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mushrooms have been exploited in human diet for centuries because of their specific taste and flavour. nowadays, they attract attention because of their beneficial effects and possible use in the prevention or treatment of diseases. it has been proven that the polysaccharide extract of pleurotus pulmonarius delays the progression of hepatocellular carcinoma ; polysaccharide from pholiota nameko has anti - inflammatory properties in rodents ; agaricus bisporus inhibits prostate tumor growth in mice ; pleurotus eryngii, grifola frondosa, and hypsizygus marmoreus protect apolipoprotein - e deficient mice from development of atherosclerosis. simultaneously, edible mushrooms are regarded as an important dietary supplement for people interested in calorie restriction, because of the low amount of fat, cholesterol, and calories in their bodies and high concentration of fiber [1, 68 ]. the therapeutic action of mushrooms is attributed to the presence of bioactive compounds such as vitamins, polysaccharides, and secondary metabolites in their fruiting bodies. some of them have antioxidant properties which are referred repeatedly to be the key aspect of their observed beneficial effects. polyphenols and carotenoids, abundant in the fruiting bodies of mushrooms, are antioxidants efficient in biological systems. polyphenols have been reported to interfere with the initiation and progression of cancer [10, 11 ], to act as antiageing, anti - inflammatory [13, 14 ], and brain - protective factors and to protect against cardiovascular diseases [16, 17 ]. apart from provitamin a properties, carotenoids are known as singlet oxygen quenchers [18, 19 ] and lipid peroxidation chain breakers. they have been reported to reduce the risk of prostate cancer [21, 22 ], digestive tract cancers [23, 24 ], and chronic diseases [2527 ]. basing on this knowledge, we decided to estimate parameters describing the antioxidant properties of extracts of dried, edible polish mushrooms : total antioxidant capacity with two most commonly used assays (trolox equivalent antioxidant capacity and ferricyanide reducing power) and the content of polyphenols and flavonoids, the main compounds contributing to the antioxidant properties of mushrooms as well as of two carotenoids (-carotene and lycopene), important from the point of view of antioxidant protection of the human organism. for some species, we distinguished subtypes, considering the polish nutritional customs (boletus edulis white - young and yellow - adult, agaricus bisporus with and without peel). sodium hydroxide, hexane, acetone, and the folin - ciolcalteu reagent were purchased from poch (gliwice, poland). the fruiting bodies of polish mushrooms were collected in 2008 in bory tucholskie (a big forest area in the northern part of poland), cleaned and air dried. 1 g of dry caps and stalks was ground into powder, then 15 ml of boiling water or methanol was added, and the material was rubbed for the next 5 min and stirred at room temperature for 15 min. then, the mixture was centrifuged at 3000 g at room temperature for 20 min. the assays used to evaluate antioxidant properties of the mushrooms are typical for such type studies to enable comparisons with literature data. the concentration of total phenolics in aqueous and methanolic extracts was estimated with the folin - ciocalteu reagent, a method most commonly used [31, 32 ]. the calibration curve was prepared with gallic acid (00.75 mg / ml). the measurement of the content of flavonoids was conducted according to the simple and reliable method described by jia.. 50 l of methanolic or aqueous extract was mixed with 700 l of deionized water and 37 l of 5% nano2. after 5-min incubation at room temperature, 75 l of 10% alcl3 was added followed by 250 l of 1 m naoh after the next 6 min. after shaking, the mixture was centrifuged (5000 g, room temperature, 15 min), and the absorbance of the supernatant was read at 515 nm against a blank. quercetin (00.4 mg / ml) was used as a standard. the concentration of -carotene and lycopene in mushroom extracts was estimated spectrophotometrically [34, 35 ]. the content of -carotene and lycopene was calculated from the equations given as follows : (1)lycopene(mg/100 ml) = 0.0458 a663 + 0.372 a5050.0806 a453,-carotene(mg/100 ml) = 0.216 a6630.304 a505 + 0.452a453. teac of mushroom extracts was estimated by a modified abts cation radical decolorization assay. this simple and cheap assay is based on the measurement of the extent of reduction of preformed abts radical cation by antioxidants. the modification used by us consists in measurement of the reduction at the wavelength of 414 nm corresponding to the main absorption peak of abts radical cation to increase the sensitivity of the assay. abts radical cation was prepared according to re. by reaction of abts with potassium persulfate and stored at 20c until use. the standard curve was prepared using trolox as a standard (020 m). the ability of the extracts to reduce potassium ferricyanide was determined according to puttaraju and coauthors. this assay was reported to be more selective for antioxidants than the abts decolorization assay and has been used by previous researchers to study mushrooms. we increased the volumes of reagents to obtain the final volume of the reaction mixture readable in a standard spectrophotometer (about 1 ml). the method was used to enable comparison with results of other studies employing this assay. 25 l of mushroom extracts was mixed with 475 l of sodium phosphate buffer (200 mm, ph 6.6) and 250 l of 1% potassium ferricyanide and incubated at 50c for 20 min. then, 250 l of 10% tca was added ; the mixture was shaken and centrifuged (5000 g, room temperature, 10 min). subsequently, 500 l of the supernatant was mixed with 500 l of deionized water and 100 l of 0.1% ferric chloride. after vigorous shaking and 10 min incubation at room temperature, the absorbance was measured at 700 nm against a blank. for the standard curve, the dependence of teac / reducing power on the concentrations of individual antioxidants analyzed is presented as pearson 's correlation coefficients, and the statistical significance of the correlation coefficients was tested with student 's t - test. the content of total phenolics differed significantly among the species employed for the experiments and ranged from 1.65 0.10 to 13.01 1.48 g / mg of dried mushrooms in the aqueous extracts and from 0.02 0.02 to 4.85 0.30 g / mg in the methanolic extracts of dried mushrooms (table 1). in general, the concentration of total phenolics was higher in aqueous than in methanolic extracts, except for the stalk of suillus bovinus. we found that the content of total phenolics was different in different parts of the fruiting bodies. in eight out of nine pairs of mushroom caps and stalks analyzed, the concentration of water - soluble phenolics was higher in the caps. the content of methanolic - soluble phenolics was higher the in caps in six pairs and in the stalks in three pairs analyzed. the highest concentration of methanolic- and water - soluble phenolics was found in the caps of white boletus edulis and xerocomus subtomentosus and the lowest in tuber mesentericum. the highest total content of flavonoids (in aqueous and methanolic extracts) was found in xerocomus badius and leccinum spp., while the lowest in tuber mesentericum and cantharellus cibarius (table 1). similarly to the experiment with polyphenols, more flavonoids were extracted with water except for a few species, for which the concentration of flavonoids was higher in the methanolic extracts or similar in aqueous and in methanolic extracts. like in the case of total phenolics, we found differences between the content of flavonoids in caps and stalks, and it should be emphasized that, in eight out of nine species, their concentration was higher in the caps. the pearson correlation coefficient between the content of total phenolics and flavonoids indicates a moderate dependence : r = 0.69 0.11 (p.001) and r = 0.59 0.14 (p.01) for aqueous and methanolic extracts, respectively. the content of -carotene differed considerably