and nefa levels were strongly reduced, even below the levels observed in the chow - fed mice (table 1). metabolic and plasma parameters in wild - type and ampk2 mice data are means se of 2730 mice for metabolic parameters and 1315 mice for other measures. ampk2 and wild - type mice were fed either a chow diet, chf, or chf+f for nine weeks. body weight gain (see supplementary fig. 1) and plasma parameters were assessed in ad libitum - fed mice after nine weeks. plasma insulin levels were also assessed in fasted mice after eight weeks. at, adipose tissue ; a.u., arbitary units ; hmw, total adiponectin, ratio of high molecular weight to total adiponectin (for levels of all molecular weight forms of adiponectin ; nd, no data ; see supplementary fig. 2) ; p < 0.05 vs. genotype chow ; p < 0.05 vs. genotype chf ; p < 0.05 vs. wild - type on respective diet. after nine weeks of dietary treatment, no change was observed in blood glucose, but elevations were observed in plasma insulin levels in response to the chf diet in ad libitum - fed mice of both genotypes. however, the increase in plasma insulin levels was less pronounced in ampk2 mice, closely reflecting the genotype - dependent differences in body weight gain (table 1). a similar pattern of changes in insulin levels was also observed in fasted mice, in which n-3 lc - pufas significantly reduced insulin levels only in wild - type animals (table 1). as expected, plasma levels of total as well as high molecular weight form of adiponectin, an adipokine associated with increased insulin sensitivity (35), were increased 1.4- and 1.2-fold, respectively, in wild - type mice in response to n-3 lc - pufa supplementation (table 1 and supplementary fig. 2, available in an online appendix) ; however, no significant increase of plasma adiponectin by n-3 lc - pufas was observed in ampk2 mice. in further experiments, hyperinsulinemic - euglycemic clamps were performed to evaluate whole - body insulin sensitivity. under basal conditions, glucose turnover rate (gto ; i.e., glucose uptake in peripheral tissues) was similar in all groups of mice (supplementary table 1). under insulin - stimulated conditions (fig. f), the amount of exogenous glucose required to maintain euglycemia during the clamp, i.e., the glucose infusion rate (gir), was 1.3-fold lower in ampk2 than in wild - type mice fed the chow diet (fig gir was decreased by the chf diet to a similar level in mice of both genotypes, manifesting diet - induced insulin resistance. this was attributed to a decreased gto and, in particular, to an impaired suppression of hepatic glucose production (hgp) by insulin, with hgp being 8.5-fold higher in the chf - fed compared with the chow - fed wild - type mice (fig. 2c). in wild - type mice, chf+f diet feeding increased gir and gto (1.9- and 1.2-fold increase, respectively) as compared with chf - fed mice, while hgp was lowered to a similar level as in the chow - fed mice. these results document the protective effects of n-3 lc - pufas from high - fat diet - induced insulin resistance in wild - type mice, namely, at the level of hgp. in contrast, none of these beneficial effects of n-3 lc - pufas was observed in ampk2 mice, in which neither the gir (fig. 2b) differed between the chf+f - fed and the chf - fed mice, whereas the rate of hgp was even higher in the chf+f - fed than in the chf - fed ampk2 mice (fig. 2c). 2d), the rate of whole - body glycogen synthesis, which reflects insulin sensitivity of muscle glucose metabolism, was dependent on both diet and genotype (fig. the rate of whole - body glycogen synthesis tended to be higher in wild - type mice than in ampk2 mice. only in the former mice was it significantly affected by dietary treatment. thus, in wild - type mice, glycogen synthesis was decreased 2.4-fold in response to the chf diet, while n-3 lc - pufas provided a partial protection from this decrease (fig. a similar pattern of changes in the glycogen synthesis rate in response to n-3 lc - pufas was observed when measured directly in the skeletal muscle (fig. thus, in accordance with the previous study (26), the results of clamp studies suggested impairment of insulin sensitivity in response to whole - body ablation of ampk2 in chow - fed mice. however, ampk2 mice seemed to be partially protected against chf - induced insulin resistance, while ampk2 was required for preservation of insulin sensitivity in the skeletal muscle and especially in the liver in response to n-3 lc - pufa feeding. gir (a), gto (b), hgp (c), whole - body glycolysis (gl - wb ; d), whole - body glycogen synthesis (gs - wb ; e) ; and glycogen synthesis in quadriceps muscle (gs - qm ; f) were measured in wild - type and ampk2 mice fed either a chow diet, chf, or chf+f for 9 weeks. p < 0.05 versus genotype chow ; p < 0.05 versus genotype chf ; p < 0.05 versus wild - type on respective diet. in addition to the ad libitum - fed mice (table 1), plasma lipid levels were also measured in fasted mice, as well as in mice subjected to hyperinsulinemic - euglycemic clamp (supplementary table 2). in contrast to the ad libitum - fed state, chf+f diet did not affect either triglyceride or nefa levels under fasting conditions. under the hyperinsulinemic - euglycemic conditions, both triglyceride and nefa levels were lower in the chf+f - fed than in the chf - fed wild - type mice (1.6-fold and 1.4-fold difference, respectively), but cholesterol levels were consistently decreased by n-3 lc - pufas independently of both the metabolic state and genotype (supplementary table 2). in ad libitum - fed mice of both genotypes, the hepatic triglyceride content was increased twofold by chf compared with the chow diet, while triglyceride accumulation was increased only 1.3-fold by chf+f diet in both genotypes, documenting a protection against hepatic triglyceride accumulation by n-3 lc - pufas (fig. 3a). under hyperinsulinemic - euglycemic conditions, n-3 lc - pufas also protected livers of wild - type mice against the chf - induced accumulation of triglycerides. however 3b). moreover, a strong correlation was found between plasma nefa levels and hepatic triglyceride content assessed under the clamp conditions in the chf+f - fed ampk2 mice (r = 0.43, p < 0.05) but not in wild - type mice (r = 0.08, p = 0.40). triglyceride concentration in the livers of ad libitum - fed mice (a) and mice killed at the end of a 3-h infusion period of the hyperinsulinemic - euglycemic clamp (b). wild - type and ampk2 mice were fed either a chow diet, chf, or chf+f for 9 weeks. the data are the means se (a, n = 1315 ; b, n = 814). p < 0.05 versus genotype chow ; p < 0.05 versus genotype chf ; p < 0.05 versus wild - type on respective diet. for the detailed fatty acid composition of triglyceride fractions in the livers of ad libitum - fed mice, see supplementary table 4. we sought to determine whether the differential effect of n-3 lc - pufas on accumulation of liver triglycerides in wild - type and ampk2 mice under the clamp conditions could be explained by hepatic lipid metabolism. in cultured hepatocytes isolated from mice following the different dietary treatments, activities of both fatty acid oxidation and de novo fatty acid synthesis were evaluated. the stimulatory effects of 5-aminoimidazole-4-carboxamide-1--d - ribofuranoside (aicar), an ampk activator, and insulin on fatty acid oxidation and synthesis are shown in fig. 4a and b, respectively (for corresponding basal metabolic activities, see supplementary table 3, available in an online appendix). in hepatocytes from both chow and chf diet - fed mice, the absence of ampk2 was associated with a trend for lower aicar - stimulated fatty acid oxidation. although hepatocytes from chf - fed mice showed reduced stimulatory effect of aicar irrespective of the genotype, chf+f feeding normalized this defect in wild - type but not in ampk2 hepatocytes (fig. 5a), suggesting ampk - dependent induction of capacity for fatty acid oxidation by n-3 lc - pufas in the liver. the stimulatory effect of insulin on de novo fatty acid synthesis was reduced in hepatocytes from chf - fed wild - type mice, whereas it was retained in the hepatocytes from chf - fed ampk2 mice (fig. chf+f feeding tended to restore the stimulatory effect of insulin only in wild - type hepatocytes (fig. the effect of differential dietary treatment on the regulation of metabolic fluxes in the liver. aicar - stimulated fatty acid oxidation (a) and insulin - stimulated de novo fatty acid synthesis (b) in cultured hepatocytes isolated from wild - type and ampk2 mice fed for 9 weeks either a chow diet, chf, or chf+f. for basal the expression of scd-1 (c) and srebp-1c (d) genes was quantified in total rna isolated from the livers of mice subjected to hyperinsulinemic - euglycemic clamp following the differential dietary treatment for 9 weeks. the data are means se (isolated hepatocytes, n = 3 in triplets ; hepatic gene expression, n = 58). p < 0.05 versus genotype chow ; p < 0.05 versus genotype chf ; p < 0.05 versus wild - type on respective diet. a.u., arbitrary units. the composition of fatty acids in hepatic diacylglycerol fraction in ad libitum - fed wild - type and ampk2 mice : total fatty acids (tfas ; a), pufas (b), monounsaturated fatty acids (mufas ; c), and saturated fatty acids (sfas ; d). animals were fed either a chow diet, chf, or chf+f for 9 weeks. < 0.05 versus genotype chow ; p < 0.05 versus genotype chf ; p < 0.05 versus wild - type on respective diet. for the detailed fatty acid composition of diacylglycerol fractions in the livers of ad libitum fed mice, see supplementary table 4. to further characterize hepatic effects of differential dietary treatment, the expression of selected genes was quantified in total rna isolated from the livers of mice subjected to hyperinsulinemic - euglycemic clamp (fig. feeding chf diet suppressed expression of lipogenic genes stearoyl - coa desaturase (scd-1) and srebp-1c in all groups (except for srebp-1c in ampk2 mice). this suppression was partially counteracted by chf+f diet in wild - type but not ampk2 mice (fig. together with the de novo fatty acid synthesis data, these results further support the ampk2-dependent improvement of liver insulin sensitivity by n-3 lc - pufas. to identify factors predisposing animals to insulin resistance in an ampk2-dependent manner, a detailed analysis of hepatic lipids in ad libitum - fed mice was performed. no major genotype - dependent differences in the contents of either ceramides or phospholipids were observed (supplementary table 4). in contrast, hepatic content of diacylglycerols was affected in a genotype- and diet - dependent manner (fig. wild - type mice fed the chf+f diet had lower diacylglycerol content than genotype - matched chf diet - fed mice, while this effect of the chf+f diet was not observed in ampk2 mice. moreover, the analysis of fatty acid composition of the diacylglycerol fraction in the liver revealed that wild - type as well as ampk2 mice fed chf diet were characterized by marked increase in the level of pufa but not monounsaturated or saturated fatty acids (fig. the increase in the pufa content tended to be smaller in the wild - type compared with ampk2 mice (1.7-fold and 2.2-fold, respectively). administration of n-3 lc - pufas completely prevented accumulation of hepatic polyunsaturated diacylglycerols in wild - type mice, whereas their level in the ampk2 animals, although decreased, was still significantly higher compared with genotype - matched chow - fed mice (fig., -linolenic acid (18:3n-3) appeared to be by far the most differentially regulated pufa in the diacylglycerol fraction in the two genotypes (supplementary table 4). in addition, chf+f diet markedly reduced hepatic content of monounsaturated diacylglycerols in wild - type but not in knockout animals (fig. hepatic diacylglycerol levels and their fatty acid composition were also analyzed in mice subjected to hyperinsulinemic - euglycemic clamp (supplementary table 5 and supplementary fig. no significant differences among the groups were observed in total diacylglycerols content or in their saturated or monounsaturated fatty acid fractions (supplementary fig. previous animal studies demonstrated that n-3 lc - pufas could counteract the development of both hepatic steatosis (8,18,36,37) and hepatic insulin resistance (8,9,16), while suppressing lipogenesis and augmenting lipid catabolism in the liver (8,13,19,21). using mice with a whole - body deletion of ampk2 and high - fat feeding, we show for the first time that ampk2 is required for the effect of n-3 lc - pufas to preserve whole - body, muscle, and especially hepatic insulin sensitivity, as well as to suppress hepatic and plasma triglycerides as well as nefa levels under hyperinsulinemic - euglycemic clamp conditions. in contrast, ampk2 was not required for protection by n-3 lc - pufas from hepatic lipid accumulation and dyslipidemia in ad libitum - fed mice. in addition to ampk2, ppar was previously identified as an important determinant of n-3 lc - pufa 's effect on lipid metabolism, especially short - term modulation of hepatic gene expression (14) and insulin sensitivity (16). thus, the reduction in hepatic triglyceride concentrations by fish oil feeding did not rescue insulin action in ppar-null mice, while hepatic diacylglycerol concentrations were decreased by fish oil in a ppar-dependent manner and were associated with a preserved hepatic insulin sensitivity (16). it is generally accepted that 1) diacylglycerols rather than triglycerides or ceramides mediate hepatic insulin resistance in mice fed a high - fat diet (19,38,39), 2) diacylglycerol - induced insulin resistance depends on activation of protein kinase c, and 3) that polyunsaturated diacylglycerols in particular are better protein kinase c activators than saturated diacylglycerol species [reviewed in refs (38,39) ]. also our results showed that chf diet - induced insulin resistance was associated primarily with the accumulation of pufa in hepatic diacylglycerols and that n-3 lc - pufa completely prevented chf diet - induced increase in pufa diacylglycerols in wild - type mice, whereas in ampk2 animals, the content of these lipids was still significantly higher compared with the control. moreover, it was only in ad libitum - fed mice but not in mice subjected to hyperinsulinemic - euglycemic clamps that the levels of hepatic diacylglycerols and their fatty acid compositions were associated with hepatic insulin sensitivity. it is possible that under clamp conditions ampk2-dependent effects of n-3 lc - pufas on liver diacylglycerols were masked by metabolic changes occurring during a 3-h infusion of insulin and glucose. the failure of n-3 lc - pufas to decrease hepatic lipids in ampk2 mice under clamp conditions could be due to primary alterations in metabolic fluxes in the liver, reflecting 1) increased de novo fatty acid synthesis, 2) decreased secretion of vldl triglycerides, or 3) reduced fatty acid oxidation. de novo fatty acid synthesis was not the responsible factor, because hepatocytes of the n-3 lc - pufa - fed ampk2 mice showed decreased insulin - stimulated de novo fatty acid synthesis and reduced expression of srebp-1c and scd-1, as compared with hepatocytes isolated from n-3 lc - pufa - fed wild - type mice, reflecting probably low insulin sensitivity of the liver in ampk2 mice. moreover, aicar - stimulated fatty acid oxidation in hepatocytes from n-3 lc - pufa - fed ampk2 mice was markedly reduced as compared with those from wild - type mice, suggesting decreased hepatic capacity for fatty acid oxidation in the absence of ampk2, which could contribute to enhanced lipid accumulation. therefore, these experiments supported a major role of hepatic ampk2 in the regulation of both insulin sensitivity and lipid metabolism by n-3 lc - pufas. however, the differential modulation of lipid accumulation by n-3 lc - pufas in the livers of wild - type and ampk2 mice under clamp conditions could also be secondary to the ampk2-dependent effects of n-3 lc - pufas in other tissues, resulting in a relatively high hepatic uptake of circulating nefa in ampk2 mice. this is supported by persistently elevated plasma levels of nefa in ampk2 mice, as well as by a significant correlation between plasma nefa levels and hepatic triglyceride content observed under clamp conditions in ampk2 but not wild - type mice fed n-3 lc - pufa - containing diet. moreover, it has been shown in humans with nonalcoholic fatty liver disease that most of hepatic triglycerides arise from circulating nefa (40). that plasma nefa levels under clamp conditions were reduced only in wild - type but not in ampk2 mice fed n-3 lc - pufas may reflect a role of ampk2 in muscle lipid uptake mediated by lipoprotein lipase (41), as well as the antilipolytic effect of ampk in adipose tissue, documented for ampk1 (42). in any case, decreased fatty acid oxidation in situ in the liver and, possibly even more importantly, abundant supply of circulating nefa could be responsible for the lack of the antisteatotic effect of n-3 lc - pufas in ampk2 mice under clamp conditions (fig.. putative involvement of ampk2 in antisteatotic action of n-3 lc - pufas in the liver. dietary intake of n-3 lc - pufas increases the capacity of hepatocytes to oxidize fatty acids in wild - type (left panels) but not in ampk2 mice (right panels). when insulin and glucose levels are high, such as during hyperinsulinemic - euglycemic clamp, wild - type mice fed n-3 lc - pufas exhibit improved hepatic insulin sensitivity and decreased plasma levels of nefas as compared with high - fat diet - fed controls. this is associated with increased expression of lipogenic genes such as srebp-1c and scd-1 and increased drive for de novo fatty acid synthesis. despite the elevated lipogenic drive under clamp conditions, the livers of wild - type mice fed n-3 lc - pufas show reduced accumulation of triglycerides. however, in ampk2 mice fed n-3 lc - pufas, hepatic triglyceride content is markedly elevated despite reduced rates of de novo fatty acid synthesis. this effect could be secondary to persisting elevated nefa levels in circulation and thus better substrate availability in ampk2 mice under clamp conditions. previous studies reported contradictory results, showing either 1) activation of ampk in rat liver (22) and murine adipose tissue (24) or 2) no changes in ampk activity in the liver, skeletal muscle, and heart of mice (43) in response to dietary n-3 lc - pufas. these discrepancies could be related to differences in dietary n-3 lc - pufa intake, nutritional state of animals, and other parameters. in accordance with the involvement of ampk2 in various effects of n-3 lc - pufas, our results document activation of ampk2 (but not ampk1) in the liver of mice by long - term n-3 lc - pufa treatment, in the absence of significant changes in either the amp to atp ratio assessed in whole liver extracts [not shown and ref (44) ] or the phosphorylation status of lkb1, an upstream kinase for ampk [not shown and ref (45) ]. in addition, no effect on ampk activity in either cultured hepatocytes (21) or embryonic kidney cells (not shown) of n-3 lc - pufas added to the cell culture medium could be detected. therefore, the activation of ampk2 by n-3 lc - pufas probably does not depend on a direct interaction between n-3 lc - pufas and ampk. on the other hand, induction of adiponectin by n-3 lc - pufas [results of this study and refs (46,47) ] could be involved, because adiponectin activates ampk in both the liver and skeletal muscle (35). adiponectin is also required for the activation of ampk upon administration of ppar agonists thiazolidinediones, whereas mice lacking adiponectin show decreased hepatic insulin sensitivity and reduced responsiveness to these compounds (48). thus, absence of ampk2 may blunt adiponectin - mediated effects of n-3 lc - pufas. in accordance with the previous study (49) moreover, the induction of adiponectin by n-3 lc - pufas in ampk2 mice was compromised (table 1 and supplementary figure 2). ampk1 and ampk2 contribute equally to total ampk activity in the liver (50). in mice with liver - specific ablation of ampk2 (27), hepatic ampk2 was essential for suppressing hepatic glucose production and maintaining fasting blood glucose levels ; however, the absence of ampk2 did not affect inhibitory action of insulin on hepatic glucose production. in our study, although fasting blood glucose levels were unaltered by whole - body ablation of ampk2, the beneficial effect of dietary n-3 lc - pufas on hepatic insulin sensitivity was clearly ampk2-dependent. differential regulation of glucose homeostasis in the above transgenic models likely reflects the complexity of whole - body (26) versus liver - specific (27) deletion of ampk2. in contrast to the previous report, showing induction of adiposity and adipocyte hypertrophy in ampk2 mice fed a lard - based high - fat diet (49), our study documented a relatively low weight gain, low adiposity, and smaller fat cells in ampk2 mice fed a corn - oil based high - fat diet. this discrepancy could be related to the differences in the composition of experimental high - fat diets ; however, our results are consistent with the elevated sympathetic tonus of ampk2 mice (26), which may stimulate energy dissipation in these animals. in any case, lower body weight of chf - fed ampk2 mice as compared with their wild - type counterparts could be related to better insulin sensitivity of the former mice, as suggested by the differences in insulinemia, results of hyperinsulinemic - euglycemic clamp, stimulatory effect of insulin on lipogenesis, and expression of lipogenic genes in the liver. in conclusion, the preservation of hepatic insulin sensitivity by n-3 lc - pufas in mice fed a high - fat diet depends on ampk2. the accumulation of diacylglycerols, which is regulated in an ampk2-dependent manner, could contribute to the modulation of hepatic insulin sensitivity in response to dietary n-3 lc - pufas. on the other hand, the ampk2-dependent acute changes in lipid metabolism and hepatic triglyceride accumulation, which are unmasked under insulin - stimulated conditions such as during hyperinsulinemic - euglycemic clamp, largely reflect the extrahepatic action of n-3 lc - pufas. our results are relevant for the development of novel strategies for prevention and treatment of the metabolic syndrome. | objectivethe induction of obesity, dyslipidemia, and insulin resistance by high - fat diet in rodents can be prevented by n-3 long - chain polyunsaturated fatty acids (lc - pufas). we tested a hypothesis whether amp - activated protein kinase (ampk) has a role in the beneficial effects of n-3 lc-pufas.research design and methodsmice with a whole - body deletion of the 2 catalytic subunit of ampk (ampk2/) and their wild - type littermates were fed on either a low - fat chow, or a corn oil - based high - fat diet (chf), or a chf diet with 15% lipids replaced by n-3 lc - pufa concentrate (chf+f).resultsfeeding a chf diet induced obesity, dyslipidemia, hepatic steatosis, and whole - body insulin resistance in mice of both genotypes. although chf+f feeding increased hepatic ampk2 activity, the body weight gain, dyslipidemia, and the accumulation of hepatic triglycerides were prevented by the chf+f diet to a similar degree in both ampk2/ and wild - type mice in ad libitum - fed state. however, preservation of hepatic insulin sensitivity by n-3 lc - pufas required functional ampk2 and correlated with the induction of adiponectin and reduction in liver diacylglycerol content. under hyperinsulinemic - euglycemic conditions, ampk2 was essential for preserving low levels of both hepatic and plasma triglycerides, as well as plasma free fatty acids, in response to the n-3 lc - pufa treatment.conclusionsour results show that n-3 lc - pufas prevent hepatic insulin resistance in an ampk2-dependent manner and support the role of adiponectin and hepatic diacylglycerols in the regulation of insulin sensitivity. ampk2 is also essential for hypolipidemic and antisteatotic effects of n-3 lc - pufa under insulin - stimulated conditions. |
chromatin compaction represents a barrier for the repair of dna double - strand breaks (dsbs). however, heterochromatin components are also required for dsb repair by homologous recombination. the bard1/hp1 interaction, required for the retention of brca1, ctip, and rad51 at dsb sites, may play a critical role in the crosstalk between chromatin compaction and dsb repair. |
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currently anti - tnf agents are widely used for cutaneous and systemic autoimmune diseases. for dermatologists most common indications for anti - tnf use are psoriasis and psoriatic arthritis. we describe a case of scedosporium apiospermum infection in a patient receiving long - term treatment with etanercept, a tnf- inhibitor. s. apiospermum is a ubiquitous filamentous fungus found in soil, polluted water, and contaminated ambient air in hospital isolation rooms. in immunocompromised patients, increasing reports of s. apiospermum infection in the past few years suggests that s. apiospermum is an emergent opportunistic pathogen. infectious diseases including tuberculosis, atypical mycobacteria, histoplasmosis, coccidioidomycosis, and various opportunistic infections in patients receiving tnf - a inhibitors have been reported in the literature and postmarketing surveillance. a pubmed search resulted in one report of scedosporium infection as a complication of infliximab therapy for ankylosing spondylitis. to the best of our knowledge, this is the first reported case of s. apiospermum infection in a patient receiving etanercept. neither he had active psoriasis lesions, but he had pitting and ridging on nails. after extensive workup, we narrowed our differential diagnosis to ankylosing spondylitis and psoriatic arthritis. he was started on methotrexate because of continued neck and lower back pain with morning stiffness. in 2004, etanercept was initiated at the dose of 50 mg / week and sulfasalazine was discontinued. he did remarkably well with etanercept in respect to back pain and morning stiffness. in 2008 as he was receiving an anti - tnf agent, we actively pursued investigation for opportunistic and rare infectious agents. a flexible endoscopic examination of the sinuses demonstrated mucopurulent ethmoid disease. tissue specimens of the right and left nasal turbinates and nasal washes were sent for evaluation and culture. the tissue isolates showed chronic inflammatory edematous respiratory tissue without any significant pathology in the underlying bones and gms stains showed no evidence of fungus. however, cultures of the specimen from the sphenoid sinuses returned positive for s. apiospermum. a mri of head and sinuses did not demonstrate any evidence for invasive rhinocerebral fungal disease. we consulted our infectious disease colleagues and the patient was started on voriconazole 200 mg bid. follow - up mri and ct of the brain and sinuses did not show any evidence for sinusitis or invasion of the adjacent anatomical structures. as there was no histopathological or radiological evidence for invasive fungal infection we felt comfortable to continue etanercept. the patient has continued to take both etanercept and methotrexate for more than 1 year with no signs of re - infection or complication. scedosporium apiospermum has been associated with mycetoma, keratitis, endophthalmitis, osteomyelitis, and brain abscesses. a wide range of pulmonary manifestations exists, ranging from simple colonization as seen in patients with cystic fibrosis to fungus ball formation in patients with cavitary lesions and invasive disease, simulating aspergillosis. as the clinical presentation of scedosporium infection, including fever, cough, and dyspnea is nonspecific, diagnosis can be based on cytology, histopathology, and isolation of the fungus in culture. culture confirmation is important as the histological appearance and clinical presentation of s. apiospermum are difficult to distinguish from that of the aspergillus species on pathological examination. in a case report of three lung transplant patients, mean - time from specimen collection to positive - culture identification for sputum culture was 4.5 days compared to 9.5 days with bronchoalveolar lavage culture. in our patient, positive culture was identified in 10 days. s. apiospermum has been shown to have intrinsic resistance to many anti - fungal agents, including fluconazole and amphotericin b, the traditional antifungal of choice for disseminated hyalohyphomycoses. much of the data on the treatment of s. apiospermum pertains to the use of voriconazole. a retrospective review of 107 patients treated for scedosporium infection with voriconazole showed 57% of patients achieved a successful response after a median of 103 days of therapy. in vitro studies have demonstrated that voriconazole is more active against s. apiospermum than either itraconazole or amphotericin b. the optimal choice and duration of therapy remain unknown. surgical debridement or drainage for limited disease in combination with antifungal therapy, as was done successfully with our patient, is recommended now. | patients on anti - tnf therapy are at increased risk for rare opportunistic infections. here we are reporting a case of scedosporium apiospermum infection in a patient treated with anti - tnf for 5 years. patients on anti - tnf need close follow - up and clinicians should be suspicious for atypical infections in these immunocompromised hosts. |
forty - four consecutive american society of anesthesiologists (asa) classes i - ii adult patients scheduled for elective inguinal hernia repair surgery under spinal anesthesia were included in this prospective randomized controlled clinical trial. the treatment group was prepared with cho and the control group fasted from midnight. during the evening before surgery, patients in the cho group ingested 800 ml of an iso - osmolar carbohydrate - rich drink [12.5% carbohydrates (glucose : 0.2 g, maltose : 0.7 g, polysaccharides : 10 g), 50 kcal/100 ml, 290 mosm / kg, ph 5.0 ; nutricia preop ; numico, zoetermeer, the netherlands ]. the patients in the control group underwent spinal anesthesia after the routine fast from midnight. nothing per os was allowed from midnight except another 400 ml of cho in the morning at least 90 minutes before spinal anesthesia in the cho group. the patients scored their subjective sense of discomfort with 100-mm visual analogue scales (vas) on five different occasions : 1) as a baseline (control) approximately 90 - 120 minutes after lunch the day before the operation ; 2) before intake of the morning drink (bi) ; 3) 40 minutes after the morning drink (40 min ai) ; 4) 90 minutes after the morning drink (90 min ai) ; and 5) 60 minutes after the spinal anesthesia (60 min sa). ten different variables were evaluated : malaise, thirst, hunger, unfitness, tiredness, nausea, pain, inability to concentrate, anxiety and depression. in order to measure blood glucose and insulin concentrations, blood samples were obtained before, and 40 and 90 minutes after the morning drink or at corresponding time points for the control group. the data except the perioperative discomfort variables are presented as means standard deviation (sd). patients characteristics were analyzed with student t - test and chi - square tests between groups. friedman 's two - way analysis of variance (anova) was used for within - group trend analysis of the vas measurements. changing patterns of glucose, insulin, mean arterial blood pressure and heart rate were evaluated by repeated measurements of anova in each group and mann - whitney u test between groups forty - four consecutive american society of anesthesiologists (asa) classes i - ii adult patients scheduled for elective inguinal hernia repair surgery under spinal anesthesia were included in this prospective randomized controlled clinical trial. the treatment group was prepared with cho and the control group fasted from midnight. during the evening before surgery, patients in the cho group ingested 800 ml of an iso - osmolar carbohydrate - rich drink [12.5% carbohydrates (glucose : 0.2 g, maltose : 0.7 g, polysaccharides : 10 g), 50 kcal/100 ml, 290 mosm / kg, ph 5.0 ; nutricia preop ; numico, zoetermeer, the netherlands ]. the patients in the control group underwent spinal anesthesia after the routine fast from midnight. nothing per os was allowed from midnight except another 400 ml of cho in the morning at least 90 minutes before spinal anesthesia in the cho group. the patients scored their subjective sense of discomfort with 100-mm visual analogue scales (vas) on five different occasions : 1) as a baseline (control) approximately 90 - 120 minutes after lunch the day before the operation ; 2) before intake of the morning drink (bi) ; 3) 40 minutes after the morning drink (40 min ai) ; 4) 90 minutes after the morning drink (90 min ai) ; and 5) 60 minutes after the spinal anesthesia (60 min sa). ten different variables were evaluated : malaise, thirst, hunger, unfitness, tiredness, nausea, pain, inability to concentrate, anxiety and depression. in order to measure blood glucose and insulin concentrations, blood samples were obtained before, and 40 and 90 minutes after the morning drink or at corresponding time points for the control group. the data except the perioperative discomfort variables are presented as means standard deviation (sd). patients characteristics were analyzed with student t - test and chi - square tests between groups. friedman 's two - way analysis of variance (anova) was used for within - group trend analysis of the vas measurements. changing patterns of glucose, insulin, mean arterial blood pressure and heart rate were evaluated by repeated measurements of anova in each group and mann - whitney u test between groups forty - four consecutive american society of anesthesiologists (asa) classes i - ii adult patients scheduled for elective inguinal hernia repair surgery under spinal anesthesia were included in this prospective randomized controlled clinical trial. the treatment group was prepared with cho and the control group fasted from midnight. during the evening before surgery, patients in the cho group ingested 800 ml of an iso - osmolar carbohydrate - rich drink [12.5% carbohydrates (glucose : 0.2 g, maltose : 0.7 g, polysaccharides : 10 g), 50 kcal/100 ml, 290 mosm / kg, ph 5.0 ; nutricia preop ; numico, zoetermeer, the netherlands ]. the patients in the control group underwent spinal anesthesia after the routine fast from midnight. nothing per os was allowed from midnight except another 400 ml of cho in the morning at least 90 minutes before spinal anesthesia in the cho group. the patients scored their subjective sense of discomfort with 100-mm visual analogue scales (vas) on five different occasions : 1) as a baseline (control) approximately 90 - 120 minutes after lunch the day before the operation ; 2) before intake of the morning drink (bi) ; 3) 40 minutes after the morning drink (40 min ai) ; 4) 90 minutes after the morning drink (90 min ai) ; and 5) 60 minutes after the spinal anesthesia (60 min sa). ten different variables were evaluated : malaise, thirst, hunger, unfitness, tiredness, nausea, pain, inability to concentrate, anxiety and depression. in order to measure blood glucose and insulin concentrations, blood samples were obtained before, and 40 and 90 minutes after the morning drink or at corresponding time points for the control group. the data except the perioperative discomfort variables are presented as means standard deviation (sd). patients characteristics were analyzed with student t - test and chi - square tests between groups. friedman 's two - way analysis of variance (anova) was used for within - group trend analysis of the vas measurements. changing patterns of glucose, insulin, mean arterial blood pressure and heart rate were evaluated by repeated measurements of anova in each group and mann - whitney u test between groups. both groups were comparable with regard to age, sex, body mass index (bmi), asa classification, duration of the anesthesia and surgery, total amount of fluid administered, and urine volume (table 1). patients characteristics the cho group showed decreasing trends for discomfort variables of hunger (p < 0.001), thirst (p < 0.001), malaise (p < 0.05) and unfitness (p < 0.01) during the perioperative period (table 2). the anxiety level was decreased significantly only at 40 and 90 minutes after the morning drink compared to baseline and before the intake of morning drink (p < 0.05). in the control group, hunger (p < 0.001) and thirst (p < 0.01) variables increased during the perioperative period (table 2). the cho group experienced less hunger (p < 0.01), thirst (p < 0.05), malaise (p < 0.001), and unfitness (p < 0.05) at 40 and 90 minutes after the morning drink and 60 minutes after the spinal anesthesia compared with the control group (table 2). furthermore, the cho group was less anxious (p < 0.05) than the control group at 40 and 90 minutes after the morning drink. plasma glucose and insulin concentrations were increased in the cho group before spinal anesthesia compared to before intake of cho (105 4 mg / dl vs. 88 3 mg / dl, and 15 4 microunits / ml vs. 6 2 microunits / ml, respectively) and the control group (p < 0.05) (figures 1 and 2). increased plasma glucose and decreased insulin were observed in the control group at 60 minutes after the spinal anesthesia compared to the baseline (128 4 mg / dl vs. 90 4 mg / dl, and 3 1 microunits / ml vs. 8 3 microunits / ml, respectively). however, the corresponding levels returned to bi levels in the cho group, leading to significant differences between the groups (p < 0.05 ; figures 1 and 2). visual analogue scale (vas) data for perioperative discomfort variables perioperative plasma glucose concentration (mg / dl) in the two groups. bi = before intake of the morning drink (or corresponding time in the fasted group) ; ai = after intake of the morning drink (or corresponding time in the fasted group) ; 60 min sa = 60 min after the spinal anesthesia ; p < 0.05 compared to bi ; bi = before intake of the morning drink (or corresponding time in the fasted group) ; ai = after intake of the morning drink (or corresponding time in the fasted group) ; 60 min sa = 60 min after the spinal anesthesia ; p<0.05 compared to bi ; p<0.05 compared to the other group. in the control group, map was lower before and 10 and 20 minutes after the spinal anesthesia compared to the cho group and before intake of morning drink (or corresponding time in the control group) (p < 0.05) (table 3). first, preoperative administration of cho increased perioperative well - being compared with overnight fasting in patients undergoing elective inguinal hernia repair surgery with spinal anesthesia. preparation with cho was more effective than overnight fasting in reducing hunger, thirst, malaise, unfitness, and, to some extent, anxiety during the perioperative period. second, administration of oral cho before the spinal anesthesia improved insulin response and stabilized the mean arterial blood pressure during the spinal anesthesia. for evaluation of perioperative discomfort, the vas method was chosen and the variables in the vas questionnaire were the same as those used in several previous studies.89 compared to the control group, patients in the cho group were less hungry and thirsty and experienced less malaise and unfitness even 60 minutes after the spinal anesthesia. this is likely to be a remaining effect of the morning dose of carbohydrates on the post - spinal anesthesia period. energy intake with carbohydrates may have secondary effects on mood by making patients feel more at ease.10 it has been demonstrated previously that a carbohydrate - rich drink has a positive effect with regard to thirst, hunger, malaise, unfitness, and anxiety and should be initiated on the evening before surgery to observe the remaining effect of the previous evening dose of carbohydrates (100 g).8911 thus, our study seemed to be in the same line with the previous investigations. however, the similarity in anxiety level after the spinal anesthesia may be explained by the fact that patients were awake and had no sedation during the surgical procedure. the positive effects of carbohydrates on the perioperative period were accompanied by increases in glucose and insulin concentrations in the cho group. the response of insulin release to carbohydrate intake can be partitioned into the first phase (0 - 10 minutes ; release phase) and the second phase (10 - 60 minutes ; de novo biosynthesis phase). obviously, the data presented in figure 2 shows the second phase of insulin response. a dose of 400 ml of this beverage given to patients ready to undergo a surgery increased serum insulin to levels seen after a standard mixed meal, thereby providing enough energy to switch the patient from the fasted to the fed state before the onset of the surgery.12 fasting from the previous evening inhibited insulin secretion and the glycolysis system was enhanced in the morning of the operation day, resulting in an increase in the plasma glucose level. in the cho group, intake of carbohydrate - rich drink prevented the increase in the plasma glucose level by endogenous release of insulin. this, in turn, resulted in a less stressful response to the surgical trauma and improved insulin sensitivity postoperatively. in patients undergoing abdominal surgery, spinal anesthesia has been shown to produce efficient afferent blockade which resulted in pronounced reduction of the metabolic - endocrine response and improved insulin sensitivity.1314 in the control group, the observed increase in glucose concentrations together with a reduction in insulin concentrations did not exactly indicate the loss of normal insulin sensitivity. it may suggest reduced release or increased turnover rate of insulin due to perioperative hypo - caloric nutrition or reduction in whole body glucose disposal, or both.15 in the cho group, intake of morning drink before the spinal anesthesia improved map just before and after the spinal anesthesia. although the present study was not designed to elucidate the factors responsible for the stable map levels in the cho group, we can only speculate on the underlying endocrine mechanisms like improved glucose and insulin responses and the patients feeling more at ease during this period due to energy intake with carbohydrates. in conclusion, preparation with oral carbohydrate before spinal anesthesia had advantages over overnight fasting during the perioperative period by increasing patient well - being, improving insulin response and stabilizing mean arterial pressure in patients undergoing elective inguinal hernia repair surgery. hya carried out the design and coordinated the study, participated in most of the experiments and prepared the manuscript. | background : the aim of this study was to investigate the role of carbohydrate - rich drink (cho) on perioperative discomfort, hemodynamic changes, and insulin response in patients undergoing surgery with spinal anesthesia.methods:forty-four adult patients were assigned to one of the two groups of 22, namely preparation with cho (cho group) or fasting from midnight (control group). ten different discomfort variables, blood glucose and insulin concentrations, and hemodynamic changes were recorded during the perioperative period.results:preparation with cho was effective in reducing hunger, thirst, malaise, unfitness, and, to some extent, anxiety (p < 0.05). plasma glucose and insulin concentrations were increased in the cho group (p < 0.05). plasma glucose increased and insulin decreased in the control group (p < 0.05). in the control group, mean arterial pressure was lower compared to the cho group (p < 0.05).conclusions : preparation with cho before spinal anesthesia is advantageous due to reducing perioperative discomfort, improving insulin response and stabilizing mean arterial pressure. |
gestational diabetes mellitus (gdm), as a common pregnancy complication, is defined as glucose intolerance with onset or first recognition during pregnancy (1). gdm is associated with adverse perinatal outcome, and increased long - term risks of type 2 diabetes mellitus, metabolic syndrome and cardiovascular disorders for both mother and offspring (2 - 6). hence, the impact of gdm on maternal and infant health is of great clinical and public health importance. the prevalence of the disorder is increasing all over the world, but most noticeable in developing countries (7). like many developing countries, bosnia and herzegovina is experiencing increased prevalence of obesity and other risks of gdm development (8 - 10). the aim of this study was to determine gdm prevalence, maternal characteristics and pregnancy outcomes for the first time among women in bosnia and herzegovina by using the who diagnostic criteria. a cross - sectional observational study was conducted in the herzegovina - neretva county, southern part of bosnia and hezegovina from october 2010 through march 2011. actually, the research was conducted at two public, ante - natal clinics in the western part of mostar city and apljina city and in the university hospital mostar as a reference institution for deliveries in southern - western region of bosnia and herzegovina. the participants were pregnant women with singleton pregnancies aged 18 years or older who participated in this research during their usual ante - natal clinic visits. in the study period, as recommended for high - risk population, all pregnant women from the western part of mostar and apljina city who consecutively attended local public antenatal clinic at 22 - 32 weeks of gestation underwent an oral glucose tolerance test (ogtt) with 75 g glucose due to one or more gdm risk factors or characteristic pregnancy complications : pre - pregnancy overweight and obesity (maternal body mass index 25kg / m), family history of diabetes type 2, previous pregnancy complicated with gdm, birth weight 4000 g, stillbirth with no clear obstetric cause or major cardiovascular / cns malformation, history of polycystic ovary syndrome, current hypertensive disorder after 20 weeks of gestation, recurrence of glucosuria, and polyhydramnios. those with no gdm risk factors were advised to participate in the study at 22 - 32 weeks of gestation, and they were informed about ogtt procedure and the possible effects in the same manner as we informed the high - risk pregnant women. gestational age was determined by last menstrual period (lmp) and according to early ultrasound examination. exclusion criteria were diabetes type 1 or 2 prior to pregnancy, multifetal pregnancy and age younger than 18. all of them completed ogtt and gave birth at the university hospital mostar where the data were finally collected for the prevalence, maternal characteristics and pregnancy outcomes (premature delivery, cesarean delivery, large for gestational age / lga, birth trauma, neonatal hypoglycemia, neonatal hyperbilirubinemia, early neonatal death). neonatal hypoglycemia was defined as glucose level < 2.2 mmol / l or < 40mg / dl. neonatal hyperbilirubinemia was defined as jaundice requiring phototherapy, except the one caused by abo or rh isoimmunisation. each participant underwent a standard 2-h ogtt, with the use of a 75 g dose of glucose, and with plasma glucose levels (pgl) measured fasting and 2-hour after ingestion. pgl was measured by means of enzymatic methods according to international federation of clinical chemistry (ifcc). statistical analysis was performed using descriptive statistics, c test, fisher s exact test, and student s t - test for independent samples or mann - whitney u test depending on the sample distribution. mean and standard deviations, as well as, median and interquartile range are reported for continuous variables. software system spss for windows (11.0, spss inc, chicago, illinois, usa) was used for statistical analysis of the obtained data. a total of 285 participants had a diagnostic ogtt and complete data regarding maternal characteristics and perinatal outcomes during the study period were obtained. the mean age of the participants was 29.8 5.6 years (mean sd), and more than half of them were primiparas (51.2%). the gdm was diagnosed in 31 (10.9%) participants according to the who diagnostic criteria (table 2). cigarette smoking and previous gdm as gdm risk factors were significantly more frequent in the gdm group (p = 0.015, p < 0.001) compared to the group of pregnancies with normal glucose tolerance. further, table 2 shows differences in pregnancy complications and adverse perinatal outcomes of the participants according to gdm status. there were significant differences in cesarean deliveries and neonatal hypoglycemia (p = 0.015, p = 0.002). maternal and newborns characteristics, and pregnancy outcomes in the group of participants (n=285) comparison of the maternal characteristics, pregnancy complications and adverse outcomes between the groups of pregnancies with normal glucose tolerance (ngt) (n = 254) and gdm (n = 31) gestational diabetes mellitus has long been recognised as a complex problem in pregnancy due to significant levels of fetal and maternal morbidity (2, 4, 11). the prevalence of the disorder is increasing all over the world, but most noticeable in developing countries (7). the main objective of this study was to asses prevalence of gdm and associated risk factors as well as pregnancy outcomes in the southern part of bosnia and herzegovina. this is the first study to estimate the prevalence of gdm in this region of developing countries. gdm prevalence in european countries is most often reported as 2 - 6% of all pregnancies (12). reported gdm prevalence is higher in the southern mediterranean seaboard but also in some other parts of europe such as ireland (10%) and finland (10 - 11%) (12,13). accordingly, our unselected population of pregnant women should be classified to the high gdm prevalence group (10.9%). this result is comparable with the studies published from asian developing countries, including bangladesh (9.6%), malaysia (11.4%) and india (13.9%) (7, 14, 15). south asian ethnic origin was observed as an independent risk of gdm and type 2 diabetes development (16, 17). it is well known that the prevalence of gdm varies widely worlwide according to the various diagnostic criteria that are of significantly different sensitivities and specificities (18). the variation may be also due to age and ethnic structure of the study population, dietary habits diversity, and socioeconomic factors. the estimated high prevalence of gdm in developing countries, including bosnia and herzegovina could be explanined by socioeconomic factors. like many developing countries, bosnia and herzegovina is experiencing increased prevalence of obesity and other risks of gdm development due to socioeconomic status and lifestyle factors (8 - 10). it is likely that stressful events, dietary habits and sedentary life styles are responsible for the increase in obesity and gdm (19). in a previous study, cullinan at al. found increased prevalence of gdm for women in the lowest socioeconomic group when compared to the highest, suggesting a strong association between socioeconomic status and gdm prevalence (20). comparing maternal characteristics according to the gdm status we observed statistically significant association between gdm and prenatal cigarette smoking. previous studies of associations between cigarette smoking and gdm are uncommon and conflicting (10). however, prevalence of cigarette smoking as a behavioural risk factor in our pregnant women population is worrying in comparison with some other reports (21). this data is consistent with the observations published in some other studies (22, 23). previous studies reported older age as an independent risk factor for gdm, as well as increasing pre - pregnancy bmi and excessive gestational weight gain (10, 22 - 24). in the present study, the mean age of gdm participants as well as the mean pre - pregnancy bmi and the mean gestational weight gain was higher, but not statistically significant. in addition, family history of diabetes type 2, history of polycystic ovary syndrome, birth weight of the previous child 4 kg and previous stillbirth as gdm risk factors were more frequent in the gdm group compared to the group of pregnancies with normal glucose tolerance, but not significantly. most of the previous studies that reported significant associations between gdm and listed maternal charasteristics (family history od diabetes type 2, increasing pre - pregnancy bmi, excessive gestational weigt gain, history of polycystic ovary syndrome, birth weight of the previous child 4 kg and previous stillbirth) had significantly higher number of participants and thus increased statistical power (10, 22 - 25). analyzing specific gdm pregnancy complications and outcomes, we found significant difference between gdm group and normal tolerance glucose group in cesarean delivery rate and neonatal hypoglycemia. these data are consistent with previous published observations (26, 27). our study had some limitations. a cross - sectional design of our research could have resulted in selection bias because only women with singleton pregnancies who had an antenatal check - up during an observed time interval and in selected clinics participated in the study. further, our study did not have sufficient statistical power for maternal characteristics as gdm risk factors and pregnancy outcomes examination due to relatively small sample. there is a general observation that the prevalence of gdm is increasing in developing countries. accordingly, it is important to increase awareness of gdm among medical professionals and pregnant women in bosnia and herzegovina. ultimately, there is a need for large well - designed study on gdm prevalence and its other features in this region of the developing countries. | background : the prevalence of gestational diabetes mellitus (gdm), as a complex problem in pregnancy, is increasing all over the world, but most noticeable in developing countries.aims:to estimate gdm prevalence and associated pregnancy features in the southern part of bosnia and herzegovina.methods:a cross - sectional observational study was conducted from october 2010 through march 2011. a total of 285 pregnant women with singleton pregnancies participated and were asigned to the study in the order they came for their usual ante - natal clinic examination. they underwent an oral glucose tolerance test (ogtt) with 75 g of glucose. information on ogtt results, maternal characteristics and pregnancy outcomes were collected from database and medical records.results:prevalence of gdm was 10.9% according to 1999 world health organisation (who) diagnostic criteria. prenatal cigarette smoking, previous gdm, cesarean delivery rate and neonatal hypoglycemia were significantly more frequent in the gdm group compared to the group of pregnancies with normal glucose tolerance (p = 0.015, p < 0.001, p = 0.015, p = 0.002).conclusion : this study presents a relatively high prevalence of gdm in bosnia and herzegovina. there is a need for large well - designed study on gdm prevalence and its other features. |
intrahepatic arterial aneurysms are rare and typically related to trauma, transplantation, iatrogenic injury, or infection we present the case of a 49-year - old man with an intraparenchymal hepatic artery aneurysm that presented as massive hemobilia following a laparoscopic cholecystectomy. the aneurysm could not be managed by interventional embolization and required a left hepatic lobectomy, which was performed laparoscopically.. the diagnosis of hepatic artery aneurysm can be most readily made by mri or ct scan. interventional embolization of the aneurysm may be effective treatment but is not always possible due to anatomic considerations. where indicated, surgical resection in a manner that preserves a maximal amount of normal hepatic parenchyma is the treatment of choice. this is the first report of laparoscopic liver resection performed for bleeding from a hepatic artery aneurysm and adds an effective treatment modality to the surgical armamentarium. hemobilia, the phenomenon of bleeding into the biliary tree, from an intrahepatic aneurysm is an extremely rare occurrence. hepatic artery aneurysms account for approximately 10% of hemobilia cases, and most are related to trauma, transplantation, surgical injury, or infection. hemobilia caused by an intrahepatic arterial aneurysm is rare and presents a challenging medical diagnosis. currently, several potential therapeutic options are available, including embolization, stenting, and resection. the choice of treatment must consider the acuity of the bleeding and the anatomic location of the aneurysm as well as the underlying cause. the role of laparoscopic liver resection in the treatment of a bleeding hepatic artery aneurysm has not been evaluated previously. we report on a 49-year - old male with massive hemobilia, a recent laparoscopic cholecystectomy, a past medical history of hypertension, and a newly diagnosed aortic dissection. we present the diagnostic approach, a review of the literature, and treatments for hemobilia caused by an intrahepatic aneurysm. in this case a 49-year - old african - american male with abdominal pain and dark tarry stools was seen at a community hospital. he had developed right upper quadrant pain and had passed several dark melanotic stools over 2 days. hospital admission laboratory tests showed his hemoglobin and hematocrit were 5.3 and 16.8, respectively. he required transfusion of several units of packed red blood cells but remained hemodynamically stable. an ultrasound demonstrated gallbladder sludge, stones, and thickening consistent with cholecystitis. within 72 hours of admission, the patient 's hematocrit became stable, and he did not require further transfusion. postoperatively, the patient had further gastrointestinal bleeding with significant drops in hemoglobin and hematocrit requiring repeated transfusions. he received 7 units of blood within 48 hours after cholecystectomy and was transferred to our hepatobiliary service for further treatment. the ct scan revealed a previously undiagnosed aortic dissection beginning just distal to the left subclavian artery and terminating at the level of the left renal artery. the scan also showed intrahepatic biliary dilatation and an accompanying left hepatic artery aneurysm with associated outpouching of the right portal vein that likely represented a venous varix (figure 1a). the mra showed a lobulated area in the left lobe of the liver, which measured 3 cm by 3 cm, highly suggestive of a portal venous varix (figure 1b). the options for treatment of the hepatic vascular / biliary abnormality included embolization by interventional techniques or hepatic resection. the arteriogram and subsequent catheter placement were technically challenging, secondary to the aortic dissection with prominent false and true lumens. the catheter was guided to the common hepatic artery and further to the left hepatic artery ; however, secondary to high - grade stenosis at the origin of the left hepatic artery aneurysm, the catheter could not be advanced, preventing embolization. intraoperative ultrasound duplex was used to identify the location of the aneurysm with the left lobe of the liver. the left portal vein and left hepatic artery were divided intrahepatically by using a 45-mm laparoscopic linear stapler, with 2.5-mm staples. ultimately, the left hepatic vein was isolated and similarly divided. the specimen contained a large aneurysm with connection to the biliary system (figure 2). the final pathology was consistent with an aneurysm that communicated with the biliary system and also demonstrated thrombi in adjacent portal vein tributaries. a hepatic artery aneurysm is very rare but potentially life - threatening. of the visceral arteries most prone to true aneurysmal formation, the splenic and hepatic arteries are most common. splenic and hepatic aneurysms account for 60% and 20% of all visceral artery aneurysms, respectively. hepatic artery aneurysms may occur anywhere along the hepatic arterial system, both external to the liver and within the liver parenchyma. seventy - seven percent of hepatic aneurysms are confined to the segment proximal to the liver, 20% have combined intra- and extraparenchymal involvement, and 3% are exclusively within the liver. excluding traumatic aneurysms, there is a male predominance (approximately 3:2). historically, most hepatic artery aneurysms have been mycotic in origin. today, nearly 50% of hepatic aneurysms traumatic hepatic aneurysms typically result from either trauma (car accidents) or are iatrogenic, resulting from a diagnostic procedure. the remainder is associated with medial degeneration and secondary atherosclerosis, with hypertension as the predominant comorbid condition. interestingly, the populations most at risk for developing hepatic aneurysms are patients with fibromuscular dysplasia or polyarteritis nodosa. decompression into the biliary tree with resulting hemobilia is more common than free rupture is into the abdominal cavity., hemobilia has increased in incidence, presumably secondary to increasing numbers of surgical and interventional radiologic procedures involving the liver. the most common symptoms associated with hemobilia are jaundice (30%), biliary colic (52%), and gastrointestinal hemorrhage (73%). the complete triad occurs in approximately 20% of symptomatic patients. once an upper gastrointestinal (ugi) bleed is suspected, endoscopy is the first - line diagnostic modality. if blood or clot is seen at the ampulla of vater, hemobilia is highly likely. however, only 12% of the upper endoscopies done to evaluate acute ugi bleeding are diagnostic. radiologic studies, such as computed tomography, abdominal sonography, magnetic resonance, and angiography, are all potentially useful in identifying hepatic arterial abnormalities. angiography can detect the source of significant hemobilia in over 90% of patients, allowing for localization of the vascular abnormality and possible therapeutic stenting or embolization. the ultimate goal is to prevent or stop active hemorrhage and relieve any associated biliary obstruction. for extrahepatic aneurysm excision, arterial reconstruction or intraarterial stenting to affect aneurysm exclusion embolization has a success rate of between 80% and 100% in some reports and a lower morbidity and mortality rate compared with that in open surgery. if embolization fails or is contraindicated, surgical intervention, such as ligation of the artery or aneurysm excision, should be considered. laparoscopic surgery for hemobilia caused by a hepatic artery aneurysm has not been described previously in the literature. in centers experienced in laparoscopic liver resection, this approach adds an additional treatment modality. in our patient, laparoscopic resection allowed for complete removal of the aneurysm, preventing further bleeding or other potential complications that could result from a vascular - biliary fistula. hemobilia secondary to intrahepatic aneurysm in a patient with newly diagnosed aortic dissection is a rare finding. embolization is a first - line therapy for intrahepatic aneurysms but could not be accomplished in this case secondary to stenosis and anatomic limitations imposed by aortic dissection. in this case, laparoscopic left lobe resection provided definitive treatment without complications. increasing familiarity with laparoscopic liver resection in centers familiar with the techniques involved | background : intrahepatic arterial aneurysms are rare and typically related to trauma, transplantation, iatrogenic injury, or infection. they account for approximately 10% of clinically significant hemobilia.case report : we present the case of a 49-year - old man with an intraparenchymal hepatic artery aneurysm that presented as massive hemobilia following a laparoscopic cholecystectomy. the aneurysm could not be managed by interventional embolization and required a left hepatic lobectomy, which was performed laparoscopically.discussion:evaluation of hemobilia requires a multidisciplinary team approach. the diagnosis of hepatic artery aneurysm can be most readily made by mri or ct scan. interventional embolization of the aneurysm may be effective treatment but is not always possible due to anatomic considerations. where indicated, surgical resection in a manner that preserves a maximal amount of normal hepatic parenchyma is the treatment of choice.conclusion:this is the first report of laparoscopic liver resection performed for bleeding from a hepatic artery aneurysm and adds an effective treatment modality to the surgical armamentarium. |
skeletal tuberculosis (tb) accounts for around 3 - 5% of all cases, of which about 10% occurs at the ankle and foot region. in most cases, tuberculosis of the foot affected a single bone, and tarsal bones are rarely affected. diagnoses of these cases are difficult and delayed because of the overlapping and vague symptoms. in the english literature, very few cases of talonavicular tuberculosis in adults have been reported. on pubmed search (using keywords talonavicular, children, tuberculosis), only one case of isolated talonavicular tuberculosis in children has been reported by birjandinejad. to the best of our knowledge a 9-year - old boy presented with a 6-month history of pain and swelling over the medial aspect of the left foot with unknown etiology. the plain radiographs of foot ap and oblique views showed diffuse osteopenia with marginal erosion of the articular surface of talus and navicular and narrowing of the talonavicular joint with prominent soft tissue medial to the talonavicular joint (figure 1). pre - treatment radiographs of the foot (ap and oblique) showing diffuse osteopenia with narrowing of the talonavicular joint and erosion of the articular surface of talus and navicular and prominent soft tissue. hematological investigations were within normal limits except for the raised erythrocyte sedimentation rate (esr) (30 mm 1st hour) and positive c - reactive protein (crp-10). we performed needle biopsy from the talonavicular joint under image intensifier and aspirated thick grayish - white material, which was sent for histopathology, culture, and acid - fast bacillus (afb) staining. on histopathological examination, epitheloid granulomas with caseating necrosis were seen which was suggestive of tuberculosis (figure 2). histopathological examination showing well formed epitheloid cell granuloma along with langerhans - type giant cell, lymphocytic infilteration, and fibrocollagenous tissue (100). anti - tuberculosis therapy (att) was started with the first - line of 4 drugs, including rifampicin, isoniazid, ethambutol, and pyrazinamide. after three months, ethambutol was stopped and the remaining 3 drugs continued for the next 3 months, followed by rifampicin and isoniazid for one year. we gave att for a longer duration because of the high prevalence of osteoarticular tuberculosis in our country. the patient responded satisfactorily with att. at the end of week 8, pain and swelling started to disappear. the patient had no pain on walking, the range of motion of the affected foot was pain - free, and there was no secondary deformity seen in the foot at the end of treatment. the plain radiological findings showed remineralization of bones with sclerosis of joint margins and reformation of the joint space (figure 3). post - treatment radiographs (ap and oblique) showing remineralization of bones with sclerosis of joint margins and reappearance of joint space of the talonavicular joint and disappearance of soft tissue. the patient was able to walk pain - free and is presently doing all of his routine activities in the last 3-year follow - up. written informed consent was obtained from the patient s legal guardian (father) for the publication of this paper and any accompanying images. the diagnosis of spinal and major joint tuberculosis such as the hip and knee is relatively easy. in contrast, the diagnosis of tuberculosis of small bones of the ankle and foot are often difficult and confusing ; leading to delayed treatment and increased risks of secondary infection. the ankle and foot are rarely affected and comprises only 1% of all tubercular infections. the most common presentation of foot tb is a granulomatous focus adjacent to a joint, which can be easily missed on initial radiographs. the x - ray findings are fairly nonspecific and usually show an osteolytic lesion with occasional evidence of central sequestration seen in cases involving calcaneum. the absence of any specific radiographic features of infection in bone and joints leads to difficulty in the diagnosis of the localized lesion. the differential diagnosis includes pyogenic osteomyelitis, bone tumour, fungal osteomyelitis, and granulomatous diseases such as gout, sarcoidosis, and amyloidosis. mittat. treated 37 patients with foot tuberculosis and observed five patterns of lesion radiologically, namely (i) cystic type : this pattern is more common in calcaneum, (ii) rhuemotoid type : appears as a coalesced mass of the bone and seen in midfoot involvement, (iii) subperiosteal type : subperiosteal scalloping of bone was noted on the outer surface of the cuboid, the base of the fifth metatarsal and talar head, (iv) kissing type : lesion was present in one joint and symmetrical scalloped lesions had developed on the adjacent articular surfaces of the bones, (v) spina ventosa type : found in the short tubular bone of the foot. tb of the foot is an uncommon entity and the reported incidence is 0.1% to 0.3%. tb of the foot can manifest as articular, soft tissue mass or an isolated bony lesion. calcaneum is the commonest tarsal bone to be involved in isolated osseous tubercular involvement, followed by talus, distal end of the first metatarsal, navicular, cuneiform, and cuboid in descending order of incidence. isolated involvement of talonavicular joint is extremely rare and this is the second case to be reported on the isolated involvement of the talonavicular joint in children. the clinical history may be unhelpful with tb of the foot as they usually have an insidious onset. the nonspecific features including pain, swelling, and localized tenderness may be present, but pyrexia or constitutional symptoms are usually absent. hematological investigations are usually within normal limits except for the raised esr and positive crp as in the present case. partial excision of the navicular bone is the surgical option for isolated navicular lesion. in the present case, the patient responded well to att and became pain - free during the course of treatment ; therefore, he was not subjected to any surgical procedure. the tuberculosis of talonavicular joint is extremely rare. the nonspecific features including pain, swelling and localized tenderness may be present in the foot. a raised esr and a positive c - reactive protein may be the only abnormal haematological investigation when there is high clinico - radiological suspicion, needle biopsy should be performed and early treatment with att should be started in endemic areas. | tuberculosis of the foot is an uncommon entity and the reported incidence is 0.1% to 0.3%. the isolated tuberculosis of talonavicular joint is exceptionally rare. in tuberculosis of the foot and ankle, the presentation is usually nonspecific. the diagnosis of tuberculosis affecting foot is difficult, especially when it is isolated. in doubtful cases, diagnosis should be confirmed by histopathological examination. unlike pulmonary kochs, osteoarticular tuberculosis should be treated with antituberculous drugs for a longer duration, preferably for 18 months. we are reporting a case of a 9-year - old boy with tuberculosis of the isolated talonavicular joint and the diagnosis was suggested on plain radiography, which was further confirmed by histopathological examination. he was treated with first - line antitubercular drugs. a good recovery was seen following the commencement of anti - tuberculosis treatment. after two years of follow - up, he was pain - free and doing all of his routine activities. in tuberculosis of the foot, diagnosis is usually delayed or missed due to vague presentation. |
local and/or regional analgesic techniques are principal components of many multimodal analgesic techniques, as they have been shown to improve pain relief as well as reduce opioid requirements, thereby reducing the potential for opioid - related adverse events.1 for many superficial and minimally invasive surgical procedures, local infiltration anesthesia can be used for pain control, while for more involved procedures, regional blockade (peripheral nerve blocks and neuraxial analgesia) with a local anesthetic may be more appropriate.13 although local anesthetics generally have favorable safety profiles,46 unintentional excessive doses of local anesthetics at effect sites can result in serious central nervous system (cns) toxicities (ranging in severity from numbness to convulsions to respiratory depression), and cardiovascular toxicities that can be potentially fatal.4,5,7 neural damage and prolonged sensory and motor deficits have also been reported in human and animal studies when large doses have been administered via epidural or subarachnoid injection.7 bupivacaine liposome injectable suspension (exparel : pacira pharmaceuticals, inc., parsippany, nj, usa) is a liposomal formulation of bupivacaine indicated for single - dose administration into the surgical site to produce postsurgical analgesia.8 bupivacaine is encapsulated in a liposomal formulation as phosphate salt, but concentration is expressed as bupivacaine free base. this needs to be taken into consideration when comparing with traditional formulations such as marcaine (astrazeneca, sodertalje, sweden) or sensorcaine (fresenius kabi usa, lake zurich, il, usa), for which concentration is expressed as bupivacaine hcl. as an example of comparison, 40 mg of bupivacaine base in the liposomal formulation is equivalent in strength to 45 mg of bupivacaine hcl.9 liposome bupivacaine utilizes depofoam technology (pacira pharmaceuticals, inc.), which consists of an aqueous suspension of multivesicular liposomes organized in a non - concentric, honeycomb - like structure.10,11 the liposomes are made up of multiple drug - containing vesicles. each vesicle is surrounded by a lipid bilayer that provides mechanical stability and allows for controlled release of drug over several days. as the outermost liposomes rupture the lipid membranes of the inner vesicles then reorganize, the vesicles enlarge, and eventually release their contents in a successive manner until all of the drug is released.1214 in clinical studies, infiltration of liposome bupivacaine into the surgical site has been shown to provide effective analgesia with reduced opioid consumption for up to 72 hours after surgery across a range of surgical models (eg, soft tissue and orthopedic surgeries).15,16 also, liposome bupivacaine is being investigated for use in peripheral nerve block.17,18 given the increased use and possibility of new indications for liposome bupivacaine, it is important to understand the safety and pharmacokinetic (pk) profiles of this formulation compared with bupivacaine hcl when administered via atypical routes to assess the potential for toxicity following inadvertent administration. this report presents results from four preclinical studies evaluating safety and pk outcomes with liposome bupivacaine following intravenous (iv), intra - arterial (ia), epidural, and intrathecal administration. all four studies used beagle dogs (canis familiaris) supplied by marshall bioresources, usa, inc., north rose, ny, usa. the study protocols were reviewed and approved by the institutional animal care and use committee prior to study initiation. all studies, except the iv / ia dose - finding study, were conducted according to the international conference on harmonisation guidelines, in accordance with good laboratory practice (glp) principles as set forth by the us food and drug administration, title 21 code of federal regulations part 58, and as accepted by regulatory authorities in the european union (organisation for economic cooperation and development principles of glp), japan (ministry of health, labour and welfare), and canada (canadian council on animal care). the objective of this study was to evaluate potential acute toxicity and determine the maximum tolerated dose (mtd) of liposome bupivacaine after iv or ia administration in five female dogs (~918 months of age ; 8.710.6 kg). for 12 days prior to the study, animals were acclimated to the laboratory environment and underwent pretreatment health checks. each conscious, telemetered animal was administered up to four doses of the following study drugs : iv and ia liposome bupivacaine 4.5 mg / kg and 9.0 mg / kg ; iv bupivacaine hcl 1.0 mg / kg, 1.8 mg / kg, and 2.5 mg / kg ; or ia bupivacaine hcl 0.1 mg / kg and 1.0 mg / kg (table 1). a minimum 3-day washout was allowed between each dose. study drug was administered by iv injection into the saphenous or cephalic veins, or by ia injection via an access port into the carotid artery. cardiovascular safety outcomes included blood pressure (bp) and electrocardiogram (ecg) waveforms at each dosing ; derived parameters were logged as 5-second means from at least 1 hour prior to each dose (baseline) to at least 2 hours after each treatment. respiratory rate was measured as the average of three assessments at 2 minutes, 10 minutes, 30 minutes, and 90 minutes postdose by counting the number of breaths in three 30-second intervals over a 5-minute period. no formal statistical evaluations were conducted. based on results from the dose - finding study, an expanded study was conducted at charles river laboratories (montreal, canada) to further characterize the acute toxicity, including potential reversibility of adverse effects after a 14-day recovery period, and pk properties of liposome bupivacaine compared with bupivacaine hcl. experiments were conducted in 40 male and 40 female dogs (67 months of age ; 6.010.7 kg). randomization was stratified by body weight, and male and female animals were randomized separately. animals were randomized to one of eight treatment groups (ten per group) and received a single dose of the following : iv saline (control) ; iv bupivacaine hcl 0.75 mg / kg and 1.5 mg / kg ; iv liposome bupivacaine 1.5 mg / kg ; iv liposome bupivacaine 4.5 mg / kg ; ia saline (control) ; ia bupivacaine hcl 0.1 mg / kg ; ia liposome bupivacaine 1.5 mg / kg ; and ia liposome bupivacaine 3.0 mg / kg and 4.5 mg / kg (table 1). iv doses were administered into the saphenous or cephalic veins ; ia doses were administered via an access port into the carotid artery. blood samples (4 ml) for pk assessments were collected before study drug administration, at 5 minutes, 15 minutes, and 30 minutes after administration, and at 1 hour, 2 hours, 4 hours, and 6 hours after study drug administration and were assayed using a validated high - performance liquid chromatography and mass spectrometry (hplc / ms) analytical procedure. key outcomes included general health / mortality, clinical signs, ecg and hemodynamic measures, pk parameters (cmax, time to cmax [tmax ], area