between the analyzed edible mushroom species, from 0.233 to 15.256 g / g of dried body (table 2). the highest content was found in methanolic extracts of the cap of tricholoma equestre and in three species of suillus. the relatively high content of -carotene was detected in the aqueous extracts of the cap of tricholoma equestre and suillus bovinus, while tuber mesentericum and the stalk of leccinum spp. the content of lycopene was far lower than the concentration of -carotene in the mushrooms studied (table 2). the highest content of lycopene was detected in the methanolic extracts of three species of suillus, s. bovinus being the richest in this compound. a high content of lycopene was found in the aqueous extracts of the cap of suillus bovinus as well. the ability to scavenge the abts cation radical ranged from 3.81 0.17 to 62.30 1.77 and from 0.42 0.48 to 20.54 1.89 trolox equivalents / g dried mushrooms for aqueous and methanolic extracts, resp. in general, we observed higher teac values for aqueous extracts (except for suillus bovinus) and differences between the analyzed parts of the fruiting bodies. our results indicate that, for the majority of species analyzed, the aqueous and methanolic extracts obtained from caps are more potent in the decolorization of abts. values of the pearson correlation coefficient point to the strong dependence between teac and the total concentration of phenolics (0.95 0.02 and 0.98 0.01 for aqueous and methanolic extracts, resp.). a statistically significant correlation was found between the concentration of flavonoids and teac as well as between the concentration of lycopene and teac of methanolic extracts, while -carotene concentration did not correlate with teac significantly. as in previous experiments, our results demonstrate differences in the ability to reduce ferricyanide between the aqueous and methanolic extracts of selected edible mushroom species as well as between the parts of their fruiting bodies (table 3). generally, the aqueous extracts were more potent in the reduction of ferricyanide (except for the stalk of suillus bovinus). however, the relation between the reducing power of the caps and the stalks was not consistent and depended on the species analyzed. simultaneously, we found a high, statistically significant correlation between the values of the reducing power and the concentration of total phenolics (r = 0.93 0.03 for methanolic and 0.92 0.03 for aqueous extracts) (table 4, figures 1(a) and 1(b)). a moderate correlation was also observed between the reducing power and the concentration of flavonoids (0.57 0.14 for aqueous extracts and 0.45 0.17 for methanolic extracts), while -carotene and lycopene did not affect this parameter significantly. studies of bioactivity of mushroom extracts suggest that the free radical scavenging ability is, at least partially, responsible for their positive action. many papers describe the antioxidative properties and the content of antioxidants in extracts of fresh, dried, and cooked edible medicinal, cultivated, and wild mushroom species [30, 3841 ]. in this study, using two in vitro assays, we evaluated the antioxidative ability of the extracts of 13 dried, edible mushroom species, which are especially popular in the polish diet. the highest abts cation radical and ferrocyanide reduction ability were determined for boletus edulis and xerocomus subtomentosus. many of in vivo studies confirm that diet supplementation with some mushroom species or mushroom extracts protects tissues against oxidative injuries [4245 ]. thus, the antioxidative ability determined in the in vitro experiments may be of relevance in in vivo systems as well. the antioxidative properties of mushroom extracts are dependent on the concentration of various antioxidants with different correlation coefficients. in our study, we found that both the teac values and the extract abilities to reduce ferricyanide showed the strongest correlation with the content of total phenolics in the dried fruiting bodies. similar observations for mushroom species were made by other authors as well [38, 39 ]. moreover, the relation between the antioxidative capacity and the concentration of polyphenols was found also for plant extracts. we found a moderate correlation between the teac / reducing power and the level of mushroom flavonoids. it needs to be emphasized that the dependence between the concentration of total phenolics and flavonoids is similar to that between teac and the flavonoids content (r of 0.69 0.11 versus 0.68 0.11 and 0.59 0.14 versus 0.58 0.14 for aqueous and methanolic extracts, resp.). we did not find any significant correlation between the -carotene content and tac or reducing power. only lycopene affected the antioxidative capacity of methanolic extracts significantly, but the correlation coefficient was relatively low (0.41 0.17). when comparing the methods of antioxidative capacity analysis employed in this study, it needs to be remarked that in the case of polyphenols, flavonoids, and lycopene, higher correlation was found between their concentrations and teac than reducing power values. these results suggest that both aqueous and methanolic mushroom extracts are more potent in the reduction of abts cation radical than in the reduction of ferricyanide. however, it should be considered that the analysis of reducing power is conducted by a more complicated procedure involving 20-min incubation at 50c at ph = 6.6, which may affect the antioxidative capacity of the extracts. nevertheless, the correlation between these two methods of estimation of reducing capacity remains relatively high (r values of 0.92 0.03 and 0.89 0.04 for aqueous and methanolic extracts, resp.). our experiments indicate that edible polish mushroom species differ in the content of the analyzed antioxidants. the differences were found between the parts of their fruiting bodies and between the aqueous and methanolic extracts as well. generally, the concentration of total phenolics was much higher in aqueous than in methanolic extracts. these results are consistent with previous reports. in the prevailing number of mushroom species, the values of total phenolics concentration in aqueous extracts of dried boletus edulis, macrolepiota procera, and cantharellus cibarius obtained in our laboratory are similar to the values reported by puttaraju and co - authors for these species collected in india. for boletus edulis, we found the average concentration of polyphenols to be 9.87 mg / g dried mushroom, while puttaraju. reported a value of 10.2 mg / g ; for macrolepiotea procera, the values were 10.3 and 10.2 mg / g, respectively, and for cantharellus cibarius 2.4 and 2.0 mg / g of dried mushrooms, respectively. however, in the case of the methanolic - soluble polyphenols more significant differences were observed. the data of khknen and co - authors indicate that total concentration of phenolics in vegetables used in their experiments ranges for vegetables from ~0.4 (pea) to ~7.4 g of gallic acid equivalents per mg dried mass (leaves of carrot). for berries and fruits, the values varied from ~11.9 g / mg (apple) to ~50.8 g / mg (crowberry) while for herbs and medicinal plants from 0.2 g / mg (wheat, grain) to ~42.1 g / mg (purple loosestrife). simultaneously, some mushroom species (suillus) were found to be a competitive source of hydrophobic antioxidants as well. the highest concentration of -carotene (~15.26 g / g of dried mushroom body) and lycopene (~15.4 g / g) were determined in the cap of suillus bovinus. for comparison, the content of carotenoids reported by ben - amotz and fishler in vegetables ranges from undetectable levels to ~52.8 g / g (persimmon) and ~532.1 g / g (pitango) in fruits and to ~1030 g / g (carrot) and ~243.1 g / g (tomato) for -carotene and lycopene, respectively. moreover, prior studies showed that phenolics and carotenoids at the concentrations found in fruits and vegetables act as antioxidants in vitro and affect the antioxidant defense in human body as well. supplementation of the daily diet with -carotene (90 mg) increased the antioxidant capacity of plasma in older woman ; enrichment of the daily diet of nonsmoking men with tomato juice (40 mg lycopene), carrot juice (22.3 mg -carotene and 15.7 mg -carotene), and spicyspinach powder (11.3 mg lutein) reduced the level of oxidatively modified dna bases in lymphocytes. 