under the concentration time curve [auc0t ], apparent terminal elimination half - life [t1/2 ]), and histopathology. two datasets were analyzed (one for each route of administration [iv and ia ]). homogeneity of the dosing groups within each dataset was assessed using ecg at the 5% significance level. the objectives of this study were to evaluate the potential local and systemic toxicity of liposome bupivacaine (including motor blockade) after epidural administration, and to examine changes to the lumbar spinal cord. the study was conducted at mpi research laboratories, mattawan, mi, usa. male dogs (58 months of age ; 6.512 kg) were acclimated to the laboratory for at least 1 week, and then randomized (six per group) to receive liposome bupivacaine 40 mg, liposome bupivacaine 40 mg plus lidocaine 1.5% (27 mg) with epinephrine 1:200,000, bupivacaine hcl 0.5% 15 mg, depofoam placebo, or saline. the liposome bupivacaine plus lidocaine / epinephrine group received lidocaine / epinephrine followed by 0.8 ml of saline, and then liposome bupivacaine 15 minutes later. lidocaine has a strong interaction with liposome bupivacaine, and co - administration accelerates release of free bupivacaine from the liposomal vesicles.19 depofoam placebo had the same lipid composition as liposome bupivacaine but without bupivacaine. at the start of the dosing procedure, dogs were anesthetized using a mixture of nitrous oxide, oxygen, and isoflurane. an incision was made, and a 22-gauge spinal needle was inserted in the epidural space under fluoroscopic guidance. treatments were given by a slow bolus 3 ml injection (0.2 ml / min ; l7s1 vertebrae). blood samples (23 ml) for pk assessment were collected before study drug administration, and at 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours, and 96 hours after study drug administration and were assayed using a validated hplc / ms analytical procedure. dogs (three per group) were necropsied after 72 hours and after a 2-week observation period. clinical signs of impaired hind limb motor function were recorded daily over 22 days to quantify onset, incidence, and reversibility of motor blockade. spinal cord tissue underlying the injection site was examined using an amino cupric silver stain method to measure the extent of neural degeneration.20 stain intensity was recorded for dorsal, lateral, or ventral funiculi (white matter tracts), or gray matter, on a scale from 1= minimal to 5= severe. an overall pathology score was assigned to each group using an average of severity scores. a parallel set of hematoxylin - and - eosin - stained cervical, thoracic, and lumbar sections was examined on days 4 and 22 to measure the extent of inflammation and tissue damage ; stain intensity was recorded on a scale from 1= minimal to 4= severe. the objectives of this study were to compare the local and systemic toxicity of liposome bupivacaine 40 mg, bupivacaine hcl 0.5% 15 mg, depofoam placebo, and saline after intrathecal injection in dogs (six per group). male dogs (58 months of age ; 6.412.2 kg) were anesthetized using the same procedure as in the epidural study and randomly assigned to treatment group. a small incision was made over the l3l4 vertebrae, and a 22-gauge spinal needle was inserted in the intrathecal space under fluoroscopic guidance. treatments were administered by a slow bolus 3 ml injection (0.2 ml / min). blood samples (23 ml) for pk assessment were collected before study drug administration, and at 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours, and 96 hours after study drug administration and were assayed using a validated hplc / ms analytical procedure. the clinical outcomes and assessments in this study all four studies used beagle dogs (canis familiaris) supplied by marshall bioresources, usa, inc., north rose, ny, usa. the study protocols were reviewed and approved by the institutional animal care and use committee prior to study initiation. all studies, except the iv / ia dose - finding study, were conducted according to the international conference on harmonisation guidelines, in accordance with good laboratory practice (glp) principles as set forth by the us food and drug administration, title 21 code of federal regulations part 58, and as accepted by regulatory authorities in the european union (organisation for economic cooperation and development principles of glp), japan (ministry of health, labour and welfare), and canada (canadian council on animal care). the objective of this study was to evaluate potential acute toxicity and determine the maximum tolerated dose (mtd) of liposome bupivacaine after iv or ia administration in five female dogs (~918 months of age ; 8.710.6 kg). the study was conducted at charles river laboratories, montreal, canada. for 12 days prior to the study, animals were acclimated to the laboratory environment and underwent pretreatment health checks. each conscious, telemetered animal was administered up to four doses of the following study drugs : iv and ia liposome bupivacaine 4.5 mg / kg and 9.0 mg / kg ; iv bupivacaine hcl 1.0 mg / kg, 1.8 mg / kg, and 2.5 mg / kg ; or ia bupivacaine hcl 0.1 mg / kg and 1.0 mg / kg (table 1). study drug was administered by iv injection into the saphenous or cephalic veins, or by ia injection via an access port into the carotid artery. cardiovascular safety outcomes included blood pressure (bp) and electrocardiogram (ecg) waveforms at each dosing ; derived parameters were logged as 5-second means from at least 1 hour prior to each dose (baseline) to at least 2 hours after each treatment. respiratory rate was measured as the average of three assessments at 2 minutes, 10 minutes, 30 minutes, and 90 minutes postdose by counting the number of breaths in three 30-second intervals over a 5-minute period. no formal statistical evaluations were conducted. based on results from the dose - finding study, an expanded study was conducted at charles river laboratories (montreal, canada) to further characterize the acute toxicity, including potential reversibility of adverse effects after a 14-day recovery period, and pk properties of liposome bupivacaine compared with bupivacaine hcl. experiments were conducted in 40 male and 40 female dogs (67 months of age ; 6.010.7 kg). randomization was stratified by body weight, and male and female animals were randomized separately. animals were randomized to one of eight treatment groups (ten per group) and received a single dose of the following : iv saline (control) ; iv bupivacaine hcl 0.75 mg / kg and 1.5 mg / kg ; iv liposome bupivacaine 1.5 mg / kg ; iv liposome bupivacaine 4.5 mg / kg ; ia saline (control) ; ia bupivacaine hcl 0.1 mg / kg ; ia liposome bupivacaine 1.5 mg / kg ; and ia liposome bupivacaine 3.0 mg / kg and 4.5 mg / kg (table 1). iv doses were administered into the saphenous or cephalic veins ; ia doses were administered via an access port into the carotid artery. blood samples (4 ml) for pk assessments were collected before study drug administration, at 5 minutes, 15 minutes, and 30 minutes after administration, and at 1 hour, 2 hours, 4 hours, and 6 hours after study drug administration and were assayed using a validated high - performance liquid chromatography and mass spectrometry (hplc / ms) analytical procedure. key outcomes included general health / mortality, clinical signs, ecg and hemodynamic measures, pk parameters (cmax, time to cmax [tmax ], area under the concentration time curve [auc0t ], apparent terminal elimination half - life [t1/2 ]), and histopathology. two datasets were analyzed (one for each route of administration [iv and ia ]). homogeneity of the dosing groups within each dataset was assessed using ecg at the 5% significance level. the objectives of this study were to evaluate the potential local and systemic toxicity of liposome bupivacaine (including motor blockade) after epidural administration, and to examine changes to the lumbar spinal cord. male dogs (58 months of age ; 6.512 kg) were acclimated to the laboratory for at least 1 week, and then randomized (six per group) to receive liposome bupivacaine 40 mg, liposome bupivacaine 40 mg plus lidocaine 1.5% (27 mg) with epinephrine 1:200,000, bupivacaine hcl 0.5% 15 mg, depofoam placebo, or saline. the liposome bupivacaine plus lidocaine / epinephrine group received lidocaine / epinephrine followed by 0.8 ml of saline, and then liposome bupivacaine 15 minutes later. lidocaine has a strong interaction with liposome bupivacaine, and co - administration accelerates release of free bupivacaine from the liposomal vesicles.19 depofoam placebo had the same lipid composition as liposome bupivacaine but without bupivacaine. at the start of the dosing procedure, dogs were anesthetized using a mixture of nitrous oxide, oxygen, and isoflurane. an incision was made, and a 22-gauge spinal needle was inserted in the epidural space under fluoroscopic guidance. treatments were given by a slow bolus 3 ml injection (0.2 ml / min ; l7s1 vertebrae). blood samples (23 ml) for pk assessment were collected before study drug administration, and at 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours, and 96 hours after study drug administration and were assayed using a validated hplc / ms analytical procedure. dogs (three per group) were necropsied after 72 hours and after a 2-week observation period. clinical signs of impaired hind limb motor function were recorded daily over 22 days to quantify onset, incidence, and reversibility of motor blockade. spinal cord tissue underlying the injection site was examined using an amino cupric silver stain method to measure the extent of neural degeneration.20 stain intensity was recorded for dorsal, lateral, or ventral funiculi (white matter tracts), or gray matter, on a scale from 1= minimal to 5= severe. an overall pathology score was assigned to each group using an average of severity scores. a parallel set of hematoxylin - and - eosin - stained cervical, thoracic, and lumbar sections was examined on days 4 and 22 to measure the extent of inflammation and tissue damage ; stain intensity was recorded on a scale from 1= minimal to 4= severe. the objectives of this study were to compare the local and systemic toxicity of liposome bupivacaine 40 mg, bupivacaine hcl 0.5% 15 mg, depofoam placebo, and saline after intrathecal injection in dogs (six per group). male dogs (58 months of age ; 6.412.2 kg) were anesthetized using the same procedure as in the epidural study and randomly assigned to treatment group. a small incision was made over the l3l4 vertebrae, and a 22-gauge spinal needle was inserted in the intrathecal space under fluoroscopic guidance. treatments were administered by a slow bolus 3 ml injection (0.2 ml / min). blood samples (23 ml) for pk assessment were collected before study drug administration, and at 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours, and 96 hours after study drug administration and were assayed using a validated hplc / ms analytical procedure. the clinical outcomes and assessments in this study were the same as those in the epidural study. the objective of this study was to evaluate potential acute toxicity and determine the maximum tolerated dose (mtd) of liposome bupivacaine after iv or ia administration in five female dogs (~918 months of age ; 8.710.6 kg). the study was conducted at charles river laboratories, montreal, canada. for 12 days prior to the study, animals were acclimated to the laboratory environment and underwent pretreatment health checks. each conscious, telemetered animal was administered up to four doses of the following study drugs : iv and ia liposome bupivacaine 4.5 mg / kg and 9.0 mg / kg ; iv bupivacaine hcl 1.0 mg / kg, 1.8 mg / kg, and 2.5 mg / kg ; or ia bupivacaine hcl 0.1 mg / kg and 1.0 mg / kg (table 1). study drug was administered by iv injection into the saphenous or cephalic veins, or by ia injection via an access port into the carotid artery. cardiovascular safety outcomes included blood pressure (bp) and electrocardiogram (ecg) waveforms at each dosing ; derived parameters were logged as 5-second means from at least 1 hour prior to each dose (baseline) to at least 2 hours after each treatment. respiratory rate was measured as the average of three assessments at 2 minutes, 10 minutes, 30 minutes, and 90 minutes postdose by counting the number of breaths in three 30-second intervals over a 5-minute period. based on results from the dose - finding study, an expanded study was conducted at charles river laboratories (montreal, canada) to further characterize the acute toxicity, including potential reversibility of adverse effects after a 14-day recovery period, and pk properties of liposome bupivacaine compared with bupivacaine hcl. experiments were conducted in 40 male and 40 female dogs (67 months of age ; 6.010.7 kg). randomization was stratified by body weight, and male and female animals were randomized separately. animals were randomized to one of eight treatment groups (ten per group) and received a single dose of the following : iv saline (control) ; iv bupivacaine hcl 0.75 mg / kg and 1.5 mg / kg ; iv liposome bupivacaine 1.5 mg / kg ; iv liposome bupivacaine 4.5 mg / kg ; ia saline (control) ; ia bupivacaine hcl 0.1 mg / kg ; ia liposome bupivacaine 1.5 mg / kg ; and ia liposome bupivacaine 3.0 mg / kg and 4.5 mg / kg (table 1). iv doses were administered into the saphenous or cephalic veins ; ia doses were administered via an access port into the carotid artery. blood samples (4 ml) for pk assessments were collected before study drug administration, at 5 minutes, 15 minutes, and 30 minutes after administration, and at 1 hour, 2 hours, 4 hours, and 6 hours after study drug administration and were assayed using a validated high - performance liquid chromatography and mass spectrometry (hplc / ms) analytical procedure. key outcomes included general health / mortality, clinical signs, ecg and hemodynamic measures, pk parameters (cmax, time to cmax [tmax ], area under the concentration time curve [auc0t ], apparent terminal elimination half - life [t1/2 ]), and histopathology. two datasets were analyzed (one for each route of administration [iv and ia ]). homogeneity of the dosing groups within each dataset was assessed using ecg at the 5% significance level. the objectives of this study were to evaluate the potential local and systemic toxicity of liposome bupivacaine (including motor blockade) after epidural administration, and to examine changes to the lumbar spinal cord. male dogs (58 months of age ; 6.512 kg) were acclimated to the laboratory for at least 1 week, and then randomized (six per group) to receive liposome bupivacaine 40 mg, liposome bupivacaine 40 mg plus lidocaine 1.5% (27 mg) with epinephrine 1:200,000, bupivacaine hcl 0.5% 15 mg, depofoam placebo, or saline. the liposome bupivacaine plus lidocaine / epinephrine group received lidocaine / epinephrine followed by 0.8 ml of saline, and then liposome bupivacaine 15 minutes later. lidocaine has a strong interaction with liposome bupivacaine, and co - administration accelerates release of free bupivacaine from the liposomal vesicles.19 depofoam placebo had the same lipid composition as liposome bupivacaine but without bupivacaine. at the start of the dosing procedure, dogs were anesthetized using a mixture of nitrous oxide, oxygen, and isoflurane. an incision was made, and a 22-gauge spinal needle was inserted in the epidural space under fluoroscopic guidance. treatments were given by a slow bolus 3 ml injection (0.2 ml / min ; l7s1 vertebrae). blood samples (23 ml) for pk assessment were collected before study drug administration, and at 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours, and 96 hours after study drug administration and were assayed using a validated hplc / ms analytical procedure. dogs (three per group) were necropsied after 72 hours and after a 2-week observation period. clinical signs of impaired hind limb motor function were recorded daily over 22 days to quantify onset, incidence, and reversibility of motor blockade. spinal cord tissue underlying the injection site was examined using an amino cupric silver stain method to measure the extent of neural degeneration.20 stain intensity was recorded for dorsal, lateral, or ventral funiculi (white matter tracts), or gray matter, on a scale from 1= minimal to 5= severe. an overall pathology score was assigned to each group using an average of severity scores. a parallel set of hematoxylin - and - eosin - stained cervical, thoracic, and lumbar sections was examined on days 4 and 22 to measure the extent of inflammation and tissue damage ; stain intensity was recorded on a scale from 1= minimal to 4= severe. the objectives of this study were to compare the local and systemic toxicity of liposome bupivacaine 40 mg, bupivacaine hcl 0.5% 15 mg, depofoam placebo, and saline after intrathecal injection in dogs (six per group). male dogs (58 months of age ; 6.412.2 kg) were anesthetized using the same procedure as in the epidural study and randomly assigned to treatment group. a small incision was made over the l3l4 vertebrae, and a 22-gauge spinal needle was inserted in the intrathecal space under fluoroscopic guidance. treatments were administered by a slow bolus 3 ml injection (0.2 ml / min). blood samples (23 ml) for pk assessment were collected before study drug administration, and at 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours, and 96 hours after study drug administration and were assayed using a validated hplc / ms analytical procedure. the clinical outcomes and assessments in this study were the same as those in the epidural study. the maximum doses at which no severe adverse events were noted were determined to be 1.8 mg / kg iv and 0.1 mg / kg ia for bupivacaine hcl based on observations of marked increases in bp and heart rate (hr), and clinical signs of decreased activity, prostration, labored breathing, tremors, and convulsions when the dose was pushed higher. the maximum dose of liposome bupivacaine at which no severe adverse events were noted was determined to be 4.5 mg / kg for both iv and ia administration ; a dose of 9 mg / kg resulted in increased bp and hr, vomiting, and convulsions in some animals. one animal was euthanized due to the severity of adverse effects. at the doses studied (iv bupivacaine hcl 0.75 mg / kg and 1.5 mg / kg, iv liposome bupivacaine 1.5 mg / kg and 4.5 mg / kg, ia bupivacaine hcl 0.1 mg / kg, and ia liposome bupivacaine 1.5 mg / kg, 3.0 mg / kg, and 4.5 mg / kg), there was no mortality or treatment - related changes in clinical or gross / microscopic pathology. one animal that received bupivacaine hcl 1.5 mg iv had sustained convulsions, and other animals treated with this dose showed excessive licking and increased respiratory rate and/or emesis. tremors and uncoordination were noted in one animal that received bupivacaine hcl 0.75 mg / kg iv. clinical signs observed with liposome bupivacaine 1.5 mg / kg or 4.5 mg / kg iv were generally mild. increased activity and vocalization were noted in two animals that received liposome bupivacaine 1.5 mg / kg iv, and no specific treatment - related cns or motor effects were observed in animals treated with liposome bupivacaine 4.5 mg / kg iv. there were no treatment - related observations with bupivacaine hcl 0.1 mg / kg ia. decreased activity, emesis, and/or uncoordination were observed in dogs treated with liposome bupivacaine 1.5 mg / kg ia. one animal treated with liposome bupivacaine 3.0 mg / kg ia had sustained convulsions, salivation, uncoordination, increased vocalization, and increased respiratory rate ; other animals treated with this dose showed uncoordination, decreased activity, tremors, salivation, and/or emesis. liposome bupivacaine 4.5 mg / kg ia was associated with convulsions, lying on side, decreased muscle tone, tremors, and/or salivation in two animals, and other animals treated with this dose showed decreased activity, uncoordination, tremors, salivation, and/or emesis. in general, these clinical signs appeared shortly after dosing and resolved within 1 hour. there were no notable effects on bp, hr, or ecg after administration of bupivacaine hcl 0.75 mg / kg iv or 0.1 mg / kg ia, or after administration of liposome bupivacaine 1.5 mg iv or ia. no male dogs treated with liposome bupivacaine 4.5 mg iv showed any adverse cardiovascular effects ; however, one female dog had significantly decreased bp followed by an increase in hr ; these effects were transient and resolved by 5 minutes postdosing. the effects of liposome bupivacaine 3.0 mg / kg ia were variable ; one female dog had significant increases in bp and hr, followed by convulsions and increased respiratory rate, another female dog had an increase in hr, and two male dogs had a tendency toward decreased bp. in general, the clinical signs described above appeared shortly after dosing and resolved within an hour. in summary, the maximum doses at which no severe adverse events were noted with ia administration were determined to be 0.1 mg / kg for bupivacaine hcl compared with 1.5 mg / kg for liposome bupivacaine. the maximum doses at which no severe adverse events were noted with iv administration were determined to be 0.75 mg / kg and 4.5 mg / kg for bupivacaine hcl and liposome bupivacaine, respectively (table 2). mean bupivacaine pk parameters are shown in table 3, and plasma bupivacaine levels after iv administration of bupivacaine hcl 1.5 mg / kg and liposome bupivacaine 4.5 mg / kg are shown in figure 1. maximum bupivacaine plasma concentrations were reached 5 minutes postdose for bupivacaine hcl and 515 minutes postdose for liposome bupivacaine. the mean cmax in animals that received iv liposome bupivacaine 4.5 mg / kg was similar to the mean cmax observed in the iv bupivacaine hcl 1.5 mg / kg group, despite the greater than threefold difference in doses used between the two treatment groups. no evidence of motor block or limb impairment was observed in animals treated with epidural liposome bupivacaine 40 mg alone. in contrast, the majority of animals treated with bupivacaine hcl 15 mg (4/6) showed limb impairment that resolved after day 1 (table 4). liposome bupivacaine 40 mg plus lidocaine / epinephrine also showed limb impairment that resolved after day 1 (5/6). these effects were consistent with a bupivacaine - related response and were not considered adverse. some impairment may have been procedural, as one of six animals in the placebo group and two of six in the saline group had transient limb impairment. there was a slight, treatment - related prolongation of activated partial thromboplastin time in dogs receiving bupivacaine hcl or liposome bupivacaine plus lidocaine / epinephrine. any red discoloration of the skin subcutis or spinal cord at the injection site was attributed to hemorrhage from needle penetration. histological findings showed no damage to the spinal cord or adjacent tissues, other than mild local reactions at the needle site as would be expected from the procedural technique. occasionally, animals that received liposome bupivacaine or placebo had minimal chronic inflammation, characterized by the presence of a few large macrophages with abundant foamy cytoplasm in adipose tissue around nerves. mean severity scores from silver staining for all treatment groups except liposome bupivacaine plus lidocaine / epinephrine were at or slightly above the minimal score, indicating that treatment - induced neural degeneration was minimal in all but the bupivacaine plus lidocaine / epinephrine group (table 5). pk results showed that plasma bupivacaine disappeared more rapidly when given as bupivacaine hcl than as liposome bupivacaine (with or without lidocaine / epinephrine) (figure 2). the depot effect of liposome bupivacaine was evidenced by a similar mean (standard deviation [sd ]) cmax in animals receiving liposome bupivacaine 40 mg and those receiving the threefold lower dose of bupivacaine hcl 15 mg (319 ng / ml vs 271 ng / ml). limb impairment occurred on the day of study drug administration in four of six dogs treated with liposome bupivacaine 40 mg ; the impairment resolved over the next 13 days (table 4). in the bupivacaine hcl 15 mg group, limb impairment was observed on the day of study drug administration in all six dogs, and took up to 8 days to resolve. no dogs in the liposome bupivacaine group experienced respiratory arrest compared with three of six dogs in the bupivacaine hcl group. there were no differences in the incidence or severity of spinal cord lesions between groups ; red skin discoloration at the injection site was presumed to be a result of hemorrhage from needle penetration. histology staining for degeneration was observed in all sections of the spinal cord, with more degeneration observed in white matter tracts than in the gray matter. mean severity scores ranged from 1.83 to 2.71, indicating that lesions were mild to moderate (table 5). there were no toxicologically relevant differences in total severity scores between groups. the pk profiles of intrathecally administered liposome bupivacaine and bupivacaine hcl are shown in figure 3. mean [sd ] peak bupivacaine plasma concentration was similar for liposome bupivacaine (442 ng / ml) and bupivacaine hcl (404 [98.3 ] ng / ml), despite administration of a threefold higher dose of liposome bupivacaine (40 mg vs 15 mg). after dose normalization, mean [sd ] cmax / dose was threefold lower with liposome bupivacaine than bupivacaine hcl (84.3 [41.7 ] ng / ml vs 256 [35.6 ] ng / ml, p 15 years in prolonged - release formulations of medications such as cytarabine (depocyt, pacira pharmaceuticals, inc.) and morphine (depodur, pacira pharmaceuticals, inc.).10,13,14 the depofoam - based formulations of cytarabine, morphine, and bupivacaine have each been shown to have safety profiles that are similar to standard formulations of the active drug.9,2224 in conclusion, results from these four preclinical studies suggest that liposome bupivacaine has a favorable safety profile compared with bupivacaine hcl, even when administered via atypical, and often accidental, routes of delivery. the difference in the safety profiles of these two formulations appears to be associated with the liposome - bound nature of the bupivacaine within the multivesicular liposomes of the liposome bupivacaine formulation, which allows for the slow release of, and lower exposure to, bupivacaine. | backgroundthis report presents results from four preclinical studies evaluating safety and pharmacokinetics (pks) of liposome bupivacaine following intravascular (intravenous [iv ], intra - arterial [ia ]), epidural, and intrathecal administration in dogs.methodsintravascular administration was initially tested in a pilot study to determine maximum tolerated doses, and then in an expanded study of systemic adverse effects and pks. an epidural study compared properties of liposome bupivacaine alone and in combination with lidocaine / epinephrine vs bupivacaine hcl. another study assessed effects after intrathecal administration.resultsin the initial intravascular studies, maximum doses at which no meaningful adverse events were observed with liposome bupivacaine were higher than for bupivacaine hcl (4.5 mg / kg iv vs 0.75 mg / kg iv, and 1.5 mg / kg ia vs 0.1 mg / kg ia, respectively). in the expanded intravascular study, there was no mortality or changes in pathology ; adverse clinical signs included convulsions, lying on side, and decreased muscle tone (all were transient). in the epidural study, liposome bupivacaine was well tolerated at doses up to the highest dose tested (40 mg), with no evidence of spinal cord damage and with less motor blockade than bupivacaine hcl 15 mg. intrathecal administration of liposome bupivacaine 40 mg was not associated with meaningful safety concerns and resulted in less motor blockade than bupivacaine hcl 15 mg. pk analyses showed that maximum plasma bupivacaine levels following administration of liposome bupivacaine (4.5 mg / kg iv and 40 mg epidural) were similar to maximum plasma bupivacaine levels following a threefold lower dose of bupivacaine hcl (1.5 mg / kg iv and 15 mg epidural).conclusionliposome bupivacaine has a favorable safety profile compared with bupivacaine hcl when administered to dogs via intravascular, epidural, and intrathecal routes. this favorable safety profile is likely related to the liposome - bound nature of bupivacaine in the liposome bupivacaine formulation. |
transcranial magnetic stimulation (tms) of human visual cortex, at an intensity above a distinct threshold, induces illusory visual perceptions called phosphenes [13, 14 ]. it is thought that phosphenes originate from early visual cortex (v1/v2) [1316 ] and depend on the integrity and excitability [17, 18 ] of the occipital region. the perceived phosphene lies within the visual hemifield contralateral to the stimulated cortical hemisphere, at a location reflecting the retinotopic organization of visual cortex. for example, tms of superior right occipital cortex elicits a phosphene in the lower - left quadrant of the visual field. this spatial specificity enabled us to use phosphene perception as a probe measure of cortical excitability, in two related experiments, under conditions of transient or sustained spatial attention. this allowed us to test directly whether spatial attention influences excitability of visual cortex, as revealed by phosphenes induced by tms of visual cortex, which bypasses the retinogeniculate pathway. attention was covertly (without displacement of gaze) directed toward a particular location (left or right) during a task involving real visual stimuli. but on some trials, instead of real visual stimuli, tms was applied, which (when sufficiently intense) produced a phosphene, either within the attended location or in the symmetric location in the opposite hemifield. this allowed us to measure whether conscious perception of tms - induced phosphenes can be influenced by spatial attention. the tms intensity needed to induce a conscious phosphene (i.e., the phosphene threshold [pt ]) is widely held to provide a measure of visual - cortex excitability [17, 18 ]. hence, if we were to find that pts are affected by spatial attention, this would imply a direct effect of attention on visual - cortex excitability as well as the corresponding awareness. at or above threshold intensity, tms phosphenes were experienced as small, brief, illusory white flashes of light, clearly localized at a particular position in the contralateral hemifield [14, 19, 20 ]. pts were determined by adjusting tms intensity as a function of the participant 's response, according to an adaptive converging - staircase algorithm [19, 21 ]. tms trials were randomly interleaved with trials in which real visual stimuli were presented for judgement at the same eccentricity in space as the possible tms phosphene. variation of the side to which covert attention was directed for the real visual task allowed measurement of pts with and without spatially congruent attention. importantly, we never gave tms simultaneously with real visual stimuli here, unlike in some other studies that used tms to suppress perception of visual stimuli [2224 ]. instead, trial types were interleaved unpredictably such that tms inputs substituted for real visual stimulation on some trials. in experiment 1, participants maintained gaze on a central point throughout, while covertly directing spatial attention toward either the left or the right hemifield on each trial, according to a randomly determined prior cue signal that preceded the presentation of the target (figures 1a1d). a briefly illuminated led (100 ms), on one side or other of the fixation point acted as an attentional cue, indicating the side on which a peripheral visual target was likely to appear for that trial. on two - thirds of trials, after a variable interval, a real, suprathreshold visual target then appeared briefly, and participants had to press a button as quickly as possible. for 80% of these visual trials, the external target was on the side to which attention had been directed (validly cued) ; for an unpredictable 20%, the target appeared in the opposite hemifield (invalidly cued). reaction times for detection of visual targets were faster to validly cued than to invalidly cued targets (358 21 ms versus 393 23 ms (mean sem), z = 2.52, p = 0.012 ; see figure 1e). this confirms that spatial attention was indeed directed to the cued location for the external task. on one - third of the trials, randomly interleaved with the valid and invalid visual trials, no external visual target appeared. instead, a single tms pulse was applied to one side or the other of the occiput, ipsilateral or contralateral to the hemifield that had been cued for attention. on these tms trials, our critical finding was that pts were significantly lower for trials on which the phosphene appeared in the hemifield that had been cued for attention (51.6% of maximum stimulator output [mso ], compared with 56.2% mso for the other side ; z = 2.52, p = 0.012 ; see figures 1f and 1 g). this indicates that transiently cued spatial attention enhances visual awareness, even for direct tms of visual cortex, which bypasses the pathway from the eye, hence precluding feedforward thalamic gating of initial afferent sensory input. it is conceivable that the heightened thresholds for the unattended noncued hemifield in experiment 1 were due, at least partly, to the fact that stimuli on the noncued side (real visual or tms - induced phosphene) were less frequent overall. surprise factor and thereby possible differences in criteria for reports associated with different probabilities of event occurrence on one side versus the other. the particular side on which the external target had to be detected was now constant throughout each block. for each block of 96 trials, participants were instructed to maintain their covert attention continuously on the lower quadrant of one particular hemifield while they retained central fixation (confirmed with eye - tracking). visual stimuli, which were presented simultaneously and bilaterally (see figures 2a2d), consisted of a variable number (one to four) of target rectangles, independently selected on the two sides for each trial. participants had to indicate, via button - press, the number of such rectangles on the attended side only. on a critical 50% of randomly interleaved trials in each block (with attention sustained to one quadrant for the external visual task), a tms pulse was delivered to the right side of the occiput instead of visual stimulation. observers had to report whether they experienced a phosphene (which now fell within the target area used for real visual stimuli in the left hemifield ; see figures 2a2d). as tms was now applied to only one hemisphere, more pt measurements could be obtained than in experiment 1, allowing fitting of full psychometric functions to the response data (see experimental procedures and figure s1 in the supplemental data available online). in half of the blocks, attention was directed constantly to the left (the side of possible phosphene appearance) for the rectangle - counting task ; in the other half, it was directed to the right. the critical finding was again that pts were significantly lower when spatial attention was congruent with the side of the phosphene (62.3% mso, compared with 69.4% mso for the other side ; z = 2.55, p = 0.011 ; figures 2e and 2f). moreover, whereas these thresholds differed reliably as a function of attention, the slopes of the underlying psychometric functions did not (see experimental procedures and figure s1). this is consistent with a genuine increase in cortical excitability as a result of spatial attention, rather than a criterion shift. recent demonstrations that attention can modulate activity in the human lateral geniculate nucleus (lgn) [25, 26 ] reopened the long - standing question of whether thalamic gating of afferent inputs may contribute to, or even be necessary for, effects of spatial attention on visually evoked activity in striate [58 ] and extrastriate cortex [6, 7, 9 ]. initial modulation of feedforward signals at the lgn might conceivably be a necessary prerequisite for effects of spatial attention to be propagated to and enhanced at subsequent cortical areas, with possible amplification at successive stages. on the other hand, many authors have previously suggested or implied that top - down influences on early visual cortex [1012, 2831 ], producing so - called baseline shifts [3234 ], might arise without thalamic mediation. typically such arguments against a critical role for the thalamus have been based on indirect evidence, from the relative timing of components in event - related potentials (erps) or from the absence of observable attentional effects in neuroimaging of the thalamus (which, in some cases, might have been attributable to use of relatively low - resolution imaging in some cases). by contrast, the present experiments were able to test the issue directly by using cortical stimulation and demonstrate a positive effect that can not be attributed to thalamic gating of initial afferents. this new evidence confirms prior proposals that top - down attentional influences (e.g., from frontal or parietal cortex) change activity and excitability of visual cortex itself (e.g., [2, 8, 28, 29 ]). it is also compatible with studies in which the relative timing of erp components was taken to indicate that attentional modulation of visual cortex is more likely to reflect top - down feedback than feedforward thalamic gating [1012, 2830 ]. what, then, should we make of the clear evidence that visual activation of the lgn itself can be influenced by spatial attention [25, 26 ] ? we suggest that this may reflect recursive corticothalamic interactions, in line with the time at which spatial attention appears to influence early visual - cortex excitability [1012, 28, 30 ]. although during normal visual perception such recursive processing may indeed contribute to spatial attention, our results show that thalamic gating of initial afferents is not a necessary precondition for attentional modulation of cortical excitability. our results further demonstrate that attention can act directly on the cortex, enhancing excitability of neural populations that process the attended region in visual space. this appears to be a very general neural mechanism that can affect the very different inputs and featural properties of our two types of stimuli (i.e., cortical tms as well as real optical stimulation) and that might apply across different degrees of attentional load, as for experiments 1 and 2 here. in conclusion, our study shows directly that spatial attention enhances excitability in visual cortex to facilitate visual awareness, even when retinal stimulation, retinogeniculate conduction of sensory signals, and thalamic gating of feedforward afferents are ruled out. in both experiments, participants wore earplugs and headphones, and stable viewing and head position were ensured with a chinrest and nose - bridge. all participants had normal or corrected visual acuity and reported no history of neuropsychiatric illness or epilepsy. there were eight participants (aged 2429 yr, mean 26.5 yr, 4 females) for experiment 1. each observer sat in a darkened room, 40 cm from a central fixation target (red led), with the tms positioned over one side of the occiput. trials were initiated by button - press. a small target light (amber led) was positioned at the center of the region of the visual hemifield contralateral to the tms coil that corresponded to the spatial location of the phosphene produced by suprathreshold tms (individual range, 1023 lateral eccentricity ; mean 11.54 standard deviation [sd ] 6.39). an identical target light was set symmetrically at the same eccentricity in the other hemifield. the task for external visual targets was to report their onset via speeded button - press. see figure 1 for the temporal sequence of events in different trial types, with timing information. on tms trials, which were evident to the participants because of the audible click produced by the coil, they had to report verbally whether or not they had perceived a phosphene. responses were entered into a pc, and tms output intensity was automatically adjusted according to the modified binary search (mobs) adaptive - converging algorithm. the button for external visual targets was inappropriately pressed on 8% 1.9% (mean sd) of tms trials. the exact number of tms trials per subject depended on how quickly the mobs algorithm converged on to a pt (mean 32 sd 9 tms trials). pts were determined separately for trials in which attention had been cued to the locus of phosphene appearance (see figure 1b) and those in which the cue had directed attention to the opposite spatial location (figure 1d). after pts had been determined for tms stimulation of one cortical hemisphere, the session was repeated with tms applied to the other hemisphere, to elicit phosphenes in the opposite hemifield., four phosphene thresholds were determined via mobs for each participant : for left and right hemifields, with each either validly or invalidly cued, respectively. in experiment 2, eleven different participants (aged 1931 yr, mean 23.5 yr, 6 females) performed a sustained spatial - attention task. they had been selected from a sample of 27 participants for reliably perceiving phosphenes in a well - circumscribed location, with eyes open, at 90% of tms stimulator output or less (see). two participants were excluded from subsequent analysis because of excessive eye movement (see supplemental data). visual stimuli were presented in a darkened room on a 21 inch computer screen, refreshing at 60 hz, at a viewing distance of 45 cm. a signal was presented at screen center for 2 s prior to the start of each block, to instruct the observer to direct attention covertly to one side for the whole of the block. the corners of each target area were demarcated by four small, gray marker dots, 2 pixels square and visible throughout the experiment, in order to help the observer sustain spatial attention on the relevant region for the visual task (see supplemental data). the target areas (each 8 square) were centered 8 below the horizontal meridian, at an eccentricity of 19 in the lower quadrants of both hemifields. in visual trials (see figures 2a and 2c), participants had to report the number of target rectangles in the attended quadrant only, by pressing one of four keys with digits 2 to 5 of the right hand. target stimuli were presented for 180 ms, between 0 and 540 ms after trial initiation. visual stimulation was bilateral, with the number of rectangles randomly selected on each side, but participants only had to judge the attended side. apart from this, there was no emphasis on speed for this difficult discrimination (response latency was similar for all trial types : left correct, 1299 ms ; left incorrect 1309 ms ; right correct, 1285 ms ; and right incorrect, 1277 ms ; friedman test, (3) = 1.93 ; p = 0.57). on tms trials (see figure 2b and 2d) subjects reported the presence or absence of a phosphene via button - press of the left index or middle finger, respectively. tms intensities were again varied from trial to trial by using mobs [19, 21 ], but whenever mobs converged prior to the end of a block the procedure was started again, in order to present as many phosphene stimuli as possible throughout a block. the number of mobs convergences per subject did not differ between experimental conditions (attend right, mean 7.00 sd 1.34 ; attend left, mean 7.18 sd 1.66). given the larger dataset in experiment 2, pts could now be determined by fitting full weibull psychometric functions to the raw data as measured with mobs, by using the matlab toolbox psignifit (see http://bootstrap-software.org/psignifit). this allowed assessment not only of pts, but also of the slopes of the underlying psychometric functions (see results and discussion and figure s1). however, note that the pts determined by psychometric curve fits for experiment 2 were highly correlated (r = 0.93 across all conditions, p < 0.001) with pts when determined by mobs convergence as in experiment 1 (demonstrating that both methods are equally valid for our purposes). importantly, the curve fits confirmed that the slopes of the psychometric functions in experiment 2 did not differ between attended and unattended sides (z = 0.53, p = 0.60), further supporting our finding of significant differences in threshold. the order of experimental blocks (two blocks each of leftward or rightward attention) and trial types was randomized. subjects were repeatedly instructed to avoid eye movements, as confirmed by eye - tracking (see supplemental data). the z values given for comparisons of attended versus unattended conditions in the main text and figure legends were derived from wilcoxon signed - ranks comparisons. tms was applied by using a 70 mm figure - of - eight coil (super rapid, magstim, dyfed, wales, uk). a two - joint holder was used to place the coil to one side or other of the occiput, approximately 4 cm above the inion, at a laterality for which phosphenes were reliably reported. the initial rising phase of the induced biphasic current (250 s duration) had a temporomedial orientation, optimal for inducing visual phosphenes. in experiment 1, the location of the real visual targets (leds) was matched in eccentricity to the center of each individual 's phosphene locus, reported before the collection of experimental data. in experiment 2, the tms coil was held in place as in experiment 1, but with the stimulation site, on the right side of the occiput, chosen to maximize spatial congruence of phosphenes with the fixed target area for external visual stimuli in the lower - left quadrant on the computer screen. note that tms - evoked phosphenes are more readily elicited for lower quadrants of the visual field. tms output intensity was varied according to the mobs algorithm [19, 21 ]. the termination criteria were a maximum of five reversals and a maximum last step size of 5% of the initial tms output range. note that in this study, no visual stimuli were presented at the same time as tms (unlike [2224 ]) because pts are known to change with visual input (e.g., as a function of its luminance contrast). indeed, this is one of the reasons why pts are widely considered to reflect excitability of visual cortex (just as tms thresholds for induced movements reflect excitability of motor cortex). sound, but this was equivalent across the different conditions of visual attention here, so it could not have confounded our results. | summaryconscious perception depends not only on sensory input, but also on attention [1, 2 ]. recent studies in monkeys [36 ] and humans [712 ] suggest that influences of spatial attention on visual awareness may reflect top - down influences on excitability of visual cortex. here we tested this specifically, by providing direct input into human visual cortex via cortical transcranial magnetic stimulation (tms) to produce illusory visual percepts, called phosphenes. we found that a lower tms intensity was needed to elicit a conscious phosphene when its apparent spatial location was attended, rather than unattended. our results indicate that spatial attention can enhance visual - cortex excitability, and visual awareness, even when sensory signals from the eye via the thalamic pathway are bypassed. |
therefore, the purpose of this work is to discuss possibilities and mechanisms of serious and grave multiple complications associated with purulent meningitis (pneumorrhachis). based on the studied case, conclusion and recommendations that would animate the debate on the topic he was admitted in the emergency department for cephalalgia, fever, and frequent easy vomiting lasting 1 week duration. symptoms were associated to a puffiness of the face and dyspnea of level i according to sadoul classification. at admission, the blood pressure was 120/70 mmhg with a heart rate of 80 beats / min and a respiratory frequency of 24 cycles / min. the neurological examination objectified signs of meningeal irritation without any motor and sensory deficit. the pleuropulmonary examination found subcutaneous emphysema in the top part of the thorax and upper limbs extending to the cervical region and the face. associated air effusion syndrome was not evidenced. afterward, a lumbar puncture was performed and revealed a blurred liquid with predominant polynuclear neutrophils (80%). g / l (with concomitant glycemia of 1.20 g / l) ; total albumin level was 0.98 g / l, and the classical cerebrospinal fluid (csf) gram s stain and culture was negative. the immunochemistry study of lumbar puncture was in favor of meningitis diagnosis, which oriented to start the antibiotherapy. the routine biological and hematological studies included blood cells count and blood culture, ionogram, and sedimentation rate were found to be normal. the patient was treated using adequate antibiotherapy consisting of 2.0 g of ceftriaxone every 12 h. ceftriaxone dose was maintained at the meningitis rate of 100 mg / kg / day. the thorax x - ray showed a pneumomediastinum in the left paratracheal border along the left edge of the heart ; the image also showed subcutaneous emphysema without associated pneumothorax [figure 1 ]. evermore, thorax ct scan confirmed all abnormalities described in the thorax x - ray and revealed the presence of air collection in the spinal canal between c7 and d6 levels [figure 2 ]. the laryngoscopy, bronchoscopy, and digestive opacification did not show any abnormality in the respiratory - digestive tracts. these invasive gestures were achieved under general anesthesia without remarkable event. the clinical evolution within 48 h was marked by the occurrence of apyrexia, coupled with disappearance of the subcutaneous emphysema and the meningeal steepness. however, the neurological status of the patient worsened gradually ; a paraparesis occurred and was followed by a tetraparesis. the patient started with presenting neurovegetative disorders with hemodynamic instability which was in favor of central affection. afterward, a spinal magnetic resonance imaging (mri) was performed and showed a cervical medullary edema extending from c2 to c6 without any sign of spinal cord compression. this result was consistent with a general clinical profile of acute transverse myelitis [figure 3 ]. the hemodynamic support consisted initially of administrating vasoactive drugs and dopamine then adrenaline since nor adrenaline was not available. the patient died within 15 days with a profile of vasoparalysis resistant to vasoactive drugs. facial thoracic x - ray showing the pneumomediastinum with a form of a left bordered paratracheal along the left edge of the heart and subcutaneous emphysema the thoracic computed tomography (ct) scan in axial slice of the dorsal spine showing a lateral pneumorrhachis, pneumomediastinum, and cutaneous emphysema spinal magnetic resonance imaging (mri) in sagittal view in t1 (a) and t2 (b) showing medullary edema extending from c2 to c6 initially described by gordon and hardman, in 1977, pneumorrhachis is defined as the presence of air in the spinal canal, either in the intradural and/or extradural spaces. it is a very rare clinical entity and mostly asymptomatic ; hence most probably underdiagnosed. the first type is associated with degenerative, malignant, inflammatory, and infectious diseases, while the last variety is generally associated to epidural anesthesia, lumbar puncture, spinal surgery, or after a chest tube insertion. the most reported hypothesis in the literature consists of a mechanism involving an increased intrabronchial pressure that leads to alveolar rupture when enough gradient pressure is produced. afterwards, the air cumulated in the mediastinum separates the mediastinal pleura from the aorta and the parietal pleura from the rachis, and penetrates the epidural space through the conjugate foramina. additional mechanisms might result from severe asthma attack, coughing, or other efforts while the glottis is closed. more rarely, spontaneous pneumorrhachis occur after grave vomiting efforts that our patient expressed ; he manifested vomiting secondary to purulent meningitis. the association of spontaneous pneumorrhachis with a pneumomediastinum is quite rare and restricted to very few cases in the literature. to the best of our knowledge, the association of spontaneous pneumorrhachis with pneumomediastinum and transverse myelitis originating of purulent meningitis was never described in the literature. in our patient, we believe that the occurrence of a tetraparesis is not secondary to a spinal cord compression caused by the pneumorrhachis itself, since the level of the intramedullary hypersignal in the mri images and the clinical profile are supporting the diagnosis of a transverse myelitis associated to pneumorrhachis. this association was never described in earlier literature. firstly, both pneumorrhachis and transverse myelitis present are distinct complications of purulent meningitis evolving independently : pneumorrhachis was caused by alveolar rupture induced by thoracic hyper pressure provoked on its turn by emesis, while transverse myelitis is an independent complication of meningitis related to infectious and/or inflammatory disorders. secondly, pneumorrhachis and transverse myelitis have been closely laid : the pneumorrhachis has facilitated and/or anticipated the occurrence of spinal cord edema through inflammatory mechanisms or by compromising the venous drainage of the epidural veins. finally, both pneumorrhachis and transverse myelitis have been probably caused by gas - forming germs. nevertheless, this last hypothesis might be excluded considering the important size of the pneumomediastinum and the absence of typical signs of infectious mediastinitis. these consist of endoscopy exploration and digestive opacification excluding any esophageal rupture, tracheal branches perforation, or laryngeal fissure the ct scan remains the diagnostic tool of choice. however, x - ray radiography is helpful for initial exploration allowing the early detection of pneumorrhachis. most reports considered the benign course of spontaneous pneumorrhachis which is usually reabsorbed spontaneously and completely without causing any symptoms, and does not recur. indeed, yoshida. reported three observations of compressive pneumorrhachis complicated by lumbar radiculopathy, which were resolved spontaneously. however, in cases of secondary pneumorrhachis, prophylactic antibiotherapy is recommended to prevent meningitis. in case of symptomatic pneumorrhachis thus, raynor and saint - louis described a case of radicular compression secondary to a pneumorrhachis that occurred 10 days after a lumbar disk surgery ; in this case, the evolution was favorable using corticosteroid therapy. exceptionally, spinal surgery is required for releasing the spinal cord from the air bubbles. to date, there are no guidelines for the treatment of this clinical entity due to its extreme scarcity. in general in addition, the control of infection is more important for the prognosis of the patient. the association of spontaneous pneumorrhachis with pneumomediastinum and transverse myelitis was never reported in the literature. therefore, particular care must be undertaken when dealing with patients of such pathological profile. further reports might better lighten the mechanisms underlying this pathological association as well as the optimal treatment. | pneumorrhachis is the presence of air in the spinal canal ; mostly, it has an iatrogenic origin. the association of this entity with spontaneous pneumomediastinum without any pneumothorax is rarely reported in the literature. the spontaneous resorption is the usual evolution. the association to acute transverse myelitis is discussed by the authors.the patient is a 21-year - old male with pneumorrhachis associated to a spontaneous pneumomediastinum was admitted at the emergency department for bacterial meningitis. the antibiotherapy has marked the clinical profile by disappearance of the meningeal signs in the 48 h after admission. in contrast, the neurological symptoms were of marked aggravation by appearance of a tetraparesis with a respiratory distress syndrome having required artificial ventilation. the computed tomography (ct) scan showed a typical hypodensity corresponding to paramedullary air extending to several thoracic segments. the spinal magnetic resonance imaging (mri) showed a high cervical medullary edema without signs of compression. the patient died within 15 days with a profile of vasoparalysis resistant to vasoactive drugs.pneumomediastinum associated to pneumorrhachis and transverse myelitis complicating purulent meningitis is a rare entity. although the usual evolution is favorable, the occurrence of serious complications is possible. |
an earless kingstar cultivar of muskmelon (cucumis melo l.) was cultivated in a greenhouse at jeollabuk - do agriculture research and extension services (jbares), iksan (latitude : 3558 ' n ; longitude : 1272 ' e), from april to august 2009. gummy stem blight was observed on the stem and leaf stalk of muskmelon during the rainy season. no chemicals were used to treat the gummy stem blight ; thus, the disease was allowed to spread naturally. skin from the diseased portion of the plant was surface - sterilized with 1% naocl for 1 min and was washed three times with sterile de - ionized water to remove the naocl. the pathogen was isolated from the inner part of the stem and cultured on potato dextrose agar (pda ; difco, sparks, md, usa) in the dark at 25 for 3 days. the end parts of hypha cultured on pda were transferred aseptically onto malt extract agar (difco) and v-8 juice agar (campbell soup co., camden, nj, usa) and incubated at 25 in the dark for 10 days. fruiting bodies (conidia and conidiophores) from the pathogen were surface - sterilized and placed on pda for growth under a 12-hr photoperiod for an additional wk to induce sporulation. a morphological examination fruiting bodies were placed on a droplet of sterile water on a microscopic slide and crushed to release the contents. the length width of the conidia from the pathogen was measured at 400 magnification and was microscopically examined for the presence of septae (nicon 80i ; nikon, tokyo, japan). the size of conidia and conidiophores was measured by using nis elements br-300 software (nikon). the morphological data were compared with previously collected data for d. bryoniae. to obtain pure mycelium, a small amount of tissue was taken from the isolate and cultured on pda in the dark at 25 for 1 wk. the pure mycelium was used for a sequence analysis of the rdna its region (its-1 region, 5.8s gene, and its-2 region). genomic dna was extracted using a dneasy plant mini kit (qiagen, valencia, ca, usa) according to the manufacturer 's instructions. amplification and sequencing of the isolate (in the region containing its1, its2, and 5.8s rdna) was performed using a pair of universal primers - its1 (5'-tccgtaggtgaacctgcgg-3 ') and its4 (5'-tcctccgcttattgatatgc-3 ') - which were also used as a positive control in a subsequent diagnostic pcr. the amplification was carried out in a 20-l reaction mixture containing 50 ng of genomic dna, 10 mm tris - hcl, 1.5 mm mgcl2, 50 mm kcl, 0.01% gelatin, 0.2 mm dntp, 200 ng of each primer, and 1 unit of taq dna polymerase (promega, madison, wi, usa). the reaction mixtures were denatured at 94 for 10 min and subjected to 35 cycles of 1 min each at 94, annealing at 56~60 for 1 min, extension at 72 for 1 min, and a final extension step of 7 min at 72. an amplified pcr product was separated on 1.5% agarose gel and purified with a dna purification kit (core - one ; core - bio, seoul, korea) according to the manufacturer 's instructions. the reactions were monitored using bigdye terminator cycle sequencing kits (applied biosytems, foster city, ca, usa) as indicated by the manufacturer and run on an abiprism 3130 automated dna sequencer (applied biosystems) as described previously. the rdna its sequence data were analyzed using the dnastar program (dnastar inc., sequence data from the ribosomal its genes of didymella spp. were used to perform a preliminary phylogenetic analysis ; 16 its sequences were obtained from the genbank database (http://www.ncbi.nlm.nih.gov/) (table 2). phylogenetic analysis based on parsimony for its, was obtained from the data by using neighbor - joining methods with mega ver. 4.0 software [14, 15 ], and the sequence distance was calculated using the tamura - nei, parameter model. the megalign program (dnastar inc.) was used to compare the percentage sequence identity from the jeonbuk isolate with that of other didymella spp. in genbank. an earless kingstar cultivar of muskmelon (cucumis melo l.) was cultivated in a greenhouse at jeollabuk - do agriculture research and extension services (jbares), iksan (latitude : 3558 ' n ; longitude : 1272 ' e), from april to august 2009. gummy stem blight was observed on the stem and leaf stalk of muskmelon during the rainy season. no chemicals were used to treat the gummy stem blight ; thus, the disease was allowed to spread naturally. skin from the diseased portion of the plant was surface - sterilized with 1% naocl for 1 min and was washed three times with sterile de - ionized water to remove the naocl. the pathogen was isolated from the inner part of the stem and cultured on potato dextrose agar (pda ; difco, sparks, md, usa) in the dark at 25 for 3 days. the end parts of hypha cultured on pda were transferred aseptically onto malt extract agar (difco) and v-8 juice agar (campbell soup co., camden, nj, usa) and incubated at 25 in the dark for 10 days. fruiting bodies (conidia and conidiophores) from the pathogen were surface - sterilized and placed on pda for growth under a 12-hr photoperiod for an additional wk to induce sporulation. a morphological examination fruiting bodies were placed on a droplet of sterile water on a microscopic slide and crushed to release the contents. the length width of the conidia from the pathogen was measured at 400 magnification and was microscopically examined for the presence of septae (nicon 80i ; nikon, tokyo, japan). the size of conidia and conidiophores was measured by using nis elements br-300 software (nikon). to obtain pure mycelium, a small amount of tissue was taken from the isolate and cultured on pda in the dark at 25 for 1 wk. the pure mycelium was used for a sequence analysis of the rdna its region (its-1 region, 5.8s gene, and its-2 region). genomic dna was extracted using a dneasy plant mini kit (qiagen, valencia, ca, usa) according to the manufacturer 's instructions. amplification and sequencing of the isolate (in the region containing its1, its2, and 5.8s rdna) was performed using a pair of universal primers - its1 (5'-tccgtaggtgaacctgcgg-3 ') and its4 (5'-tcctccgcttattgatatgc-3 ') - which were also used as a positive control in a subsequent diagnostic pcr. the amplification was carried out in a 20-l reaction mixture containing 50 ng of genomic dna, 10 mm tris - hcl, 1.5 mm mgcl2, 50 mm kcl, 0.01% gelatin, 0.2 mm dntp, 200 ng of each primer, and 1 unit of taq dna polymerase (promega, madison, wi, usa). the reaction mixtures were denatured at 94 for 10 min and subjected to 35 cycles of 1 min each at 94, annealing at 56~60 for 1 min, extension at 72 for 1 min, and a final extension step of 7 min at 72. an amplified pcr product was separated on 1.5% agarose gel and purified with a dna purification kit (core - one ; core - bio, seoul, korea) according to the manufacturer 's instructions. the reactions were monitored using bigdye terminator cycle sequencing kits (applied biosytems, foster city, ca, usa) as indicated by the manufacturer and run on an abiprism 3130 automated dna sequencer (applied biosystems) as described previously. the rdna its sequence data were analyzed using the dnastar program (dnastar inc., madison, wi, usa) and aligned by using the clustal w method. sequence data from the ribosomal its genes of didymella spp. were used to perform a preliminary phylogenetic analysis ; 16 its sequences were obtained from the genbank database (http://www.ncbi.nlm.nih.gov/) (table 2). phylogenetic analysis based on parsimony for its, was obtained from the data by using neighbor - joining methods with mega ver. 4.0 software [14, 15 ], and the sequence distance was calculated using the tamura - nei, parameter model. bootstrap analysis was performed with 1,000 replications to determine the support for each clade. the megalign program (dnastar inc.) was used to compare the percentage sequence identity from the jeonbuk isolate with that of other didymella spp. in genbank. d. bryoniae spreads during the rainy season (july to august). in early july, less than 5% of the plants examined were infected (15 of 300 plants) ; however, in early august, 18.4% of the plants were infected. gummy stem blight initially attacks the main stem and then spreads to the branches of the muskmelon. disease symptoms include stem necrosis, gummy exudation, angular water - soaked lesions on the leaves, and rotten fruit. the weight of the sticky grayish - brown mucus that oozes from the infected part of the plant eventually causes the branches of the plant to break down (fig. 1a and 1c). after some time, grayish - brown mucus turns dark brown in color (fig. black pycnidia were observed on the stems, leaves, and fruit of the muskmelon. a preliminary identification of the fungi was based on the content of conidia and conidiophores, as described in the material and methods section. the mycelia of d. bryoniae from the pda culture were white to dark gray on top and black on the bottom. after 15 days, the d. bryoniae produced a conidial mass with a white aerial mycelium at the center of colony ; few pycnidia were observed. after 10 days, the d. bryoniae colonies on the malt extract agar and v-8 juice agar varied in diameter from 8.5 to 10.5 cm and extended to the edge of the petri dish (fig. 2a). the percentage of monosepta in the conidia of d. bryoniae ranged from 0% to 10%, and the mean length width of the conidia in the pda was 70 (0.96) 32.0 (0.15) m (fig. our jeonbuk isolate had morphological characteristics similar to those reported by keinath., lee, and somai. the cultures of d. bryoniae grown on pda produced olive to dark - green or black substrate mycelium and white and hairy aerial mycelium. compared with growth on pda and malt extract agar, growth on v-8 juice agar tended to occur more frequently in concentric zones for both groups. characteristics such as the size of the conidia and the percentage of septa have been used to study the taxonomy of the genus d. bryoniae and phoma spp.. the size of the conidia and the percentage of monosepta in d. bryoniae were about the same as described previously. some reports suggest that the presence of septae may not be a useful characteristic by which to distinguish the anamorph of d. bryoniae from phoma spp. therefore, additional morphological characteristics and more sensitive methods are needed to differentiate between these closely related species. total 17 rdna its sequence data for didymella spp. were compared and analyzed for similarities. a phylogenetic analysis of the jeonbuk isolate, which was isolated from the field of jbabres, eight species of didymella spp. the size of the entire rdna its region, which was amplified using primers its1 and its4 from a d. bryoniae isolate, was 500 bp and yielded 440 aligned nucleotide positions for all species included in the alignment. the nucleotide sequences in the rdna its region of the jeonbuk isolate were compared with those of the other d. bryoniae spp. the blast analysis showed nucleotide gaps of 1/494, 2/492, and 1/478 with identities of 485/492 (98%), 492/494 (99%), 491/494 (99%), and 476/478 (99%). data from the entire rdna its sequence was analyzed using the dnastar program and was aligned using the clustal w mode (table 2). the similarity in sequence identity between the rdna its region of the jeonbuk isolate and other d. bryoniae from blast searches of genbank was 100% and was 95.0% within the group. the nucleotide sequences in the rdna its region, obtained from pure cultures of the jeonbuk isolate, were 98.2~99.8% similar to those of isolates with the accession numbers ef160074, ef160076, ef107641, ef107642, and af297228 in genbank, which were similar to the deposited sequences from the d. bryoniae isolates (table 2). the phylogenetic analysis of species related to d. bryoniae revealed that d. bryoniae is a monophyletic group distinguishable from other didymella spp., including ascochyta pinodes, mycosphaerella pinodes, m. zeae - maydis, d. pinodes, d. applanata, d. exigua, d. rabiei, d. lentis, d. fabae, and d. vitalbina. the phylogenetic analysis based on rdna its sequences clearly distinguished d. bryoniae and didymella spp. of the other 10 species, the d. bryoniae group had a bootstrap value of 97%, whereas the remaining species had a bootstrap value of 21~99% (fig. the jeonbuk isolate was identified as d. bryoniae on the basis of morphological characteristics and a comparison of rdna its sequence data with other related data from genbank and previous studies. the nucleotide sequences in the rdna its region of the jeonbuk isolate were 98.2~99.8% similar to the sequences of d. bryoniae isolates in genbank. the phylogenetic analysis showed that d. bryoniae is a monophyletic group distinguishable from other didymella spp. conducted random amplified polymorphic dna (rapd) and its sequence analyses to distinguish d. bryoniae from phoma spp. shim. also analyzed the genetic diversity of d. bryoniae on the basis of rapd profiles, which substantiated by sequence characterized amplified regions marker in korea. these results indicated that the genus d. bryoniae was not monophyletic, and d. bryoniae, d. pinodes, d. rabiei, d. lentis, and d. vitalbina were distinct based on a sequence analysis of its1, its2, and 5.8s rdna. additional studies are needed to develop biological and eco - friendly methods to control gummy stem blight in muskmelon to prevent damage caused by d. bryoniae. d. bryoniae spreads during the rainy season (july to august). in early july, less than 5% of the plants examined were infected (15 of 300 plants) ; however, in early august, 18.4% of the plants were infected. gummy stem blight initially attacks the main stem and then spreads to the branches of the muskmelon. disease symptoms include stem necrosis, gummy exudation, angular water - soaked lesions on the leaves, and rotten fruit. the weight of the sticky grayish - brown mucus that oozes from the infected part of the plant eventually causes the branches of the plant to break down (fig. 1a and 1c). after some time, grayish - brown mucus turns dark brown in color (fig. black pycnidia were observed on the stems, leaves, and fruit of the muskmelon. a preliminary identification of the fungi was based on the content of conidia and conidiophores, as described in the material and methods section. the mycelia of d. bryoniae from the pda culture were white to dark gray on top and black on the bottom. after 15 days, the d. bryoniae produced a conidial mass with a white aerial mycelium at the center of colony ; few pycnidia were observed. after 10 days, the d. bryoniae colonies on the malt extract agar and v-8 juice agar varied in diameter from 8.5 to 10.5 cm and extended to the edge of the petri dish (fig. 2a). the percentage of monosepta in the conidia of d. bryoniae ranged from 0% to 10%, and the mean length width of the conidia in the pda was 70 (0.96) 32.0 (0.15) m (fig. our jeonbuk isolate had morphological characteristics similar to those reported by keinath., lee, and somai. the cultures of d. bryoniae grown on pda produced olive to dark - green or black substrate mycelium and white and hairy aerial mycelium. compared with growth on pda and malt extract agar, growth on v-8 juice agar tended to occur more frequently in concentric zones for both groups. characteristics such as the size of the conidia and the percentage of septa have been used to study the taxonomy of the genus d. bryoniae and phoma spp.. the size of the conidia and the percentage of monosepta in d. bryoniae were about the same as described previously. some reports suggest that the presence of septae may not be a useful characteristic by which to distinguish the anamorph of d. bryoniae from phoma spp. therefore, additional morphological characteristics and more sensitive methods are needed to differentiate between these closely related species. total 17 rdna its sequence data for didymella spp. were compared and analyzed for similarities. a phylogenetic analysis of the jeonbuk isolate, which was isolated from the field of jbabres, eight species of didymella spp. the size of the entire rdna its region, which was amplified using primers its1 and its4 from a d. bryoniae isolate, was 500 bp and yielded 440 aligned nucleotide positions for all species included in the alignment. the nucleotide sequences in the rdna its region of the jeonbuk isolate were compared with those of the other d. bryoniae spp. the blast analysis showed nucleotide gaps of 1/494, 2/492, and 1/478 with identities of 485/492 (98%), 492/494 (99%), 491/494 (99%), and 476/478 (99%). data from the entire rdna its sequence was analyzed using the dnastar program and was aligned using the clustal w mode (table 2). the similarity in sequence identity between the rdna its region of the jeonbuk isolate and other d. bryoniae from blast searches of genbank was 100% and was 95.0% within the group. the nucleotide sequences in the rdna its region, obtained from pure cultures of the jeonbuk isolate, were 98.2~99.8% similar to those of isolates with the accession numbers ef160074, ef160076, ef107641, ef107642, and af297228 in genbank, which were similar to the deposited sequences from the d. bryoniae isolates (table 2). the phylogenetic analysis of species related to d. bryoniae revealed that d. bryoniae is a monophyletic group distinguishable from other didymella spp., including ascochyta pinodes, mycosphaerella pinodes, m. zeae - maydis, d. pinodes, d. applanata, d. exigua, d. rabiei, d. lentis, d. fabae, and d. vitalbina. the phylogenetic analysis based on rdna its sequences clearly distinguished d. bryoniae and didymella spp. of the other 10 species, the d. bryoniae group had a bootstrap value of 97%, whereas the remaining species had a bootstrap value of 21~99% (fig. the jeonbuk isolate was identified as d. bryoniae on the basis of morphological characteristics and a comparison of rdna its sequence data with other related data from genbank and previous studies. the nucleotide sequences in the rdna its region of the jeonbuk isolate were 98.2~99.8% similar to the sequences of d. bryoniae isolates in genbank. the phylogenetic analysis showed that d. bryoniae is a monophyletic group distinguishable from other didymella spp. conducted random amplified polymorphic dna (rapd) and its sequence analyses to distinguish d. bryoniae from phoma spp. shim. also analyzed the genetic diversity of d. bryoniae on the basis of rapd profiles, which substantiated by sequence characterized amplified regions marker in korea. these results indicated that the genus d. bryoniae was not monophyletic, and d. bryoniae, d. pinodes, d. rabiei, d. lentis, and d. vitalbina were distinct based on a sequence analysis of its1, its2, and 5.8s rdna. additional studies are needed to develop biological and eco - friendly methods to control gummy stem blight in muskmelon to prevent damage caused by d. bryoniae. | gummy stem blight is a major foliar disease of muskmelon (cucumis melo l.). in this study, morphological characteristics and rdna internal transcribed spacer (its) sequences were analyzed to identify the causal organism of this disease. morphological examination of the jeonbuk isolate revealed that the percentage of monoseptal conidia ranged from 0% to 10%, and the average length width of the conidia was 70 (0.96) 32.0 (0.15) m on potato dextrose agar. the blast analysis showed nucleotide gaps of 1/494, 2/492, and 1/478 with identities of 485/492 (98%), 492/494 (99%), 491/494 (99%), and 476/478 (99%). the similarity in sequence identity between the rdna its region of the jeonbuk isolate and other didymella bryoniae from blast searches of genbank was 100% and was 95.0% within the group. nucleotide sequences of the rdna its region from pure culture ranged from 98.2% to 99.8%. phylogenetic analysis with related species of d. bryoniae revealed that d. bryoniae is a monophyletic group distinguishable from other didymella spp., including ascochyta pinodes, mycosphaerella pinodes, m. zeae - maydis, d. pinodes, d. applanata, d. exigua, d. rabiei, d. lentis, d. fabae, and d. vitalbina. phylogenetic analysis, based on rdna its sequence, clearly distinguished d. bryoniae and didymella spp. from the 10 other species studied. this study identified the jeonbuk isolate to be d. bryoniae. |