30 min after consumption of 150 ml fruit juices (orange, melon, grape, peach, plum, apple, and kiwi), generation of reactive oxygen species in the plasma of healthy men was suppressed. similarly, the exercise - induced oxidative damage of red blood cells in athletes receiving 150 ml of chokeberry juice daily (34.5 mg anthocyanins) was lower when compared to control group (placebo). it has been documented that the chemical composition, antioxidant profile, and the concentrations of antioxidants in mushroom fruiting bodies depend on the maturation process [52, 53 ]. the analysis of the changes of the fundamental mushroom antioxidants in the methanolic extracts of lactarius piperatus in four maturity stages demonstrated that their level increases up to the second stage of maturation and then drops down dramatically. in our study, the investigation of the antioxidative capacity of immature and mature forms of boletus edulis suggests that the level of water- and methanol - soluble antioxidants may be different for various parts of the fruiting body during maturation as well. the higher concentration of water - soluble polyphenols was found in the immature form, while the stalks of mature stage of this mushroom were richer in methanol - soluble polyphenols in comparison to the immature form. thus, the aqueous extracts obtained from immature boletus edulis revealed a higher ability to reduce abts cation radical and ferricyanide, while in methanolic extracts a higher antioxidative potential was found for the stalk. simultaneously, a higher concentration of -carotene was determined in the methanolic extract of both the caps and the stalks of the mature form. the level of lycopene was relatively low in all analyzed samples. in order to take into account the polish nutritional practice, we investigated the fruiting bodies of agaricus bisporus with and without the peel. the results obtained for many fruits and vegetables indicate that their peel is characterized by high concentration of antioxidants and displays a protective action against oxidative agents in chemical and biological systems [5557 ]. our experiments proved that extracts of dried agaricus bisporus deprived of peel are slightly less effective in scavenging abts cation radical and reduction of ferricyanide. simultaneously, slightly higher levels of total phenolics and flavonoids were found in the extracts of whole constituents of the fruiting body. it can not be excluded that the higher content of antioxidants and higher antioxidative capacity of the peel of agaricus bisporus may play a physiological and, probably, protective function. certainly, the composition of antioxidants and their role is noteworthy, and further experiments including other mushroom species need to be conducted. it needs to be emphasised that commercial mushroom species employed in this study (agaricus bisporus, pleurotus ostreatus) reveal relatively weak antioxidative capacity and low total content of phenolics. likewise, it was reported previously that wild species are less energetic but possess higher concentration of polyphenols [58, 59 ]. however, in contrast to these optimistic results, it needs to be mentioned that even edible mushroom species have been described to demonstrate toxic effects as well. consumption of tricholoma equestre led to increase of serum creatine kinase, fatigue, and muscle weekness, suggesting the presence of toxin in this mushroom body which may cause rhabdomyolysis. recently, agaricus bisporus has been found to increase plasma bilirubin concentration, lentinus edodes to increase plasma creatine kinase activity, and pleurotus ostreatus to increase water intake and plasma alanine aminotransferase activity in mice. thus, the potential beneficial action of edible mushroom may be complicated by the possible disadvantageous effects. our results demonstrate that differences in the antioxidant profile and antioxidative capacity occur not only between dried mushroom species but also between different parts of the fruiting bodies. moreover, we found that polyphenols rather than other antioxidants analysed affect the antioxidative ability of dried mushroom extracts. | one of the nutritional benefits of mushrooms is the presence of bioactive secondary metabolites which have been reported to exert various beneficial effects in vivo. therefore, we selected thirteen frequently consumed species of polish mushrooms and determined the concentration of polyphenols, flavonoids, -carotene, and lycopene in aqueous and methanolic extracts of dried fruiting bodies as well as their reducing power and ability to scavenge abts cation radical. we found that the concentration of antioxidants is different in different species and in various parts of the fruiting body of mushrooms. we observed a strong correlation (r > 0.9) between the concentration of total phenolics and reducing power / scavenging effects in both aqueous and methanolic extracts, while this correlation was moderate for flavonoids. beta - carotene did not contribute discernibly to the antioxidative properties of the extracts, while lycopene had a significant contribution to the scavenging activity of methanolic mushroom extracts. |
improving informed decision making is essential for supportive end of life care [15 ]. cardiopulmonary resuscitation (cpr) preferences are the most common end of life discussion but occur infrequently and vary in content [1, 2 ]. patients may not have the basic information needed and the timing for the discussion may be inappropriate [68 ]. patient decision support focuses on providing the patient and families with practical information and resources. cpr preferences may also be overlooked or set aside by practitioners because it is a value - sensitive decision or because it is not identified as a high priority discussion [1, 3, 6, 9 ]. value - sensitive decisions would benefit from a patient decision aid, where the patient is recognized as an expert in judging his / her own values [1013 ]. patients who need to address cpr status would benefit from health care professionals comfortable and familiar with providing patient decision support. cpr status is one of the most important health decisions and requires careful consideration of all alternatives and the consequences. for seriously ill patients, cpr preferences are commonly set aside and communication between the patient, family, and health care team is lacking information and followup. this project took place at the ottawa hospital, acute monitor area (ama) unit. this six - bed unit specializes in acute care, managing patients with a variety of complex medical conditions such as chronic obstructive pulmonary disease, congestive heart failure, pneumonia, and multisystem failure disorders. the goal of this project was to implement a publically available patient decision aid for cpr status and to identify any factors which limit or encourage its use. the objectives were to identify nursing perceptions regarding patient decision support before and after using a patient decision aid regarding cpr, clarify any barriers and facilitators influencing the provision of a patient decision aid regarding cpr, and determine whether nursing perceptions of patient decision support can be positively influenced with a brief, theory - based, skill building educational intervention. this pilot project has focused on the efficacy of providing nursing specific patient decision support. a literature review was conducted to identify scholarly english publications pertaining to cardiopulmonary resuscitation preferences, end of life treatment, patient decision support, decision making, and patient decision aids in pubmed, psycinfo, cinahl, proquest nursing and allied health, and the cochrane database of systematic reviews. grey literature searches were also conducted through the registered nurses ' association of ontario (rnao), college of nurses of ontario (cno), and the ottawa hospital research institute (ohri) websites. it has been identified by heyland and colleagues through the canadian health care evaluation project (canhelp) that better planning for end of life care including enhanced relationships with physicians and improving communication and decision making needs to be addressed in many canadian hospitals. reliable information regarding the patient 's condition, understandable explanations for their situation, and addressing patients psychosocial feelings were also rated as high priorities. the cno practice guideline, guiding decisions about end - of - life care indicates that clear communication between the patient, nurse, and the interprofessional team facilitates implementation of patient 's wishes regarding end of life treatment. it is evidenced that a comprehensive and consistent way to address cpr status for seriously ill patients should be established and facilitated in the canadian hospitals. it has been determined that seriously ill patients have poor knowledge about what cpr entails and their role in the decision making process regarding their cpr status [6, 15 ]. participants in any decision may not have all the necessary information to make an informed choice and the timing of the discussion may be inappropriate [6, 8 ]. cpr in particular is a sensitive subject in which all involved in the discussion can appraise differently, even the health care team. the literature on patient decision support has increased a great deal since o'connor and colleagues published their work in 1998. patients want to be educated about their treatments and have an autonomous role in their care [5, 16 ]. there is a stress on the importance of knowing options and being able to provide that information to patients [16, 17 ]. nurses have a unique relationship with patients ; they can provide valuable support when they are faced with difficult decisions. decisional conflict means that there is uncertainty about which course of action to take [10, 18 ]. many issues contribute to decisional conflict and patients are likely to experience uncertainty to some degree when a decision is difficult to weigh. when one needs to consider risk, loss, or a challenge to their personal values conflict can arise. improving informed decision making is essential for supportive end of life care where the patient 's goals of care are clear and communicated, thus reducing uncertainty [1, 35, 20 ]. patient decision aids reduce uncertainty, improve knowledge, generate realistic expectations, and clarify personal values. these are evidence - based tools that can include outcome statistics and patient experiences for a variety of disease specific issues. they help people in making difficult decisions that are consistent with their personal values [12, 22 ]. however, shared decision making has not been embraced by all health professionals and barriers have been identified which limit the use of patient decision aids. an evaluation of nurses ' perceptions with the use of a cpr patient decision aid can clarify issues with its use and can lead to sustainable utilization of the aid [13, 24, 25 ]. the ottawa decision support framework odsf (1998) developed by annette o'connor and this concrete, midrange theory focuses on decisional needs, decision quality, and decision support. it can guide gaining decision support skills through a practical and structured approach [10, 18, 26 ]. it uses a three - step process to assess client and practitioner determinants of decisions to identify decision support needs, provide decision support tailored to client needs, and evaluate the decision making process and outcomes. the theoretical underpinnings of the odsf include decision theories in economics, psychology, social psychology, decisional conflict, and social support [32, 33 ]. the main assumption of the odsf is that patients will likely select the choice that they believe is their best alternative which aligns with their personal values [10, 26 ]. access to all the necessary resources to realize their choice through clear information when the issues are discussed is also a part of the main assumption [10, 26 ]. personal (patient) and practitioner characteristics influence the decision making process as well, but knowledge is a key. providing information for patient options include specific information on benefits and harms, clarification of values associated with each option, ways to manage the views or pressures of others involved in the decision making process, and skills on implementing decisions. decision aids clarify values and address unmet decisional needs or conflict by asking individuals to identify personal importance of issues and evaluate each risk and benefit that influences their decision [18, 34 ]. specifically, patient decision aids need to include the following elements : (1) information tailored to the patients health condition, (2) a value clarification exercise, (3) examples from other patients in similar circumstances, (4) guidance toward shared decision making, and (5) a medium such as a paper tool or interactive computer guide to present the information [12, 22 ]. patient decision aids can guide tailored decision support which focuses on patient 's needs. decision support is provided until decisional conflict is resolved and a quality decision is reached. then aiding implementation and monitoring of the decision occur [4, 10 ]. during the evaluation stage of the odsf, an understanding of the quality of decision making and outcomes informed decisions, ones that are consistent with personal values and that are determined by the patient to be the best alternative, are the result [10, 26 ]. in theory, the patient completing this process as intended will lead to overall satisfaction with their choice, quality of life, and adherence with their decision because it is informed by their values and resources [4, 34 ]. quality decisions are informed by the best available evidence and are based on the values of the patient [22, 35 ]. specifically, the odsf guided this project to address unmet decisional needs or decisional conflict where uncertainty regarding the best choice for cpr status was identified in seriously ill patients. realistic expectations were discussed, evidenced - based information was reviewed, support and resources were appraised, and patient values were considered [5, 10 ]. appraising and articulating a patient 's cpr wishes through clear communication and implementation are congruent with this framework. the criteria involved in the odsf are appropriate to the established project goal and objectives and thus are a straightforward justification for the choice of framework used. through the provision of decision support using a cpr decision aid in combination with decision coaching and counselling, the intervention design involved three steps including (1) conducting a pretest, (2) educating the nurses on patient decision support and the cpr decision aid, and (3) conducting a posttest. it was identified during discussions with nurses who work in the ama and their nurse educator (the project advisor) that frequently a patient 's cpr status is not addressed in a timely manner. it was repeatedly suggested that an improvement needs to be made to address the patient 's information and communication needs regarding cpr status. the discussions lead to an intervention focused on influencing nursing knowledge of patient decision support, uptake of a cpr decision aid, and identifying facilitators and barriers to its use [13, 23, 26, 36, 37 ]. graham and colleagues found that most health care practitioners are willing to use patient decision aids, given that adequate education and support are provided. all registered nurses who work in the ama unit were approached to participate in the education session. due to time constraints and that there were other quality improvement initiatives being implemented concurrently, it was decided that only a brief and basic education session would be offered. the education session consisted of an introduction to why the project was being implemented, what patient decision support is, and an overview of the cpr patient decision aid. taking approximately 