type 2 diabetes defined as non - insulin - dependent or adult - onset diabetes originates from the body 's inability to consume insulin which is frequently accompanied with weight gain and physical inactivity. as one of the most costly and prevalent chronic diseases worldwide, diabetes is constantly increasing due to changes in lifestyle and improvement of public health status which leads to higher rate of survival. according to the world health organization report in 2012 moreover, it results in 22% mortality from cardiovascular diseases and 16% mortality from heart attack cases. it has been predicted that the prevalence of diabetes will increase to the greatest level in the middle east among all regions by 2030. the incidence of diabetes has been estimated to be 29% in united arab emirates, 16% in oman, and 14% in phase 1 of tehran lipid and glucose study. the international diabetes federation (idf) in 2014 reported 8.6% incidence of diabetes in adults (between 20 and 79 years of age) which is equal to 4.5 million people in iran. the prevalence of diabetes with the adjusted world population was estimated to be 8% in 2010. the total cost of diabetes in that year was also estimated to be us$ 600 million. the perspective studies have the preference of revealing prevalence and incidence of disease, risk factors, and primary symptoms at the same time. the first report of diabetes incidence rate based on population, the tehran lipid and glucose study, in nondiabetic population aged over 20 years with the average of 3-year follow - up shows 11.8 per 1000 person - years. therefore, approximately, 1.2% of tehrani persons aged over 20 years are diagnosed with diabetes each year. this 9-year report showed that the incidence of diabetes was up to 1% and that there is an intense increase of prediabetes condition which is up to 4% in community population per year. the incidence of diabetes and prediabetes in a 7-year cohort study in isfahan was 18.9 and 32.3 per 1000 person - years, respectively. the incidence and prevalence of diabetes in rural population aged over 30 years in iran (kalale town) in 2005 were reported to be 0.6 and 1.13%, respectively. the crude incidence of type 2 diabetes in england, at the time interval of 19912010, was equal to 515 per 100 000 population. in american population aged over 20 years, 1.7 million individuals with new cases of type 2 diabetes and 86 million with prediabetes were diagnosed in 2012 which is higher than the statistics of 79 million people in 2010. in the follow - up of an 11-year study including aboriginal australian residents implemented on 686 individuals aged between 20 and 76 years, the incidence of diabetes was increased from 2.2 in population younger than 25 years to 39.9 per 1000 person - years in the age range of 4554 years. the first perspective study in ahvaz, capital of khuzestan province located in south west of iran, was implemented so as to assess the incidence of metabolic syndrome and diabetes in two phases of 2009 and 2014. it is worth mentioning that ahvaz being endowed with warm climate condition and diverse ethnicities of arab, fars, lor, and others is considered as a good sample of the demographic profile of the khuzestan province. the first phase of prevalence of metabolic syndrome and its related factors was implemented in 2009 in ahvaz by the diabetes research center, ahvaz jundishapur university of medical sciences. the sampling was carried out via cluster sampling method by inviting the participants to selected health centers in ahvaz. a questionnaire that includes age, sex, marital status, ethnicity, education level, family history of diabetes mellitus (dm), hypertension (htn) and obesity, smoking and parity, and previous history of gestational diabetes mellitus in women moreover, blood pressure, weight, height, body mass index (bmi) [weight (kg)/height (m) ], and abdominal and waist circumference were measured in each participant. blood pressure was measured by a standard sphygmomanometer after 15 min rest in a sitting position. the cuff was placed on the right arm at the heart level and then the device was quickly pushed until 30 mmhg above radial pulse disappearance. blood pressure was measured twice with at least 30 min interval between two measurements and mean of these two measurements was taken as blood pressure. waist circumference was measured at the midpoint between the lowest rib and the upper lateral border of the right iliac crest and hip circumference at the point of maximum hip diameter. triglyceride (tg), fasting blood sugar (fbs), cholesterol, and high density lipoprotein (hdl) were measured using an enzymatic colorimetric method with pars azmoon kit (iran) (with biotechnical instruments model bt-3000, germany). the participants were recalled by health centers executing the first phase, and the adjustment questionnaires, experiments, and anthropometric measurements were performed as in phase 1. the variables observed in this regard are as follows : diabetes and prediabetes : based on definition presented by american diabetes association in 1997, fpg 126 mg / d is considered diabetic (or the same rate with medication) and 100 fpg 88 cm in men and women, respectively.family history of diabetes : it is affliction with type 2 diabetes in at least one of the parents or brothers or sisters of the person. diabetes and prediabetes : based on definition presented by american diabetes association in 1997, fpg 126 mg / d is considered diabetic (or the same rate with medication) and 100 fpg 88 cm in men and women, respectively. family history of diabetes : it is affliction with type 2 diabetes in at least one of the parents or brothers or sisters of the person. among all of the 593 participants who took part in the second phase of the study, 282 individuals are men (47.6%) and 311 individuals are women (52.4%). the prevalence of diabetes and prediabetes was equal to 15.2 and 22.6% in 2009, respectively, which afterwards reached 20.9 and 18.3% in the second phase. the incidence of diabetes among healthy individuals present in phase 1 is 21.9 per 1000 person - years and prediabetes incidence is 40.8 per 1000 person - years. the mean age of healthy individuals in phase 1, who later became affected by diabetes, is 51.7 11.24, while that of prediabetes patients is 46.55 12.5 and that of healthy people is 38.34 12.7 years. the incidence of type 2 diabetes among prediabetes individuals present in phase 1 is 34.5 per 1000 person - years. in a 5-year period, of all prediabetics in phase 1, the mean age of healthy individuals in phase 1, who later became diabetics in the course of 5 years, is 51.7 11.24. the mean age of the ones who became prediabetics is 46.55 12.5 and that of the ones who remained healthy is 38.34 12.7 years. according to table 1, these variables are significantly different : age, bmi, family history of diabetes, and abdominal obesity among genders (p 0.013). as seen in table 2, the incidence of diabetes in ahvaz shows an ascending trend with age, and it starts increasing from 4 in 2029 years to 84.2 in 6069 years (decreasing above 70 years). the incidence of prediabetes increases from 25.2 in the range of 2029 years to 54.5 in 5059. the highest level of diabetes and prediabetes incidence was among the participants of 6069 and 70 years, respectively. the incidence of diabetes in ahvaz population starts from 10.03 in people with normal weight to 21.05 in overweight people and 46.03 of obese people in whom the risk of diabetes infliction is higher than five times. as seen in table 3, the number of people with diabetes has increased from 63 (10.8%) in phase 1 to 88 (15.1%) in phase 2, whereas the number of healthy people has decreased from 394 (67.8%) in phase 1 to 370 (63.7%) in phase 2. the variables including age, blood pressure, abdominal obesity, family history of diabetes, and bmi are closely related to the incidence of diabetes, and ethnicity is related to the incidence of prediabetes. table 4 shows the factors affecting the incidence of diabetes and prediabetes among healthy people in phase 1. in the cohort study implemented in two phases among people aged over 20 years in 2009 and 2014 in ahvaz, incidence of diabetes was calculated as 22.03 per 1000 person - years in healthy people of phase 1. the incidence of diabetes among men from ahvaz was 22.03 and in women was 21.78 (per 1000 person - years). in a cohort study with median of 6- and 9-year follow - up in population aged over 20 years in tehran lipid and glucose study (tlgs) during two time periods (20012005 and 20012009), the incidence of diabetes was 11.9 and 10.6 per 1000 person - years in nondiabetic individuals, respectively. the incidence of diabetes in ahvaz (22.03) was higher than that of the two studies in tehran (11.9 and 10.6 per 1000 person - years). there was no significant difference in the incidence of diabetes between men and women from ahvaz (p = 0.96) which is the same with the tehran study (10.2 for men and 11 for women per 1000 per person - years). the reason for higher level of diabetes incidence in ahvaz compared to tehran can be attributed to the extremely hot climate in a period of more than half of the year which limits the residents ' activity, type of nutrition, and lifestyle. besides, the time difference in both studies is another reason for higher diabetes incidence in ahvaz compared to tehran. in the 10-year cohort study in india, the overal incidence of diabetes was 22.2 (per 1000 person - years), 22.3 in men and 24.5 in women. in another cohort study from 1997 to 2005 in india, moreover, in a study including the aboriginal australian residents aged from 20 to 70 years, the incidence of diabetes was 18 (per 1000 person - years). these three reported diabetes incidences are close to that of ahvaz. in the following cohort studies the diabetes incidence was lower than the present one. the diabetes incidence in america in 2012 was 7.8 (per 1000 person - years), in england in 19912010 was 5.15 (per 1000 person - years), and was 8 in taiwan (per 1000 person - years) in 2007. the incidence of diabetes in ahvaz shows ascending trend with age ; this result is the same as that of the study in tehran. in aboriginal australians study, the incidence rates increased with increasing age, from 2.2 per 1000 person - years for those younger than 25 years to 39.9 per 1000 person - years for those aged 4555 years which is the same as ahvaz study. the incidence of diabetes of ahvaz population starts from 10.03 (per 1000 person - years) in people with normal weight to 21.05 in overweight people and 46.06 in obese people where the risk of diabetes infliction is approximately five times. moreover, it increases from 41 to 86 and 110 (per 1000 person - years) in the study in tehran which has an increasing trend similar to the present study ; however, in this study, the range between the three weights is less than that of the study in tehran. on the other hand, in aboriginal australians study, incidence of diabetes in three weights was within the range of 8.6, 21.2, and 53.2%, respectively, close to the results in this study. the incidence of diabetes in ahvaz population with family history of diabetes was 37.3 (per 1000 person - years) which is twice more than the individuals with no record of diabetes (16.5 per 1000 person - years). in comparison the incidence of diabetes in the study in tehran was 129 compared to 99 (per 1000 person - years). the family history of diabetes shows that the risk of diabetes in ahvaz was higher than 1.7 times and in tehran it was 2.4 times more than people with no family history of diabetes which means that the risk of diabetes in tehran is higher than that in ahvaz. ahvaz study shows the risk of diabetes to be 6.37 in people with abdominal obesity and tehran study shows that it is 1.4 times more compared to the people with no abdominal obesity. besides, high blood pressure in ahvaz causes the risk of diabetes to be 2.5 while it was 1.9 times higher in tehran compared to people with no high blood pressure. the risk of abdominal obesity and high blood pressure in ahvaz were higher than those in tehran. the incidence of prediabetes in this study is 40.7 per 1000 person - years, 41.7 in males and 39.9 in females per 1000 person - years. the incidence of prediabetes in isfahan study was 32.3 per 1000 person - years, which is less than this survey. each year, 4% of the total adult population in tehran change from healthy glucose to prediabetes condition. the incidence of prediabetes in tehran study was 46.1 and 36.8 per 1000 person - years in men and women, respectively. the incidence of prediabetes among males and females in ahvaz does not show a significant difference, whereas this difference in tehran study is significant, with incidence of prediabetes in men being higher than women. the study implemented in india showed that the incidence of prediabetes in men is about 31 and it is 34 in women (per 1000 person - years) which is lower compared to ahvaz with regard to both genders. the incidence of prediabetes among healthy people in phase 1 changes from 25.2 in ages 2029 years to 54.5 in ages 5059 years and also 114.3 in ages higher than 70 years as the age increases which signifies the ascending trend with age. moreover, the incidence of prediabetes among non - arab people with p = 0.03 is significantly more compared to arab ethnicity. the study shows that the incidence of diabetes among prediabetic people in phase 1 is estimated to be 40.8 (per 1000 person - years) which has great differences with the study in india (78.9 per 1000 person - years). the incidence of diabetes in the participants of phase 1 affected by prediabetes among men is more than that in women which changes from 22.2 per 1000 person - years within the age range of 2029 to 120 with age 6069 years. the multiple logistic regression shows that age, bmi 30, and family history of diabetes affect the incidence of diabetes, which is inconsistent with the 6-year cohort study in tehran. the present study shows that among people aged over 20 years in southwest of iran, the incidence of diabetes was 21.9 per 1000 person - years and the incidence of prediabetes was 40.8 per 1000 person - years which is higher than in other studies formerly implemented in iran. | background. the present study is the fourth cohort study conducted in the middle east on the evaluation of prediabetes and type 2 diabetes, implemented in ahvaz, iran. methodology. the individuals aged over twenty years who had participated in a study on the prevalence of metabolic syndrome in 2009 (phase 1) in ahvaz were invited again in 2014. the questionnaires were completed via interview, and anthropometric parameters were measured by standard method. the logistic regression and chi - square test were used for data analysis. results. in the median of five - year follow - up, a number of 593 people participated in reexamination from which 396 individuals were nondiabetic in phase 1. the incidence of diabetes and prediabetes was 21.9 and 40.6 per 1000 person - years, respectively. among phase 1 prediabetics, 16.8% were diagnosed with diabetes in a five - year period. the factors affecting the incidence of prediabetes among the people younger than 65 years include age, family history of diabetes, and gender. the age factor plays an important role in the transformation of prediabetes to diabetes. conclusion. the city of ahvaz with type 2 diabetes incidence of 13.64 per 1000 person - years is one of the areas with high incidence of diabetes in iran. |
this study was conducted to evaluate the prophylactic effect of ginger extract on ethanol - induced reproductive toxicity in male rats by measuring the total homocysteine (thcy), trace elements, antioxidant enzymes activity including glutathione peroxidase, superoxide dismutase (sod) and catalase, and malondialdehyde (mda). twenty - eight adult male sprague dawley rats were randomly divided into four experimental groups and treated daily for 28 days as follows : control, control+ginger (1 g / kg of body weight (b.w)/day by gavage), test group (ethanol 4 g / kg of b.w / day by gavage), and treated group (ethanol+ginger). at the end of the experiment, all the rats were sacrificed and their testes were removed and used for the measurement of the above factors. the results in the test group indicated that ethanol decreased antioxidant enzymes activity and increased mda and thcy compared with the control groups (p<0.05). in the treated group, ginger extract improved antioxidant enzymes activity and reduced thcy and mda level compared with the test group (p<0.05). it can be concluded that ethanol causes oxidative stress in testis and ginger extract improves the trace elements, antioxidant enzymes activity, and decreases thcy and mda. | background : ginger is a natural dietary component with antioxidant and anti - carcinogenic properties. this study was conducted to evaluate the prophylactic effect of ginger extract on ethanol - induced reproductive toxicity in male rats by measuring the total homocysteine (thcy), trace elements, antioxidant enzymes activity including glutathione peroxidase, superoxide dismutase (sod) and catalase, and malondialdehyde (mda).methods : twenty - eight adult male sprague dawley rats were randomly divided into four experimental groups and treated daily for 28 days as follows : control, control+ginger (1 g / kg of body weight (b.w)/day by gavage), test group (ethanol 4 g / kg of b.w / day by gavage), and treated group (ethanol+ginger). at the end of the experiment, all the rats were sacrificed and their testes were removed and used for the measurement of the above factors.results:the results in the test group indicated that ethanol decreased antioxidant enzymes activity and increased mda and thcy compared with the control groups (p<0.05). in the treated group, ginger extract improved antioxidant enzymes activity and reduced thcy and mda level compared with the test group (p<0.05).conclusion : it can be concluded that ethanol causes oxidative stress in testis and ginger extract improves the trace elements, antioxidant enzymes activity, and decreases thcy and mda. |
zoonotic trematode infections are public health problems in asian countries, including lao people 's democratic republic (lao pdr), vietnam, thailand, china, and korea. especially, fishborne trematodes (fbt) provoke a remarkable morbidity among local people as well as a serious damage in aquaculture industry [1 - 3 ]. fbt infections in humans are almost entirely caused by habitual consumption of raw fish containing infective larvae (= metacercariae). these infections are highly localized in riverside areas, especially where riparian populations have the raw fish eating habit. it has been known that riverside areas in southeast asia, especially the mekong river basin in vietnam, lao pdr, and thailand, are highly endemic with fbt infections [4 - 9 ]. cambodia is located in the southern part of the indochina peninsula in southeast asia, and bordered by thailand to the northwest, lao pdr to the northeast, vietnam to the east, and the gulf of thailand to the southwest. administratively, it is divided into 24 provinces including phnom penh municipality which is located in the central south region, and on the banks of the tonle sap lake, mekong, and bassac rivers. pursat province is located in the western part of the country and between the tonle sap lake and the northern end of the cardamom mountains. it has been known that many cambodian people are infected with helminth parasites, such as soil - transmitted nematodes and fbt including opisthorchis viverrini [11 - 13 ]. this was reconfirmed by fecal examinations during the korea - cambodia international collaboration project on intestinal parasite control in cambodia (2006 - 2011). with regard to fbt infections, several investigators have previously reported that o. viverrini are prevalent in some limited areas of cambodia [14 - 17 ]. reported infection of freshwater fish with o. viverrini metacercariae in areas adjacent to the lake 500 near the border of kandal and takeo provinces, southern cambodia. subsequently, adult o. viverrini specimens were recovered from humans and experimental animals, and metacercariae were detected in fish from takeo and kratie provinces. however, in other localities of cambodia, no studies have been available on fbt metacercarial infections in freshwater fish. therefore, in this study, we investigated on the infection status of freshwater fish with fbt metacercariae in phnom penh and pursat province, cambodia. we purchased a total of 184 freshwater fish (22 species) from local markets of phnom penh municipality through 3 different times (in june 2006, december 2007, and november 2010) and also purchased 36 fish (5 species) from a local market in pursat province (in june 2007) (fig. the fish were transported to the department of parasitology and institute of health sciences, gyeongsang national university school of medicine, jinju, korea under refrigeration. the fish species were identified with the aid of the fishbase website (http://www.fishbase.org/search.php) (tables 1 - 3). individual fish was finely ground with a mortar or a grinder, the ground fish meat was mixed with artificial gastric juice, and the mixture was incubated at 36 for 2 - 3 hr. the digested material was filtered with 11 mm of mesh, and washed with 0.85% saline until the supernatant became clear. the sediment was carefully examined under a stereomicroscope, and metacercariae were separately collected by their general morphological features. the collected metacercariae were categorized according to the size and morphological characteristics, and then the intensity of infection and the infection rate were calculated according to the species of fish positive for metacercariae. in fish from phnom penh municipality, 2 species of metacercariae (o. viverrini and h. yokogawai) were found. the metacercariae of o. viverrini were collected from 37 (50.0%) of 74 fish (11 of 27 species), and the average number of metacercariae per fish was 18.6. among the 11 fish species positive for o. viverrini metacercariae, barbonymus schwanefeldi was the most heavily infected, with the average intensity of infection per fish of 74.6 metacercariae (table 1). the metacercariae of h. yokogawai were detected from 23 (57.5%) of 40 fish (5 of 27 species), and the average number of metacercariae per fish was 21.0 (table 2). in fish from pursat province, 5 species of metacercariae (o. viverrini, h. yokogawai, haplorchis pumilio, centrocestus formosanus, and procerovum sp.) total 3 o. viverrini metacercariae were found in 2 fish species (henicorhynchus lineatus and puntioplites falcifer). total 51 h. yokogawai metacercariae were detected in 8 (80%) of 10 p. falcifer examined. a total of 476 h. pumilio metacercariae were collected from 9 (90%) of 10 h. lineatus and 1 (50%) of 2 pristolepis fasciata. only 1 c. formosanus metacercaria was detected in 1 (10%) of 10 h. lineatus. metacercariae were detected in all 9 (100%) anabas testudineus examined (table 3). metacercariae of 5 fbt species, namely, o. viverrini, h. yokogawai, h. pumilio, c. formosanus, and procerovum sp., were detected in freshwater fish from phnom penh municipality and pursat province, cambodia. in phnom penh municipality, 2 fbt species (o. viverrini and h. yokogawai) were found, whereas, in pursat province, 5 fbt species were detected. from previous studies in cambodia, only 2 species of fbt metacercariae, o. viverrini and h. yokogawai, were reported [15 - 17 ]. detected o. viverrini metacercariae in various species of freshwater fish from areas adjacent to the lake 500 near the border of kandal and takeo provinces, southern cambodia. sohn. found o. viverrini metacercariae in 1 fish species, puntioplites proctozysron, from takeo province. sohn. also detected o. viverrini and h. yokogawai metacercariae in freshwater fish from kratie province. therefore, by the present study, it has been confirmed for the first time that the 3 additional species of fbt, namely, h. pumilio, c. formosanus, and procerovum sp., are distributed in cambodia. the number and species of fish examined in this study were not much compared with the study of touch.. they examined a total of 1,479 fish in 30 species by the compression method. on the other hand, sohn. examined 2 fish species, p. proctozysron (n=5) and cyclocheilichthys apagon (n=10), from takeo province, and sohn., we examined a total of 184 freshwater fish (22 species) from several local markets of phnom penh municipality and 36 fish (5 species) from a market of pursat province by means of the artificial digestion method. anyhow, there may be some points to be considered which include the exact origin of the fish examined in this study. for example, fish purchased from a local market of phnom penh municipality may not have been caught from phnom penh area. those from pursat province are not enough in the number and species of fish to show the epidemiological trend of fbt metacercariae infections in the subjected area and fish species. as the second intermediate hosts of o. viverrini, 24 species of freshwater fish (barbonymus gonionotus, cyclocheilichthys apogon, cyclocheilichthys armatus, cyclocheilichthys enoplos, cyclocheilichthys furcatus, cyclocheilichthys repasson, esomus metallicus, hampala dispar, hampala macrolepidota, hypsibarbus lagleri, hypsibarbus pierrei, hypsibarbus wetmorei, labiobarbus lineatus, mystacoleucus marginatus, neolissochilus stracheyi, onychostoma elongatum, onychostoma fusiforme, osteochilus hasseltii, paralaubuca barroni, poropuntius dearatus, puntioplites falcifer, puntioplites proctozystron, puntius brevis, and puntius orphoides) have been reported in thailand and lao pdr [18 - 22 ]. in cambodia, detected o. viverrini metacercariae in 10 cyprinoid fish species, i.e., barbodes altus (= barbonymus altus), c. apagon, c. enoplos, h. dispar, h. macrolepidota, henicorhynchus siamensis, p. proctozysron, p. brevis, systomus orphoides (= puntius orphoides), and thynnichthys thynnoides, from kandal and takeo provinces. sohn. reported p. proctozysron as a fish host for o. viverrini in takeo province, and sohn. detected o. viverrini metacercariae in p. proctozysron, p. orphoides, and labeo chrysophekadion from kratie province, cambodia. in the present study, o. viverrini metacercariae were detected in 11 fish species (b. altus, b. schwanefeldi, cirrhinus jullieni, cirrhinus microlepis, henicorhynchus lobatus, h. siamensis, l. chrysophekadion, luciosoma bleekeri, osteochilus melanopleurus, p. proctozysron, and t. thynnoides) from phnom penh municipality and 2 fish species (henicorhynchus lineatus and p. falcifer) from pursat province. therefore, in cambodia, total 18 fish species (b. altus, b. schwanefeldi, c. jullieni, c. microlepis, c. apagon, c. enoplos, h. dispar, h. macrolepidota, h. lineatus, h. lobatus, h. siamensis, l. chrysophekadion, l. bleekeri, o. melanopleurus, p. proctozysron, p. brevis, p. orphoides, and t. thynnoides) have been confirmed to be the second intermediate hosts for o. viverrini [15 - 17 ]. the prevalence and intensity of o. viverrini metacercariae were relatively low in fish examined in the present study compared to lao pdr. in phnom penh municipality, the average number of o. viverrini metacercariae per fish was 18.6 in 37 positive fish (11 species). among the positive fish species. however, these results were somewhat higher than those of touch.. they detected total 789 o. viverrini metacercariae (4.8 per fish) in 163 positive fish (10 species) collected from kandal and takeo provinces, cambodia. some people in phnom penh like to eat raw fish, as has been reported in takeo province, and there may be a substantial number of opisthorchiasis patients in phnom penh areas of cambodia. distribution of h. yokogawai metacercariae has been reported in asian and middle east countries such as india, thailand, lao pdr, and egypt, involving 43 fish species [21 - 28 ]. in the present study, h. yokogawai metacercariae were detected in 5 fish species (p. proctozysron, c. jullieni, l. chrysophekadion, l. rhabdoura, and b. altus) from phnom penh municipality, and 1 species (p. falcifer) from pursat province. sohn. detected them in 3 fish species (p. proctozysron, p. orphoides, and h. siamensis) from kratie province, cambodia. metacercariae of h. pumilio have also been found in asian and middle east countries [21 - 28 ]. in china, 18 fish species were found to harbour the metacercariae in guangxi zhuang autonomous region. in vietnam, the metacercariae were detected in 17 fish species from nam dinh province and in 13 fish species from hanoi city and nam dinh province. in the present study, h. pumilio metacercariae were detected in only 1 fish species (h. lineatus) from pursat province. c. formosanus is known to be distributed in china, taiwan, japan, the philippines, india, lao pdr, and vietnam [8,20 - 22, 30 - 33 ]. their metacercariae were detected in 9 fish species from lao pdr [20 - 22 ], and in 16 fish species from vietnam. in china, sohn. reported 10 fish species as the second intermediate host in guangxi zhuang autonomous region. in the present study, c. formosanus metacercariae were found in only 1 fish (h. lineatus) from pursat province. (presumed to be p. varium) metacercariae were detected in all of 9 a. testudineus from pursat province in this study. this fish species was previously reported to be a second intermediate host for p. varium in vietnam, together with 9 more fish species (l. rohita, h. molitrix, c. mrigala, c. idella, s. curriculus, p. brachypomum, c. batrachus, b. gonionotus, and m. cephalus). in conclusion, the present study confirmed that various species of freshwater fish play the role of a second intermediate host for various kinds of fbt (o. viverrini, h. pumilio, h. yokogawai, c. formosanus, and procerovum sp.) in cambodia. epidemiological studies to detect endemic areas of human fbt infections remain to be undertaken in cambodia. | a survey was performed to investigate the infection status of freshwater fish with zoonotic trematode metacercariae in phnom penh and pursat province, cambodia. all collected fish with ice were transferred to our laboratory and examined using the artificial digestion method. in fish from phnom penh, 2 kinds of metacercariae (opisthorchis viverrini and haplorchis yokogawai) were detected. o. viverrini metacercariae were positive in 37 (50.0%) of 74 fish in 11 species (average no. metacercariae / fish, 18.6). h. yokogawai metacercariae were detected in 23 (57.5%) of 40 fish in 5 species (average no. metacercariae / fish, 21.0). in fish from pursat province, 5 kinds of metacercariae (o. viverrini, h. yokogawai, haplorchis pumilio, centrocestus formosanus, and procerovum sp.) were detected ; o. viverrini metacercariae (n=3) in 2 fish species (henicorhynchus lineatus and puntioplites falcifer), h. yokogawai metacercariae (n=51) in 1 species (p. falcifer), h. pumilio metacercariae (n=476) in 2 species (h. lineatus and pristolepis fasciata), c. formosanus metacercariae (n=1) in 1 species (h. lineatus), and procerovum sp. metacercariae (n=63) in 1 species (anabas testudineus). from the above results, it has been confirmed that various freshwater fish play the role of a second intermediate host for zoonotic trematodes (o. viverrini, h. yokogawai, h. pumilio, c. formosanus, and procerovum sp.) in cambodia. |
multiple sclerosis (ms) is a chronic inflammatory disease of the central nervous system (cns). numerous evidences strongly suggest that it is an autoimmune disease in which activated t cells enter the cns and trigger an inflammatory cascade resulting in demyelination and axonal loss. different genetic and environmental factors have shown to play a role in ms susceptibility, being class ii alleles of the major histocompatibility complex (mhc), the more closely associated genes, and infectious agents such as epstein - barr virus, the environmental factors that have been more clearly associated with ms susceptibility. conversely, high serum levels of vitamin d have a protective role, delaying the appearance of a second demyelinating event after a clinically isolated syndrome. it has been described that this action can be mediated by upregulation of regulatory t cells (treg) and b cells (breg). it was also described that ms patients suffer from a peripheral b cell tolerance defect that may be attributable to impaired treg function and that proliferation of breg induced by intestinal helminth infections ameliorates ms course. the role of regulatory cells in demyelinating diseases has been also explored in experimental autoimmune encephalomyelitis (eae), a widely used animal model of ms in which t cell mediates cns demyelination. eae can be induced in several species by immunization with myelin antigens or via adoptive transfer of myelin - reactive t cells [9, 10 ]. adoptive transfer of breg reduced significantly the severity of eae by inhibiting th1 and th17 inflammatory responses. likewise, expansion of treg diminished the infiltration of inflammatory cells into the cns and improved clinical signs of eae. these data strongly suggest that deficits in regulatory pathways may contribute to the induction of autoimmune reactivity in eae. these experimental models may be useful tools to identify immunological mechanisms mediating a protective role in demyelinating diseases. several strains of mice are resistant to develop eae as mice are asymptomatic following immunization with myelin antigens. it was argued that a poor recognition of encephalitogenic peptides by mhc molecules might contribute to resistance to eae. we explored them in two mouse strains, cd1 and c57bl/6, that are, respectively, resistant and susceptible to eae induced by the myelin oligodendrocyte glycoprotein 3555 peptide (mog3555). since cd1 is an outbred strain, these mice do not share a unique mhc haplotype, which makes this model useful to study mhc - independent mechanisms that mediate resistance to this experimental model of ms. we explored breg and treg in both strains and studied if variations in regulatory cells associated with downregulation of effector immune mechanisms. eight - to - ten - week - old female c57bl/6j mice, which are susceptible to eae induction by mog3555 immunization, were purchased from harlan (barcelona, spain). age- and sex - matched cd1 mice that are resistant to eae induction were obtained from our own conventional barrier protection breeding colony (cajal institute, csic, madrid, spain). mice were housed under standardized light- and climate - controlled conditions and were fed with standard chow and water ad libitum. experiments were done in compliance with the guidelines of animal care set by the european union (86/609/eec), and all animal protocols were approved by the cajal institute animal welfare committee (protocol n iorg0006540). for immunization c57bl/6 and cd1 mice were injected subcutaneously on day 0 with 200 l of an emulsion containing 300 g of mog3555 (cnb, csic, madrid, spain) and 800 g of heat killed mycobacterium tuberculosis h37ra (difco, bd diagnostics, md) emulsified in incomplete freund adjuvant oil (sigma - aldrich, st. in addition, the mice received 100 ng of bordetella pertussis toxin (sigma - aldrich) intraperitoneally on days 0 and 2 after immunization (p.i.). mice from both strains c57bl/6 or cd1 were immunized in the same way using the 139151 peptide of proteolipid protein (plp139151) (proteomics section, universitat pompeu fabra, barcelona, spain) for comparison with mog3555 as c57bl/6 mice do not develop eae following immunization with plp peptide. mice were monitored daily, and clinical signs of eae were graded as follows : grade 0, normal ; grade 1, flaccid tail ; grade 2, mild hind - limb weakness ; grade 3, severe hind - limb weakness ; grade 4, hind - limb paralysis ; grade 5, hind - limb paralysis and partial fore - limb weakness. on the basis of previous reports we selected day 21 p.i. as the time point in which experimental procedures were performed. mice were anaesthetized by intraperitoneal administration of eutalender (normonlab, madrid, spain). spleens were removed and splenocyte suspensions were generated by grinding spleens through a wire mesh. the splenocytes were seeded in 96-well plates at a cell density of 2 10 cells / well in iscove 's modified dulbecco 's medium (imdm ; paa laboratories gmbh, pasching, austria) supplemented with 10% hyclone fetalclone i (thermo fisher scientific, waltham, ma, usa), 50 mol / l of 2-mercaptoethanol (sigma chemical), 2 mmol / l of glutamine, 50 u / ml of penicillin, and 50 mg / ml of streptomycin ; the last three chemicals were obtained from gibco brl (paisley, uk). for splenocyte activation, we used 5 g / ml of mog3555, plp139151, or phytohaemagglutinin (pha ; sigma chemical). cells were incubated in a humidified atmosphere at 5% co2 and 37c for 3 days, the last 1820 h in the presence of 1 ci / well of [h]-thymidine (perkinelmer inc., usa). the levels of incorporated radioactivity were determined using a beta - scintillation counter (wallac, turku, finland). the stimulation index (si) was expressed as the mean of the counts per minute (cpm) of five replicates from each mouse and culture condition divided by the mean cpm of the baseline control replicates. the results are expressed as the mean value standard error of the mean (sem) of the si per group of mice. we performed this study in series of seven mice per condition. to study lymphocyte subsets, cells were resuspended at 10/ml and incubated with rat anti - mouse cd16/32 fc receptor (fcr) (cd16/cd32) during 10 minutes to avoid unspecific staining. then, they were incubated with the appropriate combinations of monoclonal antibodies (bd pharmingen, san diego, ca) for 20 minutes at 4c. cells were washed with pbs and flow - cytometry analysis was performed on facscanto ii (becton dickinson) and analyzed using the diva software (becton dickinson). as strategy for flow - cytometry analysis, an initial region was set around cells expressing intermediate to high cd45 with low - to - intermediate side scatter (p1) and then a second region was set on the forward / side scatter dot plot to exclude debris or apoptotic cells (p2). we used the following monoclonal antibodies : anti - cd4-fitc, anti - cd8a - pe, anti - cd45-cy5.5percp, anti - cd25-apc, anti - b220-fitc, anti - cd1d - pe, and anti - cd5-apc, and isotype controls conjugated with fitc, pe, cy5.5percp, and apc. splenocytes were resuspended (10 cells / ml) in complete medium [rpmi supplemented with 2 mm glutamine, 2 mg / ml gentamycin (all from gibco brl (paisley, uk)), and 10% fetal calf serum (biowhittaker technologies, md, usa) ] and the cells were cultured for four hours with pma (50 ng / ml ; sigma - aldrich, st. louis, mo), ionomycin (500 ng / ml ; sigma - aldrich), and monensin (2 m ; ebioscience, san diego, ca). for il-17a and ifn - gamma detection, fcr were blocked with anti - cd16/cd32 antibody (bd pharmingen) for 10 minutes at room temperature before cell surface staining. cells were stained with antibodies against surface antigens for 30 min at 4c using predetermined optimal concentrations of each antibody. stained cells were fixed and permeabilized using a cytofix / cytoperm kit (bd pharmingen), according to the manufacturer 's instructions, and stained with anti - il-17a - pe and anti - ifn - gamma - apc for 30 min at 4c. after washing cells, flow - cytometry analysis was performed on facscanto ii (becton dickinson) and analyzed using the diva software (becton dickinson). serum cytokines were determined by a milliplex map mouse cytokine panel (millipore, billerica, ma) following manufacturer instructions. maxisorp microtiter plates (nunc, roskilde, denmark) were coated overnight at 4c with 100 l / well of mouse mog3555 peptides (cnb, csic) at a concentration of 10 g / ml in 0.1 m carbonate buffer, ph 9.7. plates were washed with pbs containing 0.02% tween 20 and blocked with 0.1 m carbonate buffer, ph 9.7, containing 5% bsa for 1 h at 37c. then, plates were incubated with triplicate serum samples diluted at 1/200 for 1 h at 37c and washed and incubated with biotinylated anti - mouse igm (sigma - aldrich) or anti - mouse igg (jackson immunoresearch, suffolk, uk) for 1 h at 37c. after this, they were washed and incubated with streptavidin - horseradish peroxidase (roche, basel, switzerland) for 30 min at 37c. after a final wash, the reaction products were visualized using opd (sigma - aldrich) as a substrate and read at 492 nm with a microplate reader (thermo fisher, cambridge, uk). experiments were always performed in two independent series of mice using four animals per condition. we explored regulatory and effector immune mechanisms in cd1 and c57bl/6 mice immunized with mog3555, using as controls nonimmunized mice and mice immunized with plp139151, a myelin peptide that is not capable of inducing eae in any of those mouse strains. we tested the polyclonal and antigen - specific proliferative capacity of splenocytes 21 days after the immunization. a diminished proliferation showing a clear statistical trend (p = 0.056) was detected in the resistant strain when a polyclonal stimulus such as pha was used (figure 1). moreover, upon antigen - specific stimulation, the splenocytes from cd1 resistant mice did not proliferate, while those of c57bl/6 susceptible mice did (p = 0.016, figure 1). control study using irrelevant plp139151 peptide as stimulus did not render proliferation in any of the strains. all these data indicate that despite their heterogeneous mhc antigens, all cd1 resistant mice are unable to develop a t cell response against the mog3555 encephalitogenic peptide. we next aimed to explore immunological mechanisms that contribute to this phenomenon. we first examined b1 and b2 subsets (figures 2(a) and 2(b)). we did not found differences in the percentages of t cell dependant b2 cells (b220+cd5) between resistant and susceptible mice (nonimmunized or immunized with mog or plp peptides). conversely, the percentage of innate b220+cd5 + b cells was downmodulated in c57bl/6 mice after eae induction by mog3555 (p = 0.005) when compared with cd1 resistant mice immunized with the same peptide. when exploring these b cells, we detected a decrease of breg in c57bl/6 mice suffering eae (p = 0.02, figures 2(c) and 2(d)). conversely, resistant cd1 strain upregulated this subpopulation after immunization with mog3555 (p = 0.03). these differences between the two strains (p = 0.001) were only observed upon mog3555 immunization. by contrast, breg remained unchanged in either resistant or susceptible mice immunized with the plp139151 irrelevant peptide. this strongly suggests that breg are specifically triggered to prevent the autoimmune process and have an important role in inducing resistance to eae. we also explored this t cell subset (cd4 +, cd25) in our series of mice (figure 3). upon immunization with no differences were found in this t cell subset between resistant and susceptible strains when nonimmunized mice or mice immunized with the plp139151 peptide were studied. these observations indicate that treg may also play a role in resistance to eae shown by cd1 mice immunized with mog3555. we did not find differences in the levels of igm or igg anti - mog3555 antibodies or in total cd4 + and cd8 + t cell percentages between the resistant and the susceptible mice (table 1). to further discriminate different t cell responses, we analyzed th1 and th17 subsets. we explored the percentage of cd4 + t cells showing intracellular production of ifn - gamma (figure 4) and il-17 (figure 5) upon stimulation with phorbol-12-myristate-13-acetate (pma) and ionomycin. only susceptible mice that developed eae in response to mog3555 showed significant increases in the secretion of ifn - gamma (p = 0.02) and il-17 (p = 0.009) compared to resistant mice. by contrast, resistant mice were unable to mount any of these responses after the same immunization protocol. in addition, when studying serum inflammatory cytokines, we detected a significant increase of il-17 in susceptible mice immunized with mog3555 (figure 4(c)). this shows that downmodulation of regulatory b and t cells is related to the induction of th1/th17 responses in mog - induced eae. the understanding of the immune mechanisms involved in resistance / susceptibility to eae is important, as it may contribute to identification of new therapeutic targets in ms. the role of mhc by inducing defective or appropriate presentation of the encephalitogenic peptide has been previously demonstrated. however, different evidences suggest that induction of breg may also contribute to the resistance to eae. moreover, depletion of breg exacerbates eae symptoms and increases encephalitogenic t cell influx into the cns. in addition, induction of breg in mog - immunized - c57bl/6 mice treated with antibiotics ameliorates eae course, and this effect can be transferred to other c57bl/6 mice by passive transfer of these b cells. here, we aimed to explore if breg may be a mhc - independent mechanism for inducting resistance to mog - induced eae. we explored this in c57bl/6, a congenic mouse strain susceptible to mog3555-induced eae, and in cd1 an outbred strain resistant to mog3555-induced eae in an mhc - independent manner. we immunized both strains of mice with the encephalitogenic peptide and observed that cd1 splenocytes were incapable of proliferating when stimulated with mog3555. we next explored immunological mechanism associated with this phenomenon and observed that susceptible c57bl/6 mice developing the disease after mog3555 immunization downregulate breg, while cd1 resistant mice show a significant increase of these cells upon immunization with the same peptide. this strongly suggests that breg play a role in the induction of resistance to eae in cd1 mice. in addition, an increase of treg may prevent the onset of eae. in the present study we found that, in parallel with breg findings, resistant mice upregulated treg. this seems to indicate that treg also have a role in resistance to mog - induced eae. however, the immunization of c57bl/6 mice with the plp139151 peptide, which is incapable of inducing eae in this strain in an mhc - dependent manner, did not induce significant changes in breg or treg subsets. this clearly shows that when mhc molecules impede the correct peptide presentation, regulatory responses are not triggered as it happens in the outbred mice. to further study differences between resistant and susceptible mice, we explored effector responses in the same series of mice. it has been reported that antibodies may have beneficial or detrimental effects in demyelinating diseases [20, 21 ]. in mog - induced eae model they are not related to the initiation of the disease and it remains controversial if they can influence disease severity [22, 23 ]. we tested the presence of anti - mog3555 igm and igg antibodies in serum of resistant and susceptible mice. there were no significant increases in anti - mog3555 igg or igm responses in mog3555-immunized mice of either susceptible or resistant strains. this shows that igm or igg anti - mog3555 antibodies do not play a role in inducing susceptibility to mog3555-induced eae. previous reports showed that cd4 + t cells are required for the initial induction of progressive eae and that the disease is downregulated by cd8 + t cells. here, we did not find differences in total cd4 + and cd8 + t cell percentages between any of the groups of resistant and susceptible mice. to further discriminate different t cell responses, we analyzed effector th1 and th17 subsets. it has been reported that il-17 impairs the integrity of the blood brain barrier in eae and that a rise of spleen th17 cells prolongs the disease. moreover, a decrease in il-17 levels in supernatants of mog3555-activated splenocytes runs in parallel with reduced demyelination and axonal damage in mice with eae. we did not find significant differences in the percentages of cd4 + cells secreting ifn - gamma or il-17 in nonimmunized mice. conversely, we observed that susceptible mice that developed eae in response to mog3555 showed significant increases in the secretion of ifn - gamma and il-17. by contrast, resistant mice were unable to mount any of these responses after the same immunization protocol. in addition, serum levels of il-17 were increased in susceptible mice immunized with mog3555. these data confirm the importance of the induction of th1/th17 responses in mog - induced eae. in conclusion, our data show that upregulation of b and, to a lesser extent, of treg, is closely associated with mhc - independent resistance to mog - induced eae in cd1 mice and with the abrogation of both th1 and th17 responses, which have a critical role in the development of the disease. these data help to ascertain the regulatory mechanisms than can downmodulate inflammatory responses in demyelinating diseases. | background and objectives. resistant and susceptible mouse strains to experimental autoimmune encephalomyelitis (eae), an inducible demyelinating experimental disease serving as animal model for multiple sclerosis, have been described. we aimed to explore mhc - independent mechanisms inducing resistance to eae. methods. for eae induction, female c57bl/6 (susceptible strain) and cd1 (resistant outbred strain showing heterogeneous mhc antigens) mice were immunized with the 3555 peptide of myelin oligodendrocyte glycoprotein (mog3555). we studied t cell proliferation, regulatory and effector cell subpopulations, intracellular and serum cytokine patterns, and titers of anti - mog serum antibodies. results. upon immunization with mog3555, t lymphocytes from susceptible mice but not that of resistant strain were capable of proliferating when stimulated with mog3555. accordingly, resistant mice experienced a rise in regulatory b cells (p = 0.001) and, to a lower extent, in regulatory t cells (p = 0.02) compared with c57bl/6 susceptible mice. as a consequence, mog3555-immunized c57bl/6 mice showed higher percentages of cd4 + t cells producing both ifn - gamma (p = 0.02) and il-17 (p = 0.009) and higher serum levels of il-17 (p = 0.04) than resistant mice. conclusions. expansion of regulatory b and t cells contributes to the induction of resistance to eae by an mhc - independent mechanism. |
tuberculosis (tb) caused by mycobacterium tuberculosis (m. tb) still is a leading cause of morbidity and mortality worldwide. m. tb infects one - third of the world population ; however, only 10% of them progress to active tb, possibly due to complex environmental, genetic, and immunological interactions. host genetics are considered to play a critical role in the apparent differences in disease progression. toll - like receptors (tlrs), which are pathogen recognition receptors (prrs), play an essential role in host innate response in the pathogenesis of tb. currently, 11 mammalian tlrs have been identified and tlr 110 are functional in humans. tlr1, tlr2, tlr4, tlr6, tlr8, and tlr9 are thought to be involved in the recognition of m. tb. activation of tlrs may result in several possible biological outcomes such as cytokine secretion, modulation of the adaptive immune response, rapid cellular differentiation, apoptosis, and direct antimicrobial activity [68 ]. m. tb cell - surface ligands interact with tlrs that can result in nf-b activation and the production of proinflammatory cytokines, chemokines, and nitric oxide through myeloid differentiation primary response protein 88 (myd88)-dependent or independent pathways [912 ]. it has been proposed that snps of tlr4 and tlr9 genes are associated with ptb susceptibility. tlr - knockout mouse studies indicate that tlr2, tlr4, and tlr9 contribute to host resistance to m. tb infection [1317 ]. recently, a series of studies have been conducted to investigate the association between the tlr4 and tlr9 polymorphisms and the risk of pulmonary tuberculosis (ptb) in diverse populations, but the results were mixed and inconclusive. therefore, we performed a meta - analysis to evaluate the association of tlr4 and tlr9 snps with the susceptibility to ptb. a systematic search was performed for published studies on the relationship between tlr9 polymorphisms and ptb susceptibility, without language restriction. pubmed, embase, web of science, and 2 chinese databases (chinese national knowledge infrastructure and wanfang databases) were utilized to search the available articles, with the last search update on september 30, 2014. the following terms were used : toll - like receptor 4 or toll - like receptor 9 or tlr4 or tlr9, tuberculosis, and polymorphism or polymorphisms, without any limitation applied. the articles retrieved were screened and selected independently by the 2 authors (l.y. and k.x.j.) using eligibility criteria. the reference lists of selected articles and review articles were also examined to identify additional eligible studies. studies in this meta - analysis met the following inclusion criteria : (1) evaluated the relationship between tlr4 rs4986790 (asp299gly) and rs4986791 (thr399ile) and tlr9 rs352139, rs187084, and rs5743836 polymorphisms with tuberculosis susceptibility ; (2) case - control study ; (3) detailed genotype frequency data could be acquired to calculate the odds ratios (ors) and 95% confidence intervals (cis) ; and (4) original articles published in peer - reviewed journals. exclusion criteria were : (1) studies that did not specify sample origins ; (2) studies with no detailed genotype data ; (3) studies with insufficient or duplicate data ; (4) studies with same author from same country of origin. two independent investigators selected the studies according to the inclusion and exclusion criteria by screening the title, abstract, and full text. the following information was collected in duplicate by 2 investigators independently (z.l.l. and l.k.h.) according to the criteria described above : name of first author, year of publication, the characteristics of cases and controls, country of origin, the detective sample, ethnicity, genotyping methods, hardy - weinberg equilibrium, number of cases and controls, and genotype frequency in cases and controls of the selected polymorphisms. for those studies that included subjects of ptb, extra - pulmonary tuberculosis, and tuberculosis meningitis, only the data on ptb cases and healthy controls were collected. if there was a disagreement about data, the 2 investigators rechecked the original data of the included studies and had a discussion to reach consensus ; otherwise, the third investigator adjudicated the disagreements (k.x.j.). two investigators (t.z.x. and w.y.x.) assessed the methodological quality of each eligible article included in this meta - analysis according the newcastle ottawa scale (nos) based on 3 aspects : selection, comparability, and exposure, with scores ranging from 0 to 9. initially, hardy - weinberg equilibrium (hwe) for each study was assessed by chi - square test in the control groups, and p g) and rs4986791 (thr399ile, + 1196c > t) in tlr4 and rs187084 (c-1486 t), rs5743836 (c-1237 t), rs352139 (g+1174a), and rs352140 (g+2848a) in tlr9 in different ethnicities (45). rs4986790 and rs4986791 polymorphisms in tlr4 are the most widely studied and have been implicated in several diseases, including carcinomas. rs187084 and rs5743836 polymorphisms of tlr9 are located within the putative promoter region of the gene that may influence the transcriptional regulation of the tlr9 gene by altering the binding of transcription factors. in contrast, rs352139 and rs352140 in the introns may affect mrna splicing and/or enhance tlr9 gene transcription. the functional evidence supports that tlr4 and tlr9 are genetic components in the risk of ptb, and the association of several snps with ptb susceptibility has been determined. however, the recent studies on tlr4 and tlr9 gene polymorphisms with ptb sociability are still contradictory because a single case - control study only provides a small test power due to a limited sample size. in this study, we performed a systematic search in several electronic databases, including pubmed, web of science, cnki, and wanfang database, on the association of tlr4 and tlr9 polymorphisms with ptb up to december, 2014. finally, 16 articles with a total of 32 case - control studies containing a total of 14 160 participants were enrolled in this meta - analysis. after pooling independent analyses, we did not find any associations of tlr4 and tlr9 polymorphisms with the susceptibility to ptb overall under all the genetic models. however, further subgroup analysis suggested tlr4 rs4986791 was correlated with ptb risk in africans under allelic and dominant models, and tlr9 rs352139 polymorphism was correlated with ptb risk in asians under allelic, heterozygote, and dominant models. recently, several genome - wide association studies have been performed to indentify the snps that influence ptb susceptibility. however, no polymorphisms in tlr4 and tlr9 were identified to be significantly related to ptb risk, which might due to sample size, ethnicity, or geographical limitations. unfortunately, we could obtain the genotype frequencies in the gwas studies related to the polymorphisms in tlr4 and tlr9. to the best of our knowledge, the present study is the most recent meta - analysis to address the association of tlr4 polymorphisms and ptb risk. in a previous meta - analysis that enrolled the articles related to tlr4 polymorphism and ptb risk up to february 2012, the researchers found no association between tlr4 rs4986790 and rs4986791 polymorphism and ptb ; however, subgroup analysis were not performed. in the present meta - analysis furthermore, we performed subgroup analysis according to ethnicity and found a decreased risk of ptb in africans who harbor rs4986791 polymorphism, although there were few enrolled studies, suggesting more attention should be paid to the association of rs4986791 and ptb risk. in contrast, this is the first meta - analysis to investigate the association of tlr 9 polymorphisms with ptb susceptibility. firstly, the number of the included studies limited further analysis and made the results tentative. secondly, we did not perform an evaluation of potential interactions such as polymorphism - polymorphism, gene - gene, and gene - environment due to the lack of sufficient data. further well - designed case - control studies with larger sample sizes focusing on diverse populations should be conducted to confirm the results. our comprehensive conclusion from the current meta - analysis is that tlr4 rs4986791 might correlate with the decreased risk of ptb in africans, and tlr9 rs352139 polymorphism might associate with the increased risk of ptb in asians, but rs4986790, rs187084, and rs5743836 were not independent risk factors for ptb susceptibility. | backgroundfindings regarding the association of the single - nucleotide polymorphisms (snps) rs4986790 and rs4986791 in toll - like receptor 4 and rs187084, rs574386, and rs352139 in toll - like receptor 9 (tlr9) with pulmonary tuberculosis (ptb) susceptibility are inconsistent. we conducted a meta - analysis to systematically summarize and clarify the association between these snps and ptb susceptibility.material/methodsa systematic literature search for relevant studies up to december, 2014 was performed in pubmed, embase, web of science, chinese national knowledge infrastructure (cnki), and wanfang databases. information was gathered from each eligible study. odds ratios (ors) and 95% confidence intervals (95% cis) were used to pool the effect size.resultsfinally, a total of 16 case - control studies on these polymorphisms were enrolled in this meta - analysis. the meta - analysis results suggest there was no association between these polymorphisms and ptb risk ptb risk in all the genetic models overall. however, for tlr4 rs4986791, a significant increased ptb risk was found in africans, and for tlr9 rs352139 a significant increased ptb risk was found in asians after subgroup analysis by ethnicity, although the enrolled studies were limited.conclusionsthere was no association between the polymorphisms in tlr4 and 9 and ptb risk overall, but tlr4 rs4986791 and tlr9 rs352139 might be associated with increased ptb risk in africans and asians, respectively. additional well - designed, larger - scale epidemiological studies are needed to validate our results. |
exercise has long been recognized as a cornerstone of diabetes management and for the prevention of incident diabetes. although reductions in body weight or achievement of an optimal weight is often a therapeutic goal for individuals with type 2 diabetes mellitus (t2 dm), the benefits of exercise training may be better reflected in changes in body composition rather than changes in body weight. understanding body composition and how it changes is essential to the prescription and evaluation of exercise rehabilitation programs for patients with t2 dm. dual - energy x - ray absorptiometry (dxa) is an advanced technique for estimating body fat, lean soft tissue mass, bone mineral content, and bone mineral density. dxa assessment of body composition requires minimal radiation exposure, and is widely available and practical to apply as it merely requires patients to lie supine and clothed on a plinth for scan acquisition. dxa body composition software has established reliability and validity for assessment of fat, lean tissue, and muscle mass, among adults and has been the clinical gold standard for assessment of body composition for some time. however, the utility of dxa is limited due to the cost of the equipment, expertise required to acquire and analyze scans (trained operators), lack of portability, availability of funding, and their ~123 kg weight limit. these criteria combine to limit the availability of dxa in most settings with the exception of some tertiary rehabilitation centers. bioelectrical impedance analysis (bia), particularly, leg - to - leg bia (lbia) using a single frequency (50 khz), footpad electrode system for standing impedance, and body weight measurement has become a popular alternative to dxa for assessing body composition in the general population [47 ]. this lbia method has several advantages over dxa as the equipment required is relatively inexpensive, portable, requires minimal training to operate, and presents no health risk to participants. to date, one prior validation study has been done using a lbia system among individuals with t2 dm. the previous authors reported that percent of fat mass (% fm) assessed with the lbia system was comparable to that with dxa in patients with t2 dm. however, the validity of lbia for evaluation of longitudinal change in body composition following an exercise intervention among participants with t2 dm has not previously been reported. the present study aimed to compare the level of agreement between lbia and dxa assessment of ffm, % fm, and fm before and after exercise intervention among participants with t2 dm. sixty - two adult men and women with t2 dm were recruited from participants of the diabetes, exercise, and healthy lifestyle program at the toronto rehabilitation institute (tri). diagnosis of diabetes was confirmed by a fasting plasma glucose 7.0 mmoll, use of insulin, or oral hypoglycemic agents. potential participants were excluded if they had diagnosed cardiovascular disease, a diabetes - related complication including nephropathy, or retinopathy unrepaired hernia, or any other functional impairment that would preclude participation in a high intensity resistance training program. from eligible participants, only those who completed the exercise intervention program, as well as before and after intervention assessments of body composition were included in the data analysis. the study protocol was approved by the research ethics boards of the toronto rehabilitation institute (tri) and university of toronto and informed written consent was obtained from each participant. one visit assessed height, weight, and lbia. a second assessment allowed for whole body dxa scan acquisition. these visits were completed within 2 weeks of starting the exercise intervention, as well as after completion of the 6-month exercise intervention. lbia and weight were measured using a bia system (tbf-300a, tanita corporation of america, inc., arlington heights, il, usa), which consists of 4 contact electrodes (2 anterior and 2 posterior) that are mounted on the surface of a platform scale (nunez cd 1997, msse). a constant current of 500 microa at 50 khz is passed through the anterior electrode on the platform, and the voltage drop is then measured on the posterior electrode. leg - to - leg impedance of the lower extremities and body weight are measured simultaneously while the subject stands on the scale. this analyzer provides data regarding fat free mass (ffm in kilogram), percent body fat (% fm in %), and fat mass (fm in kilogram). the exact calculation formulae for ffm, % fm, and fm are proprietary. whole body dxa scans to assess whole body ffm, % fm, and fm were conducted using hologic qdr-4500a (hologic inc., waltham, ma, usa). analysis of the scans was performed using the hologic whole body software, version 12.3. for both dxa and lbia assessments, participants were measured at approximately the same time of day for both before and after tests. they were instructed to refrain from exercise for 24 hours prior to testing, and instructed to take water and medications as necessary on their usual schedule. if patients were not adherent with the premeasurement protocol, the participants were asked to come back a different day to have the measurements. a progressive resistance training (rt) program was performed as a 10-exercise circuit, repeated two or three times, twice per week. the circuit consisted of a dumbbell row, half - squat, biceps curl, lateral raise, heel raise, hamstring curl, supine fly, triceps extension, abdominal curl up, and the bird - dog for core strengthening. in addition, aerobic training (at) was performed by participants, working up to an hour in duration. initial at prescriptions of walking or cycling were given at 60% to 75% of heart rate reserve or peak oxygen consumption based on the initial graded - exercise, cardiopulmonary stress test with gas analysis (vmax series software version 12 - 3a, yorba linda, ca, usa). all participants were asked to do their at 5-days a week with one supervised at and rt session per week. statistical analyses were done in overall group except for the differences of body composition between men and women at the baseline. difference of body composition between men and women were tested by unpaired t - test. the difference between similar measures by lbia and dxa before intervention were tested by paired t - tests. linear regression was used to assess the accuracy of the lbia compared with dxa before intervention. the effects of the exercise intervention on body weight and body composition variables were tested by paired t - tests. linear regression was used to determine the level of relative agreement in changes in body composition between bia and dxa between lbia. the bland - altman approach was also used to assess the agreement between the two body composition methods for changes in body composition attributed to the intervention(s). all statistical analyses were performed with ibm spss 20 for macintosh (ibm, new york, usa). in total, 44 participants (men = 23, women = 21) were eligible for study participation (age 53.2 9.1 years, duration of known diabetes : 56.5 8.8 months, hba1c : 7.16 1.11%, oral agent treatment n = 31, insulin treatment n = 13). table 1 displays the physical and demographic characteristics of the overall participants as well as by gender at baseline. women were significantly shorter (p < 0.001) and had lighter body weight (p = 0.019) than men. additionally, the women had significantly lower ffm and greater % fm than men in both dxa and lbia (p < 0.001) (table 2). for the overall cohort, there were strong correlations between ffm, % fm, and fm assessed by lbia and that of dxa (ffm : r = 0.942, p < 0.001 ; % fm, r = 0.833, p < 0.001 ; fm : r = 0.929, p < 0.001 (figure 1). despite this good relative agreement for estimations, however, % fm and fm by lbia were significantly different from that of dxa (% fm, p = 0.047 ; fm, p = 0.003) (table 2). relative to dxa, lbia overpredicted % fm (1.5 4.9%) and fm (2.3 4.8 kg). on average, % fm and fm by lbia were 105.2% and 107.3% of the value determined by dxa, respectively. there was no significant difference between ffm by lbia and ffm by dxa (p = 0.476). the bland - altman plots showed that there were no systematic errors in assessment of ffm (p = 0.907), % fm (p = 0.100), and fm (p = 0.06) using lbia compared with dxa (figure 2). the limits of agreement between lbia and dxa ranged from 8.29 to + 8.77 kg for ffm, from 11.39 to + 9.88% for % fm, and from 11.89 to + 9.63 kg for fm. the mean weight loss during the 6 month exercise intervention was 1.99 4.72 kg (from 85.4 20.9 to 83.4 19.3 kg, p = 0.008), with a decrease in bmi of 0.69 1.53 kg / m (from 30.8 5.9 to 30.1 5.7 kg / m, p = 0.005). fm and % fm were significantly decreased after the intervention for both dxa (% fm, p < 0.001 ; fm, p < 0.001) and lbia (% fm, p = 0.036 ; fm, p = 0.024) (table 2). ffm did not show significant change by intervention for both of dxa (p = 0.649) and lbia (p = 0.113). there were no statistically significant differences in mean changes in all body composition variables between lbia and dxa (ffm, p = 0.068 ; % fm, p = 0.426 ; fm, p = 0.633). lbia assessments of changes in % fm, and fm by the intervention showed good relative agreement with those variables assessed by dxa (p < 0.001) (figure 3). there was a trend of positive relationship between changes in ffm by dxa and that of lbia, however the correlation coefficients were not significant (p = 0.053). bland - altman plots indicated that there were systematic errors in the assessment of the changes in ffm, % fm, and fm by lbia compared with dxa. thus, the more change subjects showed, the greater the disparity in ffm, % fm and fm measures from lbia and those measures from dxa (ffm, p = 0.013 ; % fm, p < 0.001 ; fm, p < 0.001) (figure 4). this study demonstrated that body composition as assessed cross - sectionally using lbia correlated well with dxa assessments in a population of adults with type 2 mellitus. in absolute terms, however, lbia significantly overestimated % fm, and fm. after 6 months of exercise intervention, we found that body fat and body fat percentage assessed by dxa decreased. the observed values are in - line with other exercise intervention studies in diabetes [10, 11 ]. in terms of measuring changes in body composition after the exercise intervention, lbia assessments of changes in ffm, % fm, and fm showed good absolute agreement with similar dxa assessments. however, bland - altman plots showed that there were systematic errors in the assessment of the changes in ffm, % fm, and fm by lbia. to the best of our knowledge, this is the first study to investigate the validity of bioelectrical impedance analysis (bia) including lbia for assessing changes in body composition following exercise intervention among individuals with t2 dm. the lbia technique demonstrated good relative agreement for assessing ffm, % fm, and fm as shown by high - correlation coefficients, although it overestimated % fm and fm. additionally the comparison between men and women in body composition evaluated by both dxa and lbia methods demonstrated that both methods can similarly detect gender difference in body composition (table 2). these results imply that lbia may be used for cross - sectional studies or assessments, but lbia and dxa should not be used interchangeably. similar overestimation of % fm by lbia has reported in female subjects with t2 dm. the bias in this cross - sectional assessment was similar or smaller than that of previous studies in people with t2 dm, healthy adults or overweight women. the primary purpose of this study was to investigate the utility of lbia for assessing changes in body composition during an exercise intervention in individuals with t2 dm. there were no statistically significant differences in the mean changes in all body composition variables between lbia and dxa. furthermore, there were strong correlations between the changes in % fm and fm measured by lbia and dxa. these results infer relatively good lbia accuracy, however, in the bland - altman plot, significant correlations were found between the mean of dxa change and lbia change and the difference between them (figure 4). this indicates that lbia tends to overestimate the changes in subjects who lost or gained more ffm (figure 4(a)), % fm (figure 4(b)), and fm (figure 4(c)). taken together, these results suggest that lbia can qualitatively assess the changes in ffm, % fm, and fm (e.g., positive changes are estimated as positive changes), while quantitatively overestimates the changes (e.g., the amount of positive change is overestimated). no prior studies have investigated whether bia methods, including hand - to - hand bia and traditional tetrapolar bia, can accurately detect quantitative changes in an individual 's body composition in the t2 dm population. however in a study with overweight and obese women, thomson. investigated the relationship between the mean of dxa change and lbia change after weight loss and the differences between them. this implied that, unlike our results in people with t2 dm, the quantitative changes in body composition can accurately be assessed by lbia in obese women. the mechanisms responsible for the overestimation of % fm and fm in the cross - sectional analysis and the systematic error in the changes in body composition are unclear. however, it is reasonable to hypothesize that the discrepancy is in part due to the population studied, that is, the original bia equations were likely derived from a generally healthy population. however, our participants were all living with t2 dm and had specific medical, anthropometric, and physiological characteristics. we are unaware of the bia equations for the specific tanita scales used in this study to calculate body ffm, % fm, and fm. but typically bia calculations are based on measurements of impedance, and then applied to a gender - specific equation to calculate either body density (bd) or ffm. in case of bd, bd is in turn used to calculate % fm according to standard densitometric formulae which assumes constant density of fat and ffm [13, 14 ]. in the case of ffm, ffm, bia assumes density of each tissue is constant between individuals, and with time during interventions. in fact, previous studies in individuals with diabetes mellitus demonstrated that glycaemia and insulin treatments induce changes in water retention and distribution, bone mass, protein, and fat mass through the anabolic effects of insulin. others have demonstrated that hydration of ffm decreases when weight decreases in individuals with t2 dm. all of these aforementioned variables may have resulted in the previously derived predictive equations derived for the general population, becoming less directly applicable to our subjects with t2 dm. these factors help explain the resulting lack of absolute agreement and systematic errors in determining changes in % fm and fm following the exercise intervention. establishment of equations for assessment of body composition in individuals with t2 dm using lbia are desired as lbia seems well suited for daily or frequent body composition assessment among individuals with t2 dm. lbia is a relatively inexpensive and portable means of assessment, and needs minimal training to acquire data. use of skin fold caliper as an alternative may be another option to assess body composition in individuals with t2 dm. the method has worked well in other patient populations when assessors are well trained. while dxa has been consistently shown to provide an accurate assessment of body composition [3, 18, 19 ], and has been used as criterion methods for comparison of other techniques [20, 21 ], some previous studies demonstrated that using dxa as a gold standard may lead to some errors [22, 23 ]. in fact, minderico. demonstrated that dxa significantly overestimated changes in fm and % fm across weight loss compared with a reference four - compartment model. 2011) demonstrated that it was difficult to predict changes in visceral fat using dxa in obese postmenopausal women. another study limitation is that a gender effect on the applicability of the lbia was not investigated in this study due to small sample size. further study is needed to determine the applicability of the lbia to each gender with a larger sample size. in conclusion, our analyses show that leg - to - leg bia could be used for cross - sectional studies or routine assessment of body composition among people with type 2 diabetes mellitus. however, leg - to - leg bia and dxa should not be used interchangeably. in longitudinal assessments, although the changes measured using lbia and dxa showed high correlations, the estimated change by leg - to - leg bia showed a systematic error, suggesting that the accuracy of lbia is less than desired, implying that leg - to - leg bia is appropriate for assessment of qualitative changes in body composition (gain or loss) but is not sufficiently sensitive to accurately quantify changes in body composition over several months among individuals ' with t2 dm. specific equations for type 2 diabetes mellitus should be established for accurate body composition assessment in this population. | we aimed to compare the level of agreement between leg - to - leg bioelectrical impedance analysis (lbia) and dual - energy x - ray absorptiometry (dxa) for assessing changes in body composition following exercise intervention among individuals with type 2 diabetes mellitus (t2 dm). forty - four adults with t2 dm, age 53.2 9.1 years ; bmi 30.8 5.9 kg / m2 participated in a 6-month exercise program with pre and post intervention assessments of body composition. fat free mass (ffm), % body fat (% fm) and fat mass (fm) were measured by lbia (tbf-300a) and dxa. lbia assessments of changes in % fm and fm post intervention showed good relative agreements with dxa variables (p < 0.001). however, bland - altman plot(s) indicated that there were systematic errors in the assessment of the changes in body composition using lbia compared to dxa such that, the greater the changes in participant body composition, the greater the disparity in body composition data obtained via lbia versus dxa data (ffm, p = 0.013 ; % fm, p < 0.001 ; fm, p < 0.001). in conclusion, assessment of pre and post intervention body composition implies that lbia is a good tool for assessment qualitative change in body composition (gain or loss) among people with t2 dm but is not sufficiently sensitive to track quantitative changes in an individual 's body composition. |