510 minutes, nurses were guided individually or in small groups through part one of the ottawa decision support tutorial, odst, and the decision support aid. after having received the education, the nurses were requested to provide patient decision support for cpr preference using the patient decision aid based on clinical opportunities and appropriateness [17, 26, 37 ]. basic knowledge regarding patient decision support is needed to work effectively with patient decision aids [13, 23 ]. with background knowledge of patient decision support, the participants needed to identify using their clinical judgment if a patient would benefit from the cpr decision aid. promotion of the patient decision aid was accomplished by going to the ama unit frequently to check on uptake and being available to answer questions. two posters were also strategically placed on the unit, acting as a reminder. each ottawa hospital form for code status was affixed with the cpr decision aid to prompt each nurse to its use. nurses were advised that if a cpr decision aid was initiated and/or completed they were to write in the interprofessional progress notes in the patient 's medical record of this. it was also asked that this information should be communicated to other team members in the patient 's daily care plan. both qualitative and quantitative measures were used to collect information. before and after intervention questionnaires were the primary means of information collection. consequently, the questionnaires developed were influenced by a study conducted by stacey and colleagues to determine factors influencing decision support by call center nurses. for this project, the number of questions was reduced due to the project scope and objectives and was modified to fit the clinical setting. the questionnaire 's design reflects the current literature on health care professionals ' perceptions of patient decision support and decision aids [12, 23 ]. the questionnaires were designed to be clear and concise using a five - point likert scale to encourage the participation in attaining data. the questionnaires also have a section to generate nurses ' views using an open - ended question format [40, 41 ]. questionnaires were used because of their ability to gather data easily and for their capacity to examine notable differences in responses after the initiated intervention [42, 43 ]. specifically responses were grouped into one of two categories, facilitators or barriers, and were grouped after each batch of questionnaires was received. qualitative reports were used to gather data on the impacts on practice and participant 's views on the project which may not be captured with the questionnaires. open - ended questions were directed to the ama nurses as appropriate, such as what do you think of patient decision support ? and what has been your experience with patient decision support ? questionnaires were given to all ama nurses who signed consent and agreed to participate in the project immediately before each education session. all nurses had an average of five weeks or more to use the patient decision aid. all participants who agreed to complete the post questionnaire were entered into a draw for two gift baskets. descriptive statistic methods were used to analyze questionnaire responses and content analysis was used in reviewing the qualitative reports. there are currently 26 nurses who are trained to work in the ama. of those nurses, 3 were on maternity leave, 2 declined to participate and 21 agreed to take part in the educational session and before intervention questionnaire (n = 21). the age range of participants was 2552 years and the average age of participants was 37.8 years. not all nurses who initially agreed to take part in the project continued their participation. sixteen agreed to participate in the after intervention questionnaire (n = 16/21). age range was 2752 years, average age was 37.8 years, 19% were male, and the average amount of nursing experience was 11.3 years. to participate in the after intervention questionnaire participants were asked to rate how strongly they agree or disagree with certain statements. the results indicate that most respondents agree or strongly agree that using a patient decision aid would be beneficial to the patient and that more education should be directed toward nurses to provide decision support. the results were negatively skewed when asked if they felt confident in providing patient decision support. most nurses responded that they agree or strongly agree that decision aids for cpr was / is useful. the following points were recognized in the questionnaire and field note results for facilitators / benefits to patient decision support and aids : a team understanding of the patient condition and status, better communication, a standardized way to present information and a knowledge tool for nurses, supported by the literature, evidenced - based information, clear understanding of what cpr is and the risks / benefits, support for when patient is not able to make their own decision (family involved). a team understanding of the patient condition and status, better communication, a standardized way to present information and a knowledge tool for nurses, supported by the literature, evidenced - based information, clear understanding of what cpr is and the risks / benefits, support for when patient is not able to make their own decision (family involved). the following points were recognized in the questionnaire and field notes results for barriers / limitations to patient decision support and aids : language barriers, cultural difference, not appropriate for all, family conflict, their lack of understanding or misconceptions, available time to discuss with patient and family, patient not emotionally ready for discussions, patient decision aid was too condition specific ; too rigid, patients / families not accepting nursing support on this (not their role). language barriers, cultural difference, not appropriate for all, family conflict, their lack of understanding or misconceptions, available time to discuss with patient and family, patient not emotionally ready for discussions, patient decision aid was too condition specific ; too rigid, patients / families not accepting nursing support on this (not their role). many nurses commented that they had limited opportunities to use the patient decision aid for cpr, but did identify that they used patient decision support for other issues. a few nurses stated that cpr status should be determined on admission to hospital and be completed routinely for all patients. there were varied views regarding evaluating cpr status on admission versus at a time of health crisis. some said it should be addressed for every patient, despite health status, and some indicated that only when death may be imminent it should be discussed. some nurses stated that they did not see this as a part of their role or something that they wish to partake in. others thought that this was completely within the nursing realm and were eager to support patients with making an informed cpr choice. most nurses agreed with the components of a shared decision making model. after reviewing the data collected it was evident that most nurses were willing to use the patient decision aid because they see it as helping the patient make informed, value - based decisions. when nurses feel they have the knowledge and skill to provide decision support they do so because they believe that they are helping the patient toward a better realization of their condition. the education session and patient decision aid intervention did not seem to significantly influence questionnaire scores, but it was observed with qualitative observations that the nurses felt more knowledgeable and confident with providing patient decision support. specific barriers to providing patient decision support were identified as cultural or language influences, time constraints, rigid application, patient 's emotional adversity, and physician preference for this role. these mirror what has been found in the literature [13, 23, 36 ]. specific facilitators identified included communication enhancement, clear and understandable knowledge base, and the ability to include family in the decision making process. there is a limited amount of literature that describes patient decision support facilitators and even less focus on family involvement. data were collected from self - report and observations, not from a validated tool ; thus obvious sources of bias were present. the information collected was helpful in this specific clinical setting but can not be generalized to others. there were only six weeks where the patient decision aid was implemented and the opportunity for its use did not come readily. most nurses welcomed this intervention but some were obviously stressed at the fact that they were approached to participate as evidenced by their body language and facial expressions. this project may have been better received during a less demanding time for nurse involvement. this project identified that cpr status specifically can be appraised by a nurse to be a difficult topic, too patient specific to use a patient decision aid, or confident that this would be used as a guide to improve patients ' knowledge of options and the provision of support. cpr status is value - sensitive topic, but it is not beyond what normally would be encountered by a practicing nurse. addressing unclear values, information needs, and resources effectively will reduce nursing contributing factors to clouding difficult decisions [5, 11 ]. patient decision aids are designed to be evidenced based and patient focused and are intended to be used as a guide. daily nursing practice should reflect a therapeutic relationship between the patient and nurse toward helping the patient make informed and supported decisions through the provision of patient decision support. based on this quality improvement project, a practice change towards supporting patients to be more educated and involved in their decision making is a priority. since nurses work in close proximity with the patient and their families and spend much time involved in their care, they are the most appropriate professionals to discuss cpr preferences using a shared - decision making model [23, 36, 44 ]. further education on when to implement a patient decision aid for values sensitive topics would be appropriate. strategically educating certain nurses on patient decision support may create an effective role for its implementation. over time challenging traditional health care roles will allow nurses to support the patient effectively through the provision of patient decision support. challenging the barriers to implementing patient decision support and enhancing the facilitating factors will eventually disclose the benefits of its use. this project identified some of those factors within the acute monitor area at the ottawa hospital. dedication and commitment to supporting patient decision support and the cardiopulmonary resuscitation decision aid will help to support patients facing these difficult situations. | the decision whether to receive cardiopulmonary resuscitation (cpr) is a decision in which the personal values of the patient must be considered along with information about the risks and benefits of the treatment. a decision aid can be used to provide patient decision support to a patient who is seriously ill and needs to consider cpr options. the goal of this project was to identify the barriers and facilitators to using a cpr decision aid, through evaluating nursing perceptions on providing patient decision support. using a needs assessment, it was determined that implementing a patient decision aid for cpr status in the acute monitor area (ama) of the ottawa hospital would be an excellent quality improvement project. the nurses who chose to participate were given an education session regarding patient decision support. questionnaires were distributed to evaluate their views of patient decision support and decision aids before and after the education session and implementation of the cpr decision aid. questionnaire results did not indicate a significant change between before or after education session and decision aid implementation. qualitative reports did indicate that nurses generally have positive attitudes toward patient decision support and decision aids. the nurses identified specific barriers and facilitators in their commentaries. this clinically relevant data supports the idea that patient decision support should be integrated into daily nursing practice. |
with the continued success of immunotherapies for various cancers in recent years 1, it is becoming widely accepted that cancer progression is not solely dependent on local tumor characteristics but also on the host inflammatory response 2. indeed, complex interactions between cancer cells and the host inflammatory response have been validated at the molecular and clinical level 3. multiple lines of evidence indicate that the systemic inflammation is linked with disease progression in cancer patients and serves as an independent prognostic indicator 4. moreover, nutritional imbalances resulting from decreased food intake in cancer patients further promote the systemic inflammatory response, thereby further contributing to poor outcomes 5. to improve and simplify the prognostic evaluation of cancer patients, considerable efforts have focused on identifying novel immunological markers 6. elevated systemic c - reactive protein (crp) is commonly used as a sensitive measure of the systemic inflammatory response and a negative prognostic indicator in cancer patients, either independently or in conjunction with other prognostic algorithms 7. furthermore, hypoalbuminemia, an indicator of malnutrition, is also associated with a negative prognosis in some malignancies 8. studies have shown that crp and albumin are independent prognostic factors in cancer patients and hypoalbuminemia and elevated crp are associated with a poor cancer prognosis 9. therefore, the significance of the combination of these parameters, which together represent both the systemic inflammatory response and nutritional imbalance, is becoming increasingly clear 10. the prognostic role of the systemic inflammatory response is well accepted and has been validated with the establishment of an immunization - based prognostic system, referred to as the glasgow prognostic score (gps)11. the gps is assayed by simply examining both serum crp and albumin levels, which measure of the systemic inflammatory response and nutritional imbalance, respectively 12. research efforts aimed at improving the prognostic value of the gps system have led to the development of modified versions of the gps system that use either adjusted cut - off values for both serum crp and albumin levels 13 or that omit hypoalbuminemia as an independent negative prognostic indicator 14. these 2 modified gps (mgps) systems have demonstrated greater prognostic value in predicting the outcome of cancer patients 15. although recent reports have shown that the gps score was an accurate indicator of cancer - specific survival in many cancers of epithelial origin, including colorectal 16, renal 17 and non - small cell lung cancers 18, the prognostic role of gps in patients with soft tissue sarcoma (sts) remains unclear. sts accounts for nearly 1% of newly diagnosed malignancies annually 19, and improving the low survival rate of sts remains an enormous challenge 20. despite advances in chemotherapy, radiation therapy and surgery, considerable efforts aimed at distinguishing which high - risk sts patients are appropriate candidates for more aggressive procedures and determining the appropriate treatment strategy for individual patients is still needed. the purpose of this study was to examine and compare prognostic value of different gps systems in patients with sts and to analyze the predictive value of the gps score in determining which patients with metastatic sts are more likely to benefit from more aggressive therapy. the study protocol was approved by the institutional review boards of sun yat - sen university cancer center. written informed consent was obtained from each patient at the time of sample acquisition and included consent for tissue analysis and for the potential use of information for medical research. the charts of 165 consecutive patients who suffered from sts between july 2007 and july 2014 were reviewed. follow - up data, including morbidity, mortality and survival, were obtained from clinical charts or by contacting the patient on an outpatient basis or by telephone. the other patients with sts were excluded from the study due either to conditions known to evoke a systemic inflammatory response or to incomplete clinical or pathological data, such as a lack of the pathological or laboratory records with crp and albumin levels. conditions known to evoke a systemic inflammatory response were defined as (i) a prior clinical history of other malignancies or neoadjuvant chemotherapy or radiotherapy, (ii) clinical evidence of active pretreatment infection or (iii) chronic active inflammatory diseases such as rheumatoid arthritis. characteristics of the patients and tumors, including age, sex (male vs. female), primary tumor size, and tumor depth (superficial : tumor does not involve the superficial fascia vs. deep), were collected at the first diagnosis of sts and at the onset of metastases. the who classification system was used as the basis for determining the pathological diagnosis and tumor grade. local treatment was defined as 1 or more procedures of surgery, radiotherapy or radiofrequency ablation. the specific treatment regimens varied and included conventional fractionated radiotherapy, stereotactic body radiation therapy (sbrt) with different radiotherapy doses and radiofrequency ablation procedures using various parameters. the response to treatment was classified according to the recist criteria (version 1.1) 22. progression - free survival (pfs) was calculated from the first treatment to the time that disease progression was recorded, and overall survival (os) was calculated from the first diagnosis to the time of death reported or the date of the last follow - up. for the 97 patients with metastases, pfs1 was defined from the date of metastases treatment to the time that disease progression was recorded, and os1 was defined as the time between the date of the metastasis diagnosis and the time of death reported or the date of the last follow - up. to evaluate gps, laboratory measurements that included crp and albumin levels were immediately evaluated from blood samples collected within 24 hours prior to first treatment (all patients) and prior to metastasis treatment (metastatic patients) according to the routine clinical practices of sysucc. serum crp and albumin levels were measured using the hitachi auto analyzer (hitachi 7600, hitachi, tokyo, japan). the inter- and intra - assay variability of crp and albumin concentrations were less than 5%, as established by routine quality control procedures. the relative gps was constructed as previously reported 23. in the traditional gps system (tgps), patients with both an elevated crp level (> 10 mg / l) and hypoalbuminemia (10 mg / l) and hypoalbuminemia (< 35 patients with neither of these abnormalities are classified into tgps0 group, and patients with only 1 of these 2 biochemical abnormalities are classified into the tgps1 group. accordingly, in the modified gps system 1 (m1gps), patients with hypoalbuminemia alone are assigned to the m1gps0 group, while all other aspects of the m1gps score are the same as with the tgps system. in the modified gps system 2 (m2gps), the cut - off values were changed to 5 mg / l for elevated crp level and 38 g / l for hypoalbuminemia. all other aspects of the m2gps system are the same as with the tgps system (table 1). we then evaluated the relationships between each gps system and the clinicopathological characteristics of the patient cohort, including prognosis and treatment procedures for metastases. the data are presented as the number (%) or median (range) unless otherwise stated. the pearson 2 test and fisher 's exact test were used to analyze categorical data, and an independent sample t - test or the mann - whitney u test was used to analyze numerical data. survival curves were calculated using the kaplan - meier method and compared by the log - rank test. data analysis was performed using the spss 18.0 software (pasw statistics 18) for windows (spss inc., a total of 165 sts patients were eligible for analysis. among 165 patients, the mean age was 47.0 years (range : 5 - 80, median 49.0) ; 72 patients were male (43.6%) and 93 were female (56.4%) ; the mean primary tumor size was 6.2 cm (range : 0.3 - 17.4, median 5.5) ; and 36 patients (21.8%) had superficial tumors while 129 patients (78.2%) had deep tumors. the tumor pathological subtypes included fibrohistiocytic tumors in 41 patients (24.8%), undifferentiated sarcomas in 93 (56.4%), smooth muscle tumors in 10 (6.1%), fibroblastic / m yofibroblastic tumors in 1 (0.6%), skeletal muscle tumors in 17 (10.3%) and adipocytic tumors in 3 patients (1.8%). in addition, the mean follow - up periods of march 2015 was 73.7 months (range : 17.2 - 533.8, median 62.4). of the 165 patients, 97 presented with metastases either at the time of the first diagnosis or after the first treatment. clinicopathological correlation of with or without local treatment in patients with metastatic sts is listed in table 2. the median os of the entire cohort was 2,581 days, and the overall 1-, 3- and 5-year os rates were 81.2%, 37.6% and 12.7%, respectively (fig. in addition, the median os1 rate for the 97 patients with metastases was 1,667 days, and the overall 1-, 3- and 5-year os1 rates were 81.4%, 39.2% and 16.5%, respectively (fig. the median pfs of the entire cohort was 662 days, and the overall 1-, 2- and 3-year pfs rates were 63.0%, 34.5% and 18.2%, respectively (fig. in addition, the median pfs1 rate for the 97 patients with metastases was 373 days, and the overall 1-, 2- and 3-year pfs1 rates were 52.6%, 21.6% and 7.2%, respectively (fig. 4a). consistent with previously observed trends, patients in the score 0 group were associated with the most favorable os and pfs rates, while patients in the score 2 group were associated with the poorest rates. 1b and 2b), m1gps (fig. 1c and 2c) and m2gps systems (fig. 1d and 2d) the variables (tgps m1gps and m2gps, age, pathological grade, primary tumor depth) were examined by multivariate analysis. multivariate analysis revealed that the m1gps [hazard ratio (hr) 9.932 ; p=0.024 ] was independently associated with pfs, and the m2gps [hazard ratio (hr) 2.228 ; p=0.044 ] was independently associated with os1 (table 4 and 6). meanwhile, the m1gps system was associated with the greatest accuracy in predicting os and pfs. receiver operating characteristic curves (roc) was performed for the survival status of os and pfs examinations. the discrimination ability of each inflammation - based prognostic score was compared by area under the roc curve (auc)(table 7 ; fig.5a and 5b). the m1gps had higher auc values at pfs (auc=0.779, p<0.001) and os (auc=0.659, p<0.001) compared with the tgps (pfs : auc=0.757, p<0.001,os : auc=0.644, p=0.001), m2gps (pfs : auc=0.717, p<0.001,os : auc=0.645, p < 0.0 01). consistent with these results, the superiority of m1gps scores to predict os and pfs were also observed in a separate analysis restricted to patients suffering from metastasis. the most significant associations observed were between os1, pfs1 and the m1gps system (table 7, fig.5c and 5d). the m1gps had higher auc values at pfs1 (auc=0.737, p<0.001) and os1 (auc=0.669, p=0.004) compared with the tgps (pfs : auc=0.732, p<0.001,os1 : auc=0.663,p=0.006), m2gps (pfs : auc=0.696, p=0.001,os1 : auc= 0.657,p=0.008). 4b - g) benefits only to patients with a score of 0, regardless of the scoring system used. for patients with a score of 2 according to any of the 3 systems, the present retrospective cohort study, which utilized clinical data, evaluated and compared the prognostic capability of 3 gps systems in patients with sts and analyzed the value of these systems in determining the appropriate treatment strategy for patients with metastatic sts. recent breakthroughs in immunotherapy have led to a major focus on exploring the relationship between the immune response and cancer 24. precise and concise markers for assaying the immune response to cancer are proving to be critical factors in cancer research 25. circulating crp levels, mediated by interleukin (il)-6, are produced locally by tumor cells in response to proinflammatory cytokines 26. studies have indisputably reported that elevated crp is a sensitive, specific, and precise prognostic factor associated with poor survival in various types of cancer 27. decreased levels of albumin, a factor associated with the systemic immune response, have also been observed in various cancers and function as a negative prognostic indicator in cancer patients 5. consequently, the gps system was established to evaluate the relationship between elevated crp and decreased albumin. the gps system is recommended in routine clinical as an objective and rational approach based on its simple, well - standardized and widely available assays and due to its well - validated prognostic value in cancer patients 9. current investigations indicate that the gps immunological - based prognostic score should be viewed as a comprehensive measure of cancer - associated systemic immunity and malnutrition 28. in clinical practice, the gps has been used to predict the prognosis of patients with multiple types of neoplasms 29. as gps is a simple, minimally invasively and low cost assay, it has the potential to serve as a routine, cost - effective and easily accessible tool for predicting outcomes in cancer patients 5. although gps has been utilized as a prognostic indicator in patients with various neoplasms, investigators are still paying considerable attention to improving its prognostic value. other studies subsequently indicated that modified cutoff values of 38 g / l for hypoalbuminemia and 10 mg / l for elevated crp provided greater accuracy in predicting prognosis 13. other studies recommended an additional modification of the gps score in which patients with hypoalbuminemia but normal crp are allocated to the gps 0 group 14. in previous report, nakamura et 30 have reported that the high - sensitivity modified glasgow prognostic score (hs - mgps) could predict the disease - specific survival and oncological outcome in adult patients with non - metastatic soft - tissue sarcoma before treatment. patients with a score of 2 before treatment had a poorer disease - specific survival and event - free survival than those with a score of 0. different from our gps systems, they indicated that modified cutoff values of 35 g / l for hypoalbuminemia and 3mg / l for elevated crp in predicting prognosis. to systematically evaluate the prognostic value of these 3 gps systems, we analyzed data from patients with soft tissue sarcoma using all 3 systems. both the kaplan - meier method and cox proportional hazards regression demonstrated that incorporating the modified cut - off values of hypoalbuminemia and elevated crp level provided superior predictive power with respect to os, and that omitting hypoalbuminemia from the risk score provided superior predictive power with respect to pfs. with respect to metastases management, a high gps was significantly associated with aggressive disease and a poor prognosis 27. moreover, the gps reflected the physical status of cancer patients with respect to the systemic immune response and nutritional imbalances. we also found that although patients with metastasis might benefit from local treatment, this does not appear to be the case with elderly patients (unpublished). similar to elderly patients, patients with a severe systemic immune response and malnutrition are less likely to benefit from local treatment 14. after validating the prognostic value of the gps systems in the patient cohort as a whole, we evaluated the strength of the 3 systems in identifying the appropriate treatment strategy for patients with metastasis. this analysis demonstrated that patients with a gps of 2 according to any of the 3 systems were not likely to benefit from local treatment. in addition, the modified gps systems provided greater accuracy in predicting prognosis in the cohort of patients with metastasis. despite the fact that institutes around the world have reported the prognostic value of gps in predicting outcomes in various cancers of epithelial origin, there have been few reports describing the value of gps in sts. as highlighted in this study, the gps might represent a novel and simple biomarker in patients with sts and potentially lead to improved clinical outcomes. for example, patients with a gps of 2 exhibited very poor survival rates and did not benefit from local treatment after the development of metastases. these findings indicate that chemotherapy might provide greater clinical benefits in patients with a gps of 2. in addition, a higher gps indicates a more intense immune response and/or more severe malnutrition. in this context, immunotherapy or nutritional support moreover, the m2gps, which employs the modified cut - off values of enhanced serum crp and hypoalbuminemia, might provide a more accurate assessment of os, whereas the m1gps that omits hypoalbuminemia as an independent prognostic factor might provide a more accurate assessment of pfs. although reports have indicated that other immunological markers, including monocyte counts, the neutrophil to lymphocyte ratio, and levels of il-2, il-4, il-6, il-10, tumor necrosis factor (tnf) and to interferon - gamma are independent prognosticators of cancer patient outcomes, gps has proven to be superior to these markers in a wide variety of cancers 31. one notable strength of this study was that we evaluated the clinical utility of gps in a cohort. thus, we evaluated local treatment as a single entity rather than stratifying patients by individual treatment regimens. we acknowledge that the reliability and consistency of the gps system might be enhanced by evaluating individual treatment regimens separately. in the current study, gps was identified as a prognostic marker and indicator for determining the appropriate treatment strategy in patients with metastatic sts. in addition, we found that the modified gps provided greater prognostic value compared with the traditional gps. our findings indicate that patients with metastatic disease and a gps of 2 are more likely to benefit from systemic treatment compared with local treatment, although dynamic changes in the gps over the course of therapy should be evaluated in future prospective studies to further clarify the clinical significance of the gps system. in addition, studies designed to determine which high - risk patients are appropriate candidates for more aggressive multimodal treatment regimens and more intensive follow - up will provide further insight into the utility of the gps system. | background : the purpose of this study was to examine and compare the prognostic value of different immunization - based scoring systems in patients with soft tissue sarcoma (sts).methods : we conducted a retrospective study evaluating a cohort of 165 patients diagnosed with sts between july 2007 and july 2014. the relative glasgow prognostic score (gps) of these patients was calculated using 3 different systems : the traditional gps system (tgps), the modified gps system 1 (m1gps), and the modified gps system 2 (m2gps). then, we evaluated the relationships between each gps system and clinicopathological characteristics. the mean follow - up for survivors in the cohort was 73.7 months as of march 2015.results : the most favorable overall survival (os) rate was associated with the score 0 groups, and the poorest progression - free survival (pfs) rate was associated with the score 2 groups, regardless of which system was used to calculate the score. specifically, the m1gps provided the greatest accuracy in predicting os and pfs. moreover, the same effect was observed in a separate analysis restricted to patients with metastases. remarkably, in patients with a score of 2 as measured by all 3 systems, local treatment resulted in a poorer prognosis compared to patients with a score of 2 who did not receive local treatment.conclusion : the gps is a valuable