indels (insertion deletion) or dips (deletion insertion polymorphisms) are short length diallelic polymorphisms, consisting of the presence or absence of short sequences (typically 150 bp). they are relatively common throughout the human genome representing 1520 % of all polymorphisms with the total number estimated at about 2 million. short amplicon size (50150 bp), low mutation rate (< 2 10), and capacity to multiplex (3040 markers) and type using a single multiplexed pcr with fluorescently labeled primers followed by capillary electrophoresis (a current technology for human identification) [35 ] are the main advantages that make indels useful in forensic genetics applications including individual identification, kinship testing, population studies and ancient dna analysis [68 ]. the investigator dipplex kit (qiagen) contain components for the simultaneous amplification and analysis of 30 biallelic autosomal indels and amelogenin. the indels are distributed over 19 autosomes at the minimum distance of 10 mbp to routinely used str and snp markers. the allele length variations of the indels are between 4 and 22 bp, and all amplicons are shorter than 160 bp. buccal swabs were anonymized and collected from unrelated volunteers along with information on the birthplace and ethnicity of the donor. signed informed consents were obtained from all the participants and this study complied with the protocol approved by the ethical committee of poznan university of medical sciences (ref : 139/13). the population sample sizes were : poles (np = 122), and taiwanese (nt = 126). the extraction of genomic dna was carried out using qiaamp dna mini kit (qiagen). the quantitation was performed using quantifiler human dna quantification kit on a 7500 real - time pcr system (applied biosystems) according to the manufacturer s specifications. the samples were then normalized to 100 pg/l and stored at 20 c until amplification. pcr conditions were applied according to the protocol recommended by the manufacturer of the investigator dipplex kit (qiagen) in pcr system 9700 (applied biosystems, usa) with a total reaction volume adjusted to 5 l containing 1.8 l nuclease - free water, 1.0 l reaction mix a, 1.0 l primer mix, 0.2 l multitaq2 polymerase, and 100 pg dna template. electrophoresis and typing were performed in 3130 genetic analyzer (applied biosystems, usa) using a 36 cm capillary array and a denaturing polymer pop-4. bto 550 (qiagen) was used as the internal lane standard spanning fragments from 60 to 550 bps. prior to the analysis, a five dye matrix standard (bt5) was established with the fluorescent labels dyes 6-fam, btg, bty, btr, and bto under the any5dye virtual filter. samples were injected for 10 s at 3 kv and electrophoresed for 1000 s at 15 kv at a run temperature of 60 c. estimates for genetic diversity (allele frequencies, heterozygosity), conformance to expectations of the hardy weinberg equilibrium (hwe) and for independence (linkage disequilibrium, ld) were obtained using gda v1.0 software. for multiple comparisons, the original significance levels achieved (p values) were transformed by the bonferroni correction procedure, i.e. 30 markers per database yield an actual significance level of 0.0016667. forensic informativeness was estimated by calculating discrimination power (dp), match probability (mp), polymorphic information content (pic), typical paternity index (tpi), and power of paternity exclusion (pe) using powerstats v1.2 spreadsheet (promega). comparison of allele frequency distributions was performed by means of a pairwise population comparison test (r c contingency test ; g. carmody, ottawa, canada). a representative dipplex profile obtained from amplification of 100 pg dna template is presented in fig. 1. in the polish population sample the indels frequency distributions showed no deviations from hwe (bonferroni corrected, 0.0025 < p < 1.0000) evaluated by randomization procedure (10,000 cycles). pairwise comparison using the exact test disequilibrium analysis with 16,000 permutation steps yielded departures from independence for 93 out of 435 pairs of indels under the analysis (0.0019 < p the departures appeared statistically insignificant when the bonferroni correction was used for the number of analysed loci. observed heterozygosity for all the systems ranged 0.35250.5164, with an average of 0.4385, which is slightly lower than the values reported for czech, german, danish, finnish, central spain, and the basque country populations. in the taiwanese population sample the indels frequency distributions showed no deviations from hwe (0.0032 < p < 1.0000) except for dlh39 (p = 0.0005). there were no statistically significant departures from independence between any pair - wise combination of indels (0.0018 < p < 0.0597) (data not shown). observed heterozygosity for all the systems ranged 0.12700.6191, with an average of 0.4079, which corresponds to the values reported for asian - americans, and african - americans. the highest dp loci were hld114 (dp = 0.660) for poles and hld118 (dp = 0.656) for taiwanese. based on data of the 30 indels the combined mp value among poles amounts 7.98 10 which is more than two orders of magnitude lower than the value calculated for the taiwanese population (1.22 10). both parameters however, indicate a favourable value of a random match comparable with that of ampflstr sgm kit [18, 19 ]. the combined values of pe are 0.9900 versus 0.9884, correspondingly (table 1). 1representative dipplex profile obtained from amplification of 100 pg dna templatetable 1population data and forensic efficiency parameters for 30 dipplex indels in polish (np = 122) and taiwanese (nt = 126) population sampleshldchromosomal locationgenbank snp idpolestaiwanese-/dip / phohepicmppetpi-/dip / phohepicmppetpi1777q31.1rs16110480.42210.08590.40980.48990.370.3520.1150.840.50790.59710.47620.50190.370.3640.1680.952452q31.1rs23079590.46720.70440.47540.49990.370.3660.1670.950.32530.04170.52380.44080.340.4490.2091.0531317q36.2rs16110010.38110.68750.45080.47670.360.3820.1480.910.70630.83100.42860.41650.330.4320.1320.884706q16.1rs23076520.50000.11130.42620.50210.380.3460.1310.870.34130.23550.39680.45140.350.3900.1120.835616q13rs16109050.45090.00250.36070.49720.370.3400.0960.790.47620.47290.53970.50090.370.3980.2251.09611117p11.2rs13050470.47540.01810.39340.50080.370.3400.1100.820.83330.10960.23810.27890.240.5690.0410.667585q14.1rs16109370.57381.00000.49180.49110.370.3820.1800.980.56350.58660.52380.49390.370.3960.2091.058564q25rs23082920.33200.69440.42620.44810.350.4010.1250.860.38101.00000.47620.47350.360.3920.1680.95911820p11.1rs164380.59020.09750.40980.48570.370.3590.1250.860.09530.01390.12700.17300.160.7250.0130.57109211q22.2rs171744760.58200.18440.42620.48860.370.3600.1310.870.53170.14940.42860.50000.370.3490.1320.88119312q22rs23075700.46311.00000.50000.49930.370.3780.1881.000.42060.46640.52380.48930.370.4000.2091.05129914q23.1rs23081630.40571.00000.48360.48420.370.3850.1740.970.15870.00320.19050.26810.230.5970.0270.6213889q22.32rs81905700.56970.36780.45080.49230.370.3640.1480.910.45240.03010.39680.49740.370.3440.1120.831410115q26.1rs23074330.41800.58560.45900.48860.370.3700.1540.920.53970.28040.44440.49780.370.3600.1430.9015675q33.2rs13050560.39340.04500.38520.47750.360.3610.1100.820.34130.69110.42860.45140.350.3970.1320.8816838p22rs23080720.52870.85190.51640.50040.370.3850.2021.030.57940.14570.55560.48930.370.4200.2411.131711417p13.3rs23075810.68850.39000.39340.43070.340.4100.1100.820.71431.00000.41270.40980.320.4350.1220.8518482q11.2rs283699420.43850.19880.43440.49450.370.3560.1360.880.66671.00000.44440.44620.350.4070.1430.901912422q12.3rs64810.36060.43280.42620.46310.350.3850.1310.870.46410.28310.55560.50150.370.4080.2411.132012221q22.11rs81785240.54510.71060.51640.49800.370.3880.2021.030.83330.74860.26980.27890.240.5620.0520.682112522q11.23rs163880.57790.85250.47540.48860.370.3790.1740.970.52380.59900.47620.50090.370.3650.1680.9522645q12.3rs16109350.46310.13750.42620.49870.370.3470.1250.860.14291.00000.25400.24590.210.5980.0460.6723817q21.3rs178799360.55740.46970.45900.49540.370.3640.1540.920.27780.00690.30160.40280.320.4340.0640.722413622q13.1rs163630.53281.00000.49180.49990.370.3730.1800.980.56350.00620.61910.49390.370.4640.3141.31251333p22.1rs20672350.48770.14630.43440.50180.370.3490.1360.880.64291.00000.46030.46100.350.3980.1550.93269713q12.3rs172388920.50410.07280.41800.50200.370.3640.1480.910.61110.71000.46030.47720.360.3820.1550.9327401p32.3rs23079560.49590.21060.44260.50210.370.3530.1450.900.35710.12500.39680.46100.350.3880.1160.84281281q31.3rs23079240.54510.01750.38520.49800.370.3410.1070.820.65080.07750.38100.45630.350.3800.1060.8229391p22.1rs178784440.58200.00590.35250.48420.370.3470.0830.760.82540.00050.19050.28940.250.5760.0270.6230848q24.12rs30814000.46310.15500.43440.50120.370.3640.1480.910.26980.03900.31750.39560.320.4400.0710.73-/dip/ frequency of deletion allele, p probability value for hwe, ho observed heterozygosity, he expected heterozygosity, pic polymorphic information content, mp match probability, pe power of exclusion, tpi typical paternity index representative dipplex profile obtained from amplification of 100 pg dna template population data and forensic efficiency parameters for 30 dipplex indels in polish (np = 122) and taiwanese (nt = 126) population samples -/dip/ frequency of deletion allele, p probability value for hwe, ho observed heterozygosity, he expected heterozygosity, pic polymorphic information content, mp match probability, pe power of exclusion, tpi typical paternity index a pairwise testing for heterogeneity using the -test was applied to compare allelic distributions. minor or no significant differences were found between the polish sample and czech, danish, finnish, and american - caucasian data sets. correspondingly, the comparison between the taiwanese sample and asian - americans yielded no significant differences (0.032 < p. on the other hand, among differences revealed between the poles and the taiwanese at 14 indels (p < 0.05), these at hld131, hld111, hld118, hld99, hld48, hld122, hld64, hld81, hld39, and hld84 remained significant after the critical value was corrected for multiple testing (table 2). it is noteworthy that the same loci significantly accounted for diversity between caucasian and asian samples, based on north american datasets published elsewhere. table 2p values of population differentiation tested by an exact test and population specific fst indices per polymorphic locus (absolute values)hldp valuefst polesfst taiwaneseaverage fst770.2020.01080.01070.0107450.0430.03710.03760.03731310.0000.18930.18970.1895700.0220.04630.04680.046660.7710.00270.00280.00281110.0000.24500.24680.2459580.8870.03310.03310.0331560.4600.00130.00110.00121180.0000.42600.42820.4271920.4770.00110.00100.0011930.5690.00040.00030.0004990.0000.13610.13810.1371880.1200.02320.02320.02321010.0890.02530.02520.0253670.4630.00170.00190.0018830.4770.00110.00120.00121140.7580.00260.00240.0025480.0010.09620.09660.09641240.1510.04400.04370.04391220.0000.17320.17510.17411250.3940.00190.00180.0019640.0000.21320.21540.2143810.0000.14520.14590.14551360.6700.00220.00210.00211330.0320.04380.04410.0440970.1180.01890.01910.0190400.0460.03460.03490.03471280.1130.01890.01920.0190390.0000.12870.13050.1296840.0000.07330.07420.0737italicised significant differentiation test p values, after bonferroni correction p values of population differentiation tested by an exact test and population specific fst indices per polymorphic locus (absolute values) italicised significant differentiation test p values, after bonferroni correction wright s fst was analysed to measure population substructure effects. amova results revealed that most of the molecular variation was due to variation within the analysed populations (92.54 %) rather than among them, with average fixation index values of 0.0743 and 0.0749 (poles and taiwanese, respectively). our findings correspond to those presented by other authors who used amova to compare the allelic frequencies for each dipplex locus in populations of europe, africa, asia and north america [16, 17 ]. moreover, in our analysis individual indels displayed noticeable disparities in fixation index spanning from 0.0004 to 0.0003 (hld93) to 0.4260 and 0.4282 (hld118) for poles and taiwanese, respectively (table 2). the individual mutation rate of a locus is one of the factors that may explain the observed discrepancy. however, when compared with mutation rates of 1010 for strs [22, 23 ], snps have essentially mutation rates estimated at as low as 10. from the point of view of forensic genetics, markers with high heterozygosity and very low fst are potentially advantageous due to relatively high discrimination efficiency irrespective of population of origin [24, 25 ]. high heterozygosity enhances the polymorphism information at each snp and low fst diminishes the chance of interpopulation effects. some snps are reported to have remarkably little variation in allele frequency around the world. on the other hand, ancestry informative single - nucleotide polymorphisms (aisnps) are required to show low heterozygosity and high allele frequency divergence between different ancestral or geographically distant populations (fst values). these genetic markers are especially useful in establishing the high probability of an individual s biogeographical ancestry [27, 28 ]. we have selected eight indels (hld131, hld111, hld118, hld99, hld122, hld64, hld81, hld39) with fst higher than 0.1 between poles and taiwanese as potential aisnps for further analyses. | the investigator dipplex kit (qiagen) contain components for the simultaneous amplification and analysis of 30 biallelic autosomal indels and amelogenin. the objective of this study was to estimate the diversity of the 30 markers in polish (np = 122) and taiwanese (nt = 126) population samples and to evaluate their usefulness in forensic genetics. all amplicon lengths were shorter than 160 base pairs. the dipplex genotype distributions showed no significant deviation from hardy weinberg rule expectations (bonferroni corrected) except for dlh39 in the taiwanese population. among the poles and the taiwanese the mean observed heterozygosity values are 0.4385 and 0.4079, and the combined matching probability values are 7.98 1014 and 1.22 1011, respectively. the investigated marker set has been confirmed as a potential extension to standard short tandem repeat - based kits or a separate informative system for individual identification and kinship analysis. eight indels have been selected as possible ancestry informative single - nucleotide polymorphisms for further analyses. |
periodontal disease is a chronic inflammatory condition induced by dental biofilm that results in pocket formation, alveolar bone destruction and tooth loss as evidenced by studies in animals and humans. periodontal disease has been divided in two main categories : gingivitis (gingival inflammation without loss of connective tissue attachment) and periodontitis. periodontitis is characterized by an apical migration of the junctional epithelium, which in most patients increased probing depths (pds) or the formation of periodontal pockets accompany the development of periodontitis. the majority of patients with periodontitis will have the chronic form of the disease (most prevalent in adults over 30 years of age, slow to moderate rate of progression, substantial deposits of subgingival calculus, periodontal pockets, and bone destruction) that are categorized in slight : 1 - 2 mm of clinical attachment loss (cal) ; moderate : 3 - 4 mm cal ; or severe : > 5 mm cal, localized or generalized (> 30% of sites involved). the final goal of periodontal therapy is to eliminate the inflammation and to achieve periodontal tissue regeneration, especially in intraosseous defect, destroyed by the disease process. innumerous treatment modalities have been proposed to re - establishing periodontal health for patients with intraosseous defects associated to periodontitis, such as : proper plaque control, open - flap debridement, or single flap approach, regenerative procedures with bone grafts, guided tissue regeneration (gtr), and orthodontic movement. albeit the treatment of these defects by surgical techniques are commonly used, there are significant benefits of treating intraosseous defects by means of conservative approaches, especially related to the cost - benefit outcome and absence of gingival flap that could result in delayed healing, wound dehiscence, loss of primary closure, lower filling osseous defect and postsurgical infection. in addition, esthetics may be compromised by gingival recession and loss of interproximal papillary tissue. for these reasons, new conservative techniques especially designed to optimize the elimination of intraosseous defect have been developed by means of orthodontic movement, but at least in part, this protocol has not been validated in patients. orthodontic treatment has been proposed to achieve a more favorable osseous contour, especially in 1-walled defects in which predictability of regenerative periodontal therapy is poor. there are some possibilities to modify the osseous contour with orthodontic movement : intrusion, extrusion, movement of a tooth into an osseous defect, alignment and leveling, and moving a tooth in the opposite direction, away from the osseous defect. thus, orthodontic therapy can reduce or even eliminate the intraosseous 1-walled defect without the necessity of surgical procedures. orthodontic movements in patients with reduced alveolar bone do not increase the damage to the periodontium if good plaque control and good oral hygiene is offered by the patient and the periodontist. in the presence of active periodontal disease and inflammation, orthodontic forces may cause a more severe periodontal bone resorption. in a study by re., the periodontal - orthodontic combined treatment, in patients with reduced periodontium, was capable to maintain the long - term stability after 12 years follow - up in 276 patients. this result was corroborated by another study that showed the use of continuous and light orthodontic forces, without gingival inflammation, resulted in reduction of pd, clinical attachment gain and radiological bone fill. here, we proposed a new therapy for the treatment of 1-walled intraosseous defect by means of orthodontic movement toward the osseous defect. to the best of our knowledge, this is the first long - term case report that describes an orthodontic movement to eliminate the periodontal bone defect without surgical or regenerative procedures in a patient with localized severe chronic periodontitis. a 47-year - old caucasian woman was referred to the department of periodontology for the treatment of her left maxillary central incisor. the periapical radiographs, standardized by means of parallel technique, showed a localized vertical bone defect on the mesial region her left central incisor [figure 1a ]. periodontal examination with transgingival probing, under local anesthesia, revealed a 1-walled periodontal defect with 9 mm of pd, bleeding on probing (bop), with signs of class ii tooth mobility, and normal fremitus position in this site [figure 1b ]. the patient had never been treated for periodontitis and based on clinical and radiographic examinations the diagnosis was severe localized chronic periodontitis. the initial treatment plan was nonsurgical basic periodontal therapy to control the disease and reduce pd. (a) initial periapical radiographic showing the 1-walled intraosseous defect a in the mesial aspect of the central incisor. (b) initial clinical appearance of the left central incisor, showing the 9 mm probing depth periodontal pocket. note the bleeding on probe initially, a periodontal examination was performed including plaque scores, full - mouth bleeding, assessment of pd and cal [table 1 ]. to evaluate the qualitative changes in the gingival soft tissue, the gingival index was then accessed and scored at 3 (moderate inflammation and spontaneously bleeding), as described previously. then, the treatment involved oral hygiene instructions (ohi), supragingival and subgingival scaling followed by root planning with mini - gracey curettes (mini five gracey curette, hu - friedy, chicago, il, usa) and chemistry control with 0.12% chlorhexidine. 2 months after the periodontal therapy, the patient exhibited good plaque control, healthy gingival tissues and 7 mm pd reduction, resulting in a final pd of 2 mm [figure 2a and table 1 ]. the patient was followed during 3 months and as an expected result of periodontal treatment, a black triangle between the central incisors was created due to the apical displacement of the gingival interproximal papilla, compromising the patient esthetics, and the cal was 9 mm [figure 2b ]. (a) 3 months after basic periodontal therapy with supra and subgingival scaling and root planning. (b) 3 months after basic periodontal therapy resulting in apical displacement of gingival margin leading to a compromised patient esthetics. (c and d) initial orthodontic movement toward the intraosseous defect the periodontal clinical indexes at the beginning of the therapy (baseline), at the end of the periodontal treatment, after orthodontic movement and after 6-year of follow - up to improve the esthetics of the gingival tissues between the central incisors and to achieve a more favorable osseous contour, orthodontic movement was proposed and accepted by the patient. the patient showed a class i cephalometric pattern with good facial relationship and a normal profile without skeletal deviations. the orthodontic treatment began by the placement of fixed appliances in the maxillary arch with brackets and archwires. the maxillary right and left canine received a cuspid to cuspid titanium - molybdenum alloy 0.017 0.025 (3 m unitek, monrovia, ca, usa) wire with progressively artistic bend incorporated, designed to produce a tipping root movement toward the intraosseous defect [figure 2c and d ]. the movement was accomplished by applying equal moments of approximately 15 in both central incisors. after 13 months of active orthodontic treatment, acceptable esthetics results were achieved, and the intraosseous defect was partially filled, as evidenced by a decrease in the cal from 9 mm to 5 mm, resulting in 4 mm of bone gain [figure 3a and b, table 1 ]. at the end, the patient received a permanent retention apparatus, placed in her right and left central incisors, to prevent orthodontic relapse after treatment. (a) 8 months after orthodontic movement by means of root movement toward the intraosseous defect. note the partial filling of the papilla between the central incisors. (b) periapical radiographic 8 months after active orthodontic therapy showing the partial intraosseous defect filling. (c) 6 years follow - up after periodontal and orthodontic therapy for the treatment of localized periodontal intraosseous defect. (d) final radiographic result of an intraosseous defect treated with periodontal and orthodontic therapy. compare with the initial radiography following the orthodontic treatment, the patient was seen monthly during the first 4 months to evaluate the periodontal status of the patient (gingival index, plaque scores, and bop), followed by professional prophylaxis and ohi [table 1 ]. six years of follow - up showed normal pd, no bop, no plaque scores, and the intraosseous defect was almost completely eliminated [table 1 ]. the black triangle was reduced and the patient was rather pleased with the absence of sensitivity and spontaneous bleeding and good esthetics results. clinical success was achieved in this case involving basic periodontal therapy and orthodontic treatment by means of root movement toward the intraosseous defect [figure 3c and d ]. new surgical approaches have been developed, in recent years, to optimize wound healing, to minimize the surgical trauma and to improve primary closure in the reconstructive procedures of periodontal intraosseous defects. commonly, treatments of intraosseous defect involve scaling and rooting planning with or without surgical flap. in this last case, additional reconstructive procedures are indicated, such as : bone grafts, gtr and biological agents to allow the intraosseous defect fill and periodontal tissue regeneration. however, the predictability in these approaches only is found for 2- or 3-walled defects. on the other hand, orthodontic movement has been evaluated only in few cases for the treatment of deep intraosseous defect with open - flap surgery and orthodontic intrusion, but not with orthodontic movement and basic periodontal therapy. thus, we propose a novel approach to obtain periodontal regeneration of 1-walled intraosseous lesion by means of orthodontic movement toward the bone defect. the results of this clinical case indicate that orthodontic movement improve the healing process after basic periodontal therapy as demonstrated by reduction of pd, a significantly improve in the cal, the new bone formation filling the periodontal defect evidenced by radiological examination, and for improvement in the esthetics of the interproximal papillary area. besides, this approach can be beneficial because allow better clinical conditions to plaque control resulting in reduced inflammation, favors the incidence of occlusal forces, and enhances the osseous contour. unlike the findings of a previous study in dogs, where the authors observed small level of vertical bone apposition compared to the control group, using orthodontic movement in 1-walled bone defect, our results showed an increased bone gain, filling the intraosseous defect due to the movement of the root into the lesion. the clinical results obtained in the present case can be attributed to the orthodontic movement that induced a slowly displacement of the root toward to the base of the osseous defect, allowing new bone formation, reduction of gingival recession and better tooth position, and could be stated that stretching the periodontal fibers resulted in reduction of the downgrowth of the epithelial cells. moreover, orthodontic movement increased the periodontal ligament cells turnover enhancing the repopulating of the root surface. in addition, good plaque control made by the patient and by the periodical visits to the periodontist, and due to a longer stabilization period of the orthodontic appliance were determining factors for achieving the results presented in this clinical report. previous study showed that proper oral hygiene maintenance during orthodontic therapy, no injury to the supporting teeth will occur. however, if periodontal inflammation is present, and if oral hygiene is poor controlled, orthodontic treatment will result in increased risk for bone resorption. as stated by corrente., absence of gingival inflammation are considered to be the most important factors related to the long - term success in patients with reduced periodontium. patients with periodontal disease commonly show interproximal bone loss, which result in papillary height defect. an interesting finding of this clinical case is the significant esthetics improvement of the interproximal papillary area between the central incisors. this result can be explained by the intraosseous defect filling and apical displacement of the contact point between central incisors after the orthodontic movement, which provided an adequate distance from the contact point to the alveolar crest. according to tarnow., when the distance from the base of the contact point to the bone crest was 3 - 5 mm, the papilla was present in 98% of cases. however, when the distance was > 6 mm usually the interdental papilla was absent. in this particular case, the distance from the contact point to the bone crest allowed the papilla filling of almost the entire space between the central incisors. basic periodontal therapy, good plaque control, light orthodontic forces, and root movement into the defect, can be a viable alternative for the treatment of intraosseous lesions in the esthetics region resulting in radiological bone fill, clinical attachment level gain, reduction of gingival recession, and pd. however, it should be stated that light orthodontic forces, good plaque control made by the patient, periodical maintenance visits to the periodontist, and a permanent retention orthodontic apparatus are necessary to achieve good results. | extensive intraosseous lesions represent a clinical challenge for the periodontist. sites with bone defects have been shown to be at higher risk of periodontitis progression in patients who had not received periodontal therapy. thus, the aim of this case report was to describe a novel approach for the treatment of 1-walled intraosseous defect by combining nonsurgical periodontal therapy and orthodontic movement toward the bone defect, avoiding regenerative and surgical procedures. a 47-year - old woman underwent the proposed procedures for the treatment of her left central incisor with 9 mm probing depth and 1-walled intraosseous defect in its mesial aspect. initially, basic periodontal therapy with scaling and root planning was accomplished. two months later, an orthodontic treatment was planned to eliminate the intraosseous lesion and to improve the interproximal papillary area. orthodontic root movement toward the osseous defect was performed for 13 months with light forces. after 6 years postoperative it was concluded that combined basic periodontal therapy and orthodontic movement was capable of eliminating the intraosseous defect and improve the esthetics in the interproximal papillary area between the central incisors. |
neurocysticercosis is the most common parasitic infection of central nervous system and result from infection with the intermediate stage of taenia solium. in india, cysticercosis is highly prevalent and cns involvement is seen in 60%90% of infested patient. cerebrum and cerebellum are common sites but may involve brainstem, basal ganglion, thalamus, and lateral sinus. seizures are the commonest presenting feature in majority but can present with headache, focal deficits, hydrocephalus, and raised intracranial pressure. we report a case of pontomedullary neurocysticercosis presenting with left vi and vii cranial nerve palsy of lower motor neuron type and contralateral hemiplegia, i.e., millard gubler syndrome, which is not reported till date. a 7 year girl presented in outpatient department with history of vomiting and deviation of angle of mouth to right side for 3 days. there was no history of fever, headache, seizures, altered sensorium, diplopia, nasal regurgitation of feeds, trauma, ear discharge, rash, recent vaccination, and history of contact with an open or treated case of tuberculosis. examination of patient revealed palsy of vi and vii cranial nerve of left side with right - sided grade iii / v weakness, upper motor neuron type, of upper and lower limbs. her right - sided babiniski 's sign was positive with exaggerated deep tendon reflexes on same side. his complete blood count cbc, x - ray chest, blood glucose, electrolytes, liver and renal function tests were normal. mantoux test was negative at 72 h. cerebrospinal fluid study showed cells 8/mm protein ; 48 mg / dl, and glucose was 68 mg / dl. contrast - enhanced computed tomograph (cect) of cranium revealed single - ring enhancing lesion, with eccentric dot and perilesional edema at pontomedullary area of brain stem suggestive of inflammatory granuloma with possibility of neurocysticercosis [figure 1 ]. magnetic resonance imaging (mri) of brain showed a well - defined ring enhancing lesion with thick rim noted in pontomedullary area of brain stem [figure 2 ], which appeared hypointense on t1-weighted and hyperintense on t2-weighted images. flair images showed suppression of internal signal with mild perilesional edema ; further suggestive of neurocysticercosis in its colloidal - vesicular stage. coronal cect image showing solitary ring enhancing lesion, with eccentric scolex, in pontomedullary area mri of cranium showing thick cyst wall enhancement with mild perilesional edema, depicting colloidal vesicular stage of cysticercosis in pontomedullary area patient was administered 20% mannitol (1 gm / kg / dose i.v. 8 hourly for 48 h), dexamethasone (0.5 mg / kg / day for 2 days), and then prednisolone 2 mg / kg / day along with acetazolamide at 75 mg / kg / day. on 3 day of steroid, oral albendazole at 15 mg / kg / day steroid was stopped after 7 days and patient was discharged on oral albendazole which was to continue for 28 days and advice of physiotherapy. at discharge, there was no vomiting but deviation of angle of mouth and right - sided weakness persisted. she was called for follow - up after 15 days and found to have no deviation of angle of mouth and weakness. patient is doing well at 1 and 3 months of follow - up with no clinical evidence of cranial nerve palsy and hemiparesis. unusual manifestations are stroke, visual loss, ataxia, dystonia, dementia, and hydrocephalus. intraventricular (5%10%) and meningeal cysticerosis are associated with hydrocephalus, signs of meningeal irritation, and raised intracranial pressure. we here report a case of neurocysticercosis presenting as left vi and vii cranial nerve palsy of lower motor neuron type and contralateral hemiplegia, i.e., millard gubler syndrome, which is unreported in literature. ct scan and mri are useful in anatomical localization of cysts, but mri cranium is more sensitive in picking up active lesions. our case fulfilled the imaging absolute diagnostic criteria (histologic demonstration of the parasite from biopsy of a brain or spinal cord lesion, cystic lesions showing the scolex on ct or mri, and direct visualization of subretinal parasites by funduscopic examination). therefore, all patients with multiple cysts should receive treatment with steroid to reduce intracranial pressure and edema ; thereafter cysticidal drug, i.e., albendazole. our patient improved and became asymptomatic with steroid treatment and albendazole. recognizing this clinical entity would avoid unnecessary antituberculous treatment and surgical intervention. cysticercosis is common in tropical countries including india but neurocysticercosis of brain stem is rare. | neurocysticercosis is a common childhood neurological illness in india. a variety of presentations have been reported in the literature, including weber syndrome. neurocysticercosis, manifesting as millard gubler syndrome, have not been reported in literature. therefore, we report a child presented to us with millard gubler syndrome due to pontomedullary neurocysticercosis and was treated successfully. |
bloodstream recurrent infections have been reported for a great variety of opportunistic bacteria, with coagulase - negative staphylococci being the most common. the majority of the relapsing sepsis infections are either catheter related or are caused by indwelling devices that serve as an excellent solid phase for sticky biofilms, the source for transient bacterial translocation into the vessel lumen. molecular fingerprinting, as well as biochemical analysis and serotyping have shown that the majority of the sepsis episodes might be caused by the same strain even when adequate antimicrobial therapy is provided. taking into account the multistrain and multispecies complexity of biofilm, it remains unclear why sepsis episodes are often caused by exactly the same strain and what kind of leads this strain may have compared to other altogether, the patient experienced seven episodes of e. coli bacteraemia in just over a year. all the episodes can be considered as relapses caused by the extended - spectrum -lactamase (esbl) strain or by the ampc hyperproducer. finally, the focus of these bloodborne infections was found in his spleen and pancreas cauda, which was resected. of interest, the underlying disease, autoimmune pancreatitis of type 1 with igg4 hyperglobulinaemia, had probably contributed to the formation of the cyst that harboured infection. a 47-year - old man came to finland as a refugee from southeast asia. in the immigration health inspection, he was diagnosed with a toxocara canis infection and treated with mebendazole. soon after arrival, he complained of epigastric pain and fever ; he was diagnosed with acute pancreatitis, which was treated conservatively. pancreatitis episodes, often complicated with septicaemias, recurred several times during the following years. three years after the first pancreatic episode, the patient was hospitalized again for fever and multiple liver abscesses. drainage of abscesses was not feasible because of the small size of the multiple abscesses and the challenging anatomic site. an infection of the pancreatic duct stent was suspected, and the stent was removed. instead, toxocara canis serology remained positive, compatible with his early infection. during the following months, the patient was hospitalized several times for recurrent pancreatitis and relapsing episodes of sepsis. abscess formation continued even though the patient received various antimicrobials (table 2, table 3). in addition to resection of the pancreatic cauda, the enlarged spleen was removed, and cholecystectomy was performed. histopathologic examination of the pancreas revealed extensive fibrosis, multiple cysts (12 cm in diameter) and inflammatory cells such as neutrophils, macrophages, lymphocytes and plasma cells that were partly immunopositive for igg4. the hilum of the spleen revealed purulent inflammation, but the splenic parenchyma and gallbladder were structurally normal. computed tomographic scan revealed numerous degenerative cystic lesions in the spleen that most probably had formed as a result of prolonged bacterial infection (fig. 1). a pseudocyst filled with a grey fluid was found between the pancreatic cauda and the spleen. all other tissue cultures were negative in bacteriologic, mycobacteriologic, virologic and parasitologic examinations. these findings finally confirmed suspected end - stage chronic autoimmune pancreatitis associated with the igg4 syndrome. during convalescence, the patient had a relapse of esbl e. coli sepsis but recovered completely. three other e. coli isolates (table 1) had an ampc phenotype and were negative for ampc genes (cit, dha, mox, fox, ebc, acc), thus apparently overproducing the chromosomal ampc. in order to investigate the clonality of the strains the culture isolates were analysed with pulsed - field gel electrophoresis using a protocol based on pulsenet for e. coli (http://www.cdc.gov/pulsenet/pathogens/ecoli.html). briefly, genomic dna was digested with xbai (new england biolabs), and separated in a chef dr iii (bio - rad). the esbl isolates were found to be the same strain (one- to two - band differences in the banding patterns). the ampc overproducers were also similar (a one - band difference in one isolate) ; however, they were different from the esbl clone (fig. 2). soon after the surgical intervention, our patient experienced a new episode of esbl sepsis a complication that might have been avoided if previous bacteriologic data had been taken into account so perioperative carbapenem prophylaxis could have been implemented. the reported case emphasizes that the primary prevention of bloodstream relapses caused by gram - negative bacteria is the surgical elimination of the focus of infection. being encapsulated in a cyst, the bacteria are well protected against the bactericidal action of antimicrobials even in organs with good vascularization such as the pancreas. relapses of bacteraemia with enterobacteria can occur in patients with underlying gastrointestinal or autoimmune disorders, as in our case. these episodes can be caused by resistant bacteria, which should be considered during perioperative protection after the first sepsis episode. | bloodstream recurrent infections have been reported for a variety of opportunistic bacteria. these are often either catheter related or are caused by indwelling devices. a case of relapsing sepsis with two escherichia coli strains carrying extended - spectrum -lactamase and derepressed ampc genes is reported. the patient had seven episodes of bloodstream infections within 1 year and was diagnosed with chronic autoimmune pancreatitis and igg4 hypergammaglobulinaemia. abscesses were found in his spleen and pancreas cauda, which was finally resected. relapses of bacteraemia with resistant enterobacteria should be considered during perioperative protection. surgical removal of the infective focus could be curative. |
our study was performed on the basis of the guidelines set both by the u.s. national institute of health and the recommendations of the committee on animal research at our institution. ten pigs (n = 10, 30 - 35 kg) were subjected to 90 minutes of occlusion of the left anterior descending coronary artery, and this was followed by 90 minutes of reperfusion. after performing a left lateral thoracotomy along the fifth intercostal space, the left anterior descending coronary artery was isolated distal to the first diagonal branch, and a snare loop made with 4 - 0 silk was placed in a slender plastic tube. occlusion or reperfusion of the left anterior descending coronary artery was produced by simply fastening or releasing the snare loop. the hearts were rapidly excised and placed in a bath of cold (4) cardioplegic solution. the hearts were stored in formalin solution for at least 18 hour to let the possible early ventricular geometry changes occur before mr imaging, as was previously suggested (9, 14). the dt - mri were acquired from the ten excised hearts by using a philips 3 t achieva scanner (philips medical system, best, netherlands) and an eight - channel head coil. the formalin - fixed porcine heart specimens were suspended in a cylinder filled with formalin to avoid tissue - air susceptibility artifacts. after the scout images were acquired on the four - chamber and two - chamber views, the short axis images of the left ventricle were obtained at the midventricular level for dt - mri. the sensitivity encoding (sense)-based echo - planar imaging technique was applied to shorten the image acquisition time and to alleviate any image distortion caused by susceptibility artifacts from the single - shot echoplanar imaging sequences. the sense factor was chosen to be 2.4 in this study as a trade off between image distortion due to the high echo - planar imaging factor and the sense artifacts caused by a high sense factor, as was previously suggested (14). with a b - value of 800 s / mm, the diffusion tensor images were obtained, respectively, for 6, 15 and 32 diffusion gradient directions at the same midventricular level of each specimen. the imaging parameters were as follows : te = 55 ms, tr = 5000 ms, number of slices = 5, slice thickness = 1.13 mm, slice gap = 0 mm and the number of excitations = 1. three - dimensional reconstruction of the myocardial fibers was done by using the commercially available pride software package (philips medical systems, best, netherlands). for each of the short - axis slices, the numbers of tracked fibers, the fractional anisotropy (fa) and the length of the tracked fibers in the left ventricle were measured using the pride software by an experienced cardiac radiologist who has 12 years of experience with quantitative analysis. the fiber distribution was investigated with the fa magnitude and directional thresholds set at 0.15 and 40, respectively (14). the image quality of the fiber tractography was assessed by two radiologists for qualitative analysis. they scored each image on a 3-point scale : 1 (poor), 2 (moderate) and 3 (good). the definition of each rating score is as follows : ' poor ' was visualization of the myocardium with frequent discontinuity except for in the infarcted myocardium ; ' moderate ' was visualization of the myocardium with partial discontinuity except for in the infarcted myocardium ; ' excellent ' was visualization of myocardial fibers without or with minimal discontinuity except in the infarcted myocardium. the average of the scores for each image was used for qualitative analysis. for statistical analysis among the three paired data sets, all quantitative and qualitative comparisons were analyzed by using friedmann 's test and the wilcoxon - signed rank test for post - hoc consideration. spss version 12.0 (spss, chicago, il) was used for the statistical calculation. our study was performed on the basis of the guidelines set both by the u.s. national institute of health and the recommendations of the committee on animal research at our institution. ten pigs (n = 10, 30 - 35 kg) were subjected to 90 minutes of occlusion of the left anterior descending coronary artery, and this was followed by 90 minutes of reperfusion. after performing a left lateral thoracotomy along the fifth intercostal space, the left anterior descending coronary artery was isolated distal to the first diagonal branch, and a snare loop made with 4 - 0 silk was placed in a slender plastic tube. occlusion or reperfusion of the left anterior descending coronary artery was produced by simply fastening or releasing the snare loop. the hearts were rapidly excised and placed in a bath of cold (4) cardioplegic solution. the hearts were stored in formalin solution for at least 18 hour to let the possible early ventricular geometry changes occur before mr imaging, as was previously suggested (9, 14). the dt - mri were acquired from the ten excised hearts by using a philips 3 t achieva scanner (philips medical system, best, netherlands) and an eight - channel head coil. the formalin - fixed porcine heart specimens were suspended in a cylinder filled with formalin to avoid tissue - air susceptibility artifacts. after the scout images were acquired on the four - chamber and two - chamber views, the short axis images of the left ventricle were obtained at the midventricular level for dt - mri. the sensitivity encoding (sense)-based echo - planar imaging technique was applied to shorten the image acquisition time and to alleviate any image distortion caused by susceptibility artifacts from the single - shot echoplanar imaging sequences. the sense factor was chosen to be 2.4 in this study as a trade off between image distortion due to the high echo - planar imaging factor and the sense artifacts caused by a high sense factor, as was previously suggested (14). with a b - value of 800 s / mm, the diffusion tensor images were obtained, respectively, for 6, 15 and 32 diffusion gradient directions at the same midventricular level of each specimen. the imaging parameters were as follows : te = 55 ms, tr = 5000 ms, number of slices = 5, slice thickness = 1.13 mm, slice gap = 0 mm and the number of excitations = 1. three - dimensional reconstruction of the myocardial fibers was done by using the commercially available pride software package (philips medical systems, best, netherlands). for each of the short - axis slices, the numbers of tracked fibers, the fractional anisotropy (fa) and the length of the tracked fibers in the left ventricle were measured using the pride software by an experienced cardiac radiologist who has 12 years of experience with quantitative analysis. the fiber distribution was investigated with the fa magnitude and directional thresholds set at 0.15 and 40, respectively (14). the image quality of the fiber tractography was assessed by two radiologists for qualitative analysis. they scored each image on a 3-point scale : 1 (poor), 2 (moderate) and 3 (good). the definition of each rating score is as follows : ' poor ' was visualization of the myocardium with frequent discontinuity except for in the infarcted myocardium ; ' moderate ' was visualization of the myocardium with partial discontinuity except for in the infarcted myocardium ; ' excellent ' was visualization of myocardial fibers without or with minimal discontinuity except in the infarcted myocardium. the average of the scores for each image was used for qualitative analysis. for statistical analysis among the three paired data sets, all quantitative and qualitative comparisons were analyzed by using friedmann 's test and the wilcoxon - signed rank test for post - hoc consideration. spss version 12.0 (spss, chicago, il) was used for the statistical calculation. the results of the quantitative analysis are summarized in table 1. by increasing the numbers of diffusion - sensitizing gradient directions from 6 to 15 and to 32 on dt - mri, the mean fa and standard deviation were significantly reduced (p < 0.01). for the evaluation of fiber tracking, the number of tracked fibers was significantly increased (p < 0.01) and the length of the tracked fibers was also increased (p < 0.01) with the increased number of the diffusion - sensitizing gradient directions (figs. 1 - 3). in the qualitative analysis, the image quality of the fiber tractography was significantly increased according to the increased number of the diffusion - sensitizing gradient directions (p < 0.01) (table 2). our result showed that higher numbers of diffusion - sensitizing gradient directions can provide more detailed information about the myocardial fiber structure on in vitro dt - mri at 3 t. to the best our current knowledge, no consensus exists for the optimal number of diffusion - sensitizing gradient directions for dt - mri. jones (19) insisted that the mean diffusivity, fa and tensor orientation requires a dt - mri sampling scheme in which there are at least 30 diffusion - sensitizing gradient directions by using monte carlo simulations. our study also suggested that one should use as many unique sampling orientations as time will allow for applications such as fiber tractography. (13) demonstrated that dt - mri in the cervical spinal cord with using 15 diffusion - sensitizing gradient directions showed significantly better quality than that using 6 directions. yet dti using 32 directions did not show a significant improvement of quality over that using 15 directions. they suggested that the increased numbers of diffusion - sensitizing gradient directions in vivo dt - mri require longer scan times and there may be a greater risk for motion artifacts. this is a first report on using a higher number of diffusion - sensitizing gradient directions for cardiac dt - mri. most studies have used low diffusion resolution with employing 6 diffusion - sensitizing gradient directions for cardiac dt - mri. wu. (14) recently used dt - mri with medium diffusion resolution with employing 15 diffusion - sensitizing gradient directions for the evaluation of myocardial fiber pathways in canine heart samples fixed in formalin. they revealed that the long fiber pathways were found to predominantly run circumferentially with small helix angles, and this dominated the fiber architecture in the myocardium. however, they only investigated the myocardial fiber pathway distribution and they did not analyze the effect of medium diffusion resolution for the dt - mri quality for the evaluation of myocardial anisotropy and fiber tracking. in this study, we demonstrated the effect of high diffusion resolution by using 32 diffusion - sensitizing gradient directions for in vitro dt - mri. we suggest that high resolution dt - mri may be used as a tool for delineating and monitoring the detailed myocardial fiber structure after a myocardial infarction has responded to various treatments such as stem cell therapy. although the advantages of mri at 3 t over the 1.5 t systems have not been established for the use of cardiac dt - mri, a better depiction of fiber tracts at 3 t compared with 1.5 t has been demonstrated with performing high spatial resolution dt - mri in brain (20 - 22). the sensitivity - encoded (sense) mr technique, as a parallel imaging method, is now feasible with using a commercial machine and with no dedicated hardware. using the sense technique, the duration of the echo train is reduced by faster filling of the k - space and so this results in a wider bandwidth in the phase - encoded direction ; therefore, the susceptibility artifacts are reduced (23, 24). to the best of our knowledge, there is no consensus concerning the optimum method for evaluating the quality of dt - mri. (25) investigated the optimum imaging parameters of dt - mri in brain, and they quantitatively evaluated the numbers of reconstructed fibers and the visual image quality. in a similar fashion, we evaluated the qualities of the dt - mri with using different numbers of diffusion - sensitizing gradient directions by quantifying the numbers and length of the tracked fibers, as well as by visually comparing the image quality of the fiber tractography. our results demonstrated that the numbers and length of the tracked fibers were significantly increased with the increased number of diffusion - sensitizing gradient directions, which suggests that the image quality of the fiber tractography was significantly improved. however, the validity of using fiber tracking as a means to determine the accuracy of dt - mri measurements was not proven experimentally. without loss of generality, the dti - derived fa index (26), which is a commonly used rotationally invariant index of diffusion anisotropy, is examined as a function of the left ventricular wall depth and the circumferential and longitudinal locations. jiang. (27) reported that the diffusion anisotropy measured by dt - mri in the fixed sheep myocardium remained relatively constant from the epicardium to the midwall and then it decreased steadily toward the endocardium. (12) also demonstrated that in vivo dt - mri of the postinfarct myocardium in patients with myocardial infarction revealed a significant decrease in fa, indicating there was altered tissue integrity. furthermore, the redistribution of the fiber architecture correlated with the infarct size and the left ventricular function. therefore, exactly measuring the fa is important for characterizing such tissue parameters as the extracellular volume fraction as well as the fiber orientation on cardiac dt - mri. chang. (28) revealed, by using the histogram method, that the image quality of the fa map of a brain was significantly improved as the number of diffusion - sensitizing gradient directions increased. they also reported that that the histogram showed that the fa values and their variance were increased as the number of diffusion gradient directions decreased, and this was probably due to the overestimation of the fa values. it was also shown in our study that the mean fa and its standard deviation were significantly reduced with the increased number of diffusion - sensitizing gradient directions. first, we have no direct histopathological correlations to validate our findings. however, it is difficult to precisely quantify the numbers and the length of myocardial fibers on histopathological exam. the increased scan time may have produced more motion - related artifacts on the in vivo dt - mri. optimizing the number of diffusion - sensitizing gradient directions should be considered as a tradeoff between increasing the image quality and minimizing the scan time for in vivo dt - mri. advanced mr technology will be required to overcome such problems as the low signal - to - noise ratio and the long scan time for in vivo dt - mri. third, we did not consider several important parameters such as the b - value, the numbers of excitations and the slice thickness for cardiac dt - mri. further study will be needed to combine several individual parameters for optimizing the acquisition of in vivo dt - mri before it can be applied to clinical settings. finally, we did not separately evaluate the alterations in tissue integrity, such as the fa of both the infarcted and normal myocardium, because the purpose of our study was only to evaluate the effect of the number of diffusion - sensitizing gradient directions on the image quality of dt - mri. from this study, we conclude that the image quality of in vitro dt - mri is significantly improved as the number of diffusion - sensitizing gradient directions is increased. | objectivewe wanted to evaluate the effect of the number of diffusion - sensitizing gradient directions on the image quality for evaluating myocardial anisotropy and fiber tracking by using in vitro diffusion tensor mr imaging (dt - mri).materials and methodsthe dt - mr images, using a sense - based echoplanar imaging technique, were acquired from ten excised porcine hearts by using a 3 t mr scanner. with a b - value of 800 s / mm2, the diffusion tensor images were obtained for 6, 15 and 32 diffusion - sensitizing gradient directions at the midventricular level. the number of tracked fibers, the fractional anisotropy (fa), and the length of the tracked fibers were measured for the quantitative analysis. two radiologists assessed the image quality of the fiber tractography for the qualitative analysis.resultsby increasing the number of diffusion - sensitizing gradient directions from 6 to 15, and then to 32, the fa and standard deviation were significantly reduced (p < 0.01), and the number of tracked fibers and the length of the tracked fibers were significantly increased (p < 0.01). the image quality of the fiber tractography was significantly increased with the increased number of diffusion - sensitizing gradient directions (p < 0.01).conclusionthe image quality of in vitro dt - mri is significantly improved as the number of diffusion - sensitizing gradient directions is increased. |
elucidation of protein protein interactions contributes to the characterization of the function of novel proteins and hence the genes that encode them. methods for investigation of protein protein interactions include biophysical, computational, biochemical and genetic approaches. for the identification of multi - protein complexes and to determine their association and dissociation rates together with sites of interactions, methods based on surface plasmon resonance (spr) (5) and different types of mass spectrometry (ms) have been employed (68). these methods are proven to be useful, although purification, sequencing and identification of novel proteins can be limiting especially when present only in small quantities. computational methods based on various principles, including correlated changes of amino acid sequence between interacting protein domains (9), properties related to interface topology, solvent accessible area (asa) (10,11) that estimates sites of interaction and primary structure and associated physiochemical properties (12), each can predict interactions. while these predictive methodologies are informative in so much as they provide an indication of the affinity of a given protein for another protein, they all require further experimental validation this system exploits the fact that transcription factors are comprised of two functional domains, a dna binding domain and a transcription activation domain. the dna binding domain recognizes a specific dna sequence whilst the activation domain facilitates the recruitment of polymerase ii associated transcription complex and initiates transcription of the downstream gene. in this system the protein domains are separated from each other until brought together by two interacting proteins. this system has been widely used to screen pray - expression libraries for proteins that interact with a bait protein. the system is prone to contamination by false positives (interactions that are difficult to validate) and false negatives (interactions that are not detected). bait proteins that alone activate or repress the expression of the reporter gene can also prove problematic. to counter a number of inefficiencies associated with the system proteins of interest are fused to c- and n - terminal domains of ubiquitin. in the event of protein protein interactions an active ubiquitin ubiquitin is recognized by ubiquitin specific protease, which leads to the release of reporter protein. additional technologies, based on the yeast two - hybrid screen are being developed all the time. these include systems based on signalling (15) and dual - bait (16). each of these modifications has a major limitation as many mammalian proteins are exposed to significant post - translational modifications, important in protein protein complex formation and function. in an attempt to overcome these difficulties mammalian versions of the two - hybrid screen have been developed (1720) but these have been single reporter functions and are hence susceptible to variation between samples of levels of transfection and transcription. we have developed a dual reporter assay system based on yeast two - hybrid screen, which comprised two autonomous units of gene expression. the upstream unit is expressed regardless of protein protein interactions. in the absence of interacting proteins the downstream unit is switched off. in the event of an interaction, the downstream expression unit thus, the ratio of the two reporter activities can be used as a reference value to detect mutations or trans - acting factors that might affect protein protein interactions. construction of the reporter plasmids (ptn114 and ptn110) was based on pbluga (21), which contains reading frames for the -galactosidase and luciferase genes. to construct ptn114, a deactivation activation (d / a) unit was cloned into xhoi / sali and bglii / bamhi sites of pbpluga. the unit comprises a translation termination signal for the upstream reporter followed by a 3 end trimming and polya tail addition site (aataaa), an upstream activation sequence of saccharomyces cerevisiae (22) linked to a tata box sequence from adenovirus e1b minimal promoter (23), six copies of binding sites for gal4 transcription factor (24) and a synthetic mrna splicing signal for effective expression of the downstream reporter, a 5-utr and an in - frame start codon for the downstream reporter. a dna fragment containing the activation sequences, tata box, gal4 binding site and the splicing signal that comprised nucleotides 1535 of pgal / lacz (invitrogen) was amplified using uasf(xho) 5-ccgctcgagggtgaaataaagtcgacccgagctcttacgcggg-3 and uasr(bglii) 5-gaaagatcttgccatgtcttcgatctgcagaattcc-3. for cloning purposes the internal xhoi site was replaced by a bamhi site using site directed mutagenesis (25). the construct ptn111 was made by cloning the gal4 dna binding domain (residues 1147) (26) with a n - terminal t7 epitope tag into aflii and kpni sites of pcdna3.1 (invitrogen). the coding region of limk1 gene (nm_002314) was sub - cloned into kpni and xbai sites of ptn111. construct ptn112 was constructed by cloning the vp16 transcriptional activation domain (27) into xbai and apai sites of pcdna3.1 (invitrogen). the coding sequences of bmpr - ii [nm_001204 and nm_033346 for long (lf) and short forms (sf), respectively ] and d485 g mutant were cloned into bamhi and ecori sites of ptn112. activation unit into xhoi and bamhi sites of pdsred - tn24-gfp (siskoglou and nasim, unpublished data). this vector was similar to ptn24 (28) except that the genes encoding -galactosidase and luciferase were replaced by genes encode for fluorescence proteins and the sv40 promoter was replaced by the cmv promoter. the d / a unit is similar to that used in ptn114 except that an additional sv40 polya site comprising nucleotides 14981648 of the dsred - express - c1 (clonetech) was introduced at the 5 end of the upstream activation sequence. (human embryonic kidney) cells were transiently transfected with relevant plasmids using gene jammer (stratagene). cells were harvested 48 h after transfection and -galactosidase and luciferase activities were measured using dual light system (applied biosystems) as described elsewhere (28,29). for live cell imaging, cells were grown as before and the images were taken under a fluorescence microscope (te 300, nikon). for single - cell expression, cells expressing both green and red fluorescences were selected using a facscan flow cytometer (becton dickinson) and the fluorescence intensities were measured using cellquest software. construction of the reporter plasmids (ptn114 and ptn110) was based on pbluga (21), which contains reading frames for the -galactosidase and luciferase genes. to construct ptn114, a deactivation activation (d / a) unit was cloned into xhoi / sali and bglii / bamhi sites of pbpluga. the unit comprises a translation termination signal for the upstream reporter followed by a 3 end trimming and polya tail addition site (aataaa), an upstream activation sequence of saccharomyces cerevisiae (22) linked to a tata box sequence from adenovirus e1b minimal promoter (23), six copies of binding sites for gal4 transcription factor (24) and a synthetic mrna splicing signal for effective expression of the downstream reporter, a 5-utr and an in - frame start codon for the downstream reporter. a dna fragment containing the activation sequences, tata box, gal4 binding site and the splicing signal that comprised nucleotides 1535 of pgal / lacz (invitrogen) was amplified using uasf(xho) 5-ccgctcgagggtgaaataaagtcgacccgagctcttacgcggg-3 and uasr(bglii) 5-gaaagatcttgccatgtcttcgatctgcagaattcc-3. for cloning purposes the internal xhoi site was replaced by a bamhi site using site directed mutagenesis (25). the construct ptn111 was made by cloning the gal4 dna binding domain (residues 1147) (26) with a n - terminal t7 epitope tag into aflii and kpni sites of pcdna3.1 (invitrogen). the coding region of limk1 gene (nm_002314) was sub - cloned into kpni and xbai sites of ptn111. construct ptn112 was constructed by cloning the vp16 transcriptional activation domain (27) into xbai and apai sites of pcdna3.1 (invitrogen). the coding sequences of bmpr - ii [nm_001204 and nm_033346 for long (lf) and short forms (sf), respectively ] and d485 g mutant were cloned into bamhi and ecori sites of ptn112. activation unit into xhoi and bamhi sites of pdsred - tn24-gfp (siskoglou and nasim, unpublished data). this vector was similar to ptn24 (28) except that the genes encoding -galactosidase and luciferase were replaced by genes encode for fluorescence proteins and the sv40 promoter was replaced by the cmv promoter. the d / a unit is similar to that used in ptn114 except that an additional sv40 polya site comprising nucleotides 14981648 of the dsred - express - c1 (clonetech) was introduced at the 5 end of the upstream activation sequence. hek-293 (human embryonic kidney) cells were transiently transfected with relevant plasmids using gene jammer (stratagene). cells were harvested 48 h after transfection and -galactosidase and luciferase activities were measured using dual light system (applied biosystems) as described elsewhere (28,29). for live cell imaging, cells were grown as before and the images were taken under a fluorescence microscope (te 300, nikon). the images were analysed using openlab software (improvision). for single - cell expression, cells expressing both green and red fluorescences were selected using a facscan flow cytometer (becton dickinson) and the fluorescence intensities were measured using cellquest software. we have constructed a set of reporter constructs in which genes encoding -galactosidase and luciferase are fused with a recombinant fragment containing the translation termination signal for the upstream reporter, a polyadenylation site, an upstream activation sequence linked to a tata box sequence from adenovirus e1b minimal promoter, six copies of binding sites for gal4 transcription factor, a pre - mrna splicing signal, a 5-untranslated region (5-utr) and in - frame start codon for the downstream reporter. upon transfection into the mammalian cells, transcription from the sv40 promoter leads to the production of a pre - mrna which contains a translation termination signal and a polyadenylation signal. efficient processing and termination (transcription and translation) would result in the production of -galactosidase protein. in the event of protein protein interactions, transcription of the luciferase gene would be activated, the mrna would be exported to the cytoplasm resulting in the production of luciferase protein. since luciferase activity would be produced only after protein protein interactions, whereas -galactosidase is expressed constantly, the ratio of luciferase and -galactosidase activities would indicate the proportion of cytoplasmic rna derived from protein protein interactions. to test the ability of the assay system, hek 293 cells were transfected with a reporter plasmid ptn114. a second plasmid (ptn110) was generated as identical to ptn114 except for the absence of signals for polyadenylation and splicing. cells transfected with either plasmid produced only -galactosidase activity (figure 2b). upon co - transfection with plasmids encode for the murine p53 (pcr2.1/p53 produced a gal4-p53 fusion protein) and sv 40 large t antigen (pcr2.1/lgt produced a vp16-lgt fusion protein), the plasmid ptn114 was able to produce both -galactosidase and luciferase activities, whereas ptn110 failed to show any luciferase activity (figure 2a and b). co - transfection with plasmids containing the p53 and the polyoma viral coat protein (cp) (pcr2.1/vp16-cp generated by invitrogen produced a vp16-cp fusion protein), failed to produce a significant amount of luciferase activity (figure 2a). the luciferase and -galactosidase activities were highly variable between experiments whereas when the data was expressed as the ratio of luc gal activities, the variability was significantly reduced (figure 2c). the presence of luciferase protein was confirmed by western blotting using luciferase hrp conjugate antibody (ab cam, uk) (data not shown). mutations in the type ii receptors for bone morphogenetic protein (bmpr - ii), a member of the tgf- receptor family underlie the majority of inherited forms of primary pulmonary hypertension (pph) (30) and familial pulmonary arterial hypertension (31). bmpr - ii is a multi - domain protein containing extracellular, transmembrane, kinase and cytoplasmic tail domains. the mutations are dispersed all over the functional domains and are likely to impinge upon receptor mediated function. a number of proteins have been shown to interact with bmpr - ii (8). the interactions between lim kinase 1 (limk1) and bmpr - ii were initially discovered by yeast two - hybrid screening and confirmed by immunoprecipitation in mammalian cells (32). we wished to examine whether we could employ the reporter system to examine the interactions of limk1 and bmpr - ii. this would provide the basis of a rapid assay system to investigate the effects of mutations in the bmpr2 on receptor - mediated function. we employed the system to screen mutants of bmpr - ii where protein protein interactions might be abrogated. we constructed a series of constructs where the coding sequence of lim kinase 1 gene was fused with a n - terminal dbd and the bmpr2 gene was fused with an activation domain at the c - terminal end. both proteins were either t7 or myc tagged so that they could be easily purified. co - transfection of plasmids encoding both proteins along with the reporter plasmid (ptn114) into the mammalian cells enabled both proteins to interact with each other giving rise to a luc gal ratio (figure 2d). co - transfection of plasmids encode for the limk1 and sf of the bmpr2 or missense mutation produced significantly reduced luc gal ratio (figure 2d). the expression of limk1 and bmpr - ii proteins was confirmed using antit7 and anti - myc antibodies, respectively, by means of western blotting (data not shown). in order to establish a system for high - throughput screening of libraries (chemical, peptide, cdna, etc.) a dual fluorescence reporter was constructed using genes encoding red (dsred express) and green fluorescence proteins (gfp). dsred express and gfp were chosen as their excitation peaks are 488 and 557 nm, respectively and therefore they can be easily distinguished under a fluorescence microscope. in addition, each is a highly stable protein, exhibiting no detectable photoinstability with high signal - to - noise ratio (33) and a shorter maturation time than that of other fluorescence proteins including dsred. upon transfection into the mammalian cells the reporter plasmid produced only the upstream reporter (dsred express) (figure 3a). co - transfections of plamids containing either p53 or lgt gene were unable to initiate transcription of the green fluorescence protein. overexpression of both genes (in this case p53 and lgt) initiated transcription of the gfp. quantification of the fluorescence ratio indicated that the extent of interactions was uniform in a population of cells (figure 3b). significant amount of green fluorescence intensity was observed when cells were transfected with both p53 and lgt genes but not with the lgt alone (figure 4a and b), a finding consistent with the data obtained from fluorescence microscopy. since the conventional methods based on the yeast two - hybrid system for assaying protein protein interactions in mammalian cells are susceptible to numerous variables, we developed a system that bypassed the variables that affect single reporter assays. one of the important features of this system is that a synthetic deactivation activation unit is created which comprises signals for transcription, polyadenylation, processing and translation. signals for polyadenylation, transcription and translation termination are introduced such that the upstream reporter is expressed. upon protein protein interactions the pol ii transcription machinery is recruited upstream of the second reporter, hence enabling the production of the second reporter. we have previously showed that there is a good correlation between the level of transcript and luciferase galactosidase ratio (28). we have analysed the inter - experiment variability for expression of the reporter proteins and found substantial scatter of the data points. however, by taking advantage of constitutive expression of reporter a, we can correct the expression of reporter b to this reference standard. hence, the ratio of two reporter activities show marked reduction in the signal variability (figure 2c). we have tested the dual - light reporter in several ways to show that it reflects the efficiency of protein protein interactions. the highest ratio of luciferase and galatosidase activities was observed when co - transfection was carried out using plasmids encoding the p53 with lgt and the bmpr2 with limk1 (figure 2c and 2d), as these proteins interact with each other (32,34,35). the lowest ratio was observed when either the reporter (ptn114) was transfected alone or co - transfected with plasmids encoding the p53 with cp and the limk1 with short or mutant form of bmpr2. we observed that the interaction between limk1 and bmpr - ii was weaker than that of lgt and p53, possibly due to mislocalization of bmpr - ii. luciferase activity was completely abolished when the polyadenylation and splicing signals were ablated (ptn110). co - transfections of the p53 and lgt had no discernible effects possibly due to transcriptional interference, a process that is known to affect the expression of downstream gene (36). in this case, upon transcription from the sv40 promoter a gal luc fusion rna was produced. the presence of translation termination signal at the end of -galactosidase gene prompted the production of -galactosidase protein. we would assume that in the case of ptn114, transcription termination occurred at poly a sites located at the end of both reporter genes (37). both rnas were polyadenylated, bound by poly a binding protein (pabp), circularized by interacting with translation initiation complex (38) and thereby acted as autonomous units of expression. a potential complication of the approach was that of transcriptional interference, which might affect recruitment of transcription complex for the second reporter. this could be easily addressed by inserting multiple termination sites (36) at the end of first reporter as transcription termination occurs through a non - processive mechanism (39). we have not determined the level of promoter suppression by which the expression of the downstream reporter could be affected but again this could be tackled by interposing mar / sar elements (40) between the adjacent units. although we have not encountered the problems of autoactivation of library screening (41), it is unlikely to affect the usefulness of the method, as long as a second independent method (i.e. pull - down, immunoprecipitation) is used to validate the most interesting candidates that emerge from a screen. identification of a small molecule that dimerizes two proteins can be used for induction of cell proliferation (42) and apoptosis (43). on the contrary, small molecule antagonists of proteins can be useful in developing antibiotic, antiviral and antiparasitic drugs (44). the advantages of the dual fluorescence reporter are that the assay is measured directly on the intact cell and therefore does not require cell wall disruption or addition of a substrate and the sensitivity of this assay is comparable with that reported for luc gal assay. in addition the dual fluorescence assay could be performed in 96- or 386-well format and would be useful to screen chemical or peptide libraries to identify dimerizers or antagonists that affect specific protein protein interactions. in this case it would be advantageous to set up a stable mammalian cell line by incorporating the fluorescence reporter. the maturation time of the dsred express is shorter than that of its dsred counterpart, which makes the reporter system useful in investigating protein interactions in the early stages of developing embryos. although we have only shown that this system would be useful in screening mutations of bmpr - ii that are involved in limk interactions, there are a number of other human diseases for which it would be beneficial to isolate drugs capable of targeting selected protein protein interactions. this system in its present form would be useful, for example, in identifying proteins that interact with selenoprotein n. mutations in the selenoprotein n gene have been implicated in muscular dystrophy (45). the classical two - hybrid screen to identify the interacting proteins is not suitable because yeast is known to contain no selenocysteine incorporation machinery. other methods, such as pull - down or immunoprecipitation, would be difficult due to the fact that selenocysteine incorporation in mammalian cell line is an inefficient process (46). we earlier developed a dual - reporter based assay system (28) to determine rna processing efficiency in mammalian cells. in this system, two reporter activities fused with splicing signals were linked to single gene expression unit. in the event of splicing, the expression unit produced a fusion protein with two reporter functions whereas inefficient splicing produced a protein with single reporter function. the system presented here is based on two autonomous gene expression units where one expression unit is expressed regardless of the expression of the other unit. we have shown here the usefulness of this principle in upgrading a classical two - hybrid screen. we suggest that this principle may be beneficial for other systems where the activities are limited to single reporter functions. the dual - light reporter system for determining protein protein interactions is based on two reporter genes, which are fused via a recombinant fragment containing a synthetic deactivaion / activation (d / a) unit. the unit comprises polyadenylation signal(s), six copies of gal4 dna binding site, a tata box, a synthetic splicing unit. upon transfection into mammalian cells the reporter a is transcribed under the control of sv40 promoter, while transcription of the reporter b is switched off. in the event of protein protein interactions, transcription of the reporter b analyses of the gal luc reporter system to determine protein protein interactions in mammalian cells. the reporter construct along with relevant plasmids was co - transfected into hek 293 cells and activities of luciferase (a) and -galactosidase (b) were measured. the ratio of both reporter activities was normalized to a value of 100 (c) with p53 and lgt and (d) with limk1 and bmpr2. (a) the expression of gfp was suppressed in the absence of interacting proteins as shown, where cells were transfected with (i) reporter alone, or co - transfected with (ii) p53 and (iii) lgt. the expression of the gfp is activated when cells were co - transfected with plasmids encoding the p53 and cp (iv and v). (b) fluorescence intensities of both proteins were measured and normalized as mentioned in figure 2. mean and standard deviations were derived from a pool of 30 to 40 randomly selected cells. cells with both fluorescence activities were selected using a fluorescence activated cell sorter and their intensities were determined (a) and normalized (b). the mean and standard deviations were derived from 300 to 400 cells. | methods for determining protein protein interactions in mammalian cells typically rely on single reporter functions and are susceptible to variations between samples particularly in regard to levels of transcription, processing and translation. a method has been developed for determining protein protein interactions in mammalian cells, which bypasses these variables confounding single reporter assays. the approach utilizes two units of gene expression linked to reporter functions that are interposed by a deactivation activation unit in such a way that the downstream expression unit is switched off. hence upstream expression occurs regardless of protein protein interaction, leading to the production of the upstream reporter. in the event of protein protein interactions, the downstream expression unit is switched on leading to dual reporter read outs. thus, the ratio of the two reporter activities provides a measure to determine the efficiency of protein protein interactions. to access the system we screened a mutant of bmpr2 where the interaction between bmpr - ii and limk is abrogated. bmpr - ii is a type ii receptor of the tgf superfamily and plays a key role in the pathogenesis of familial pulmonary arterial hypertension. this system has potential for high - throughput screening of libraries (peptide, chemical, cdna, etc.) to isolate agents that are capable of interfering with highly selective protein protein interaction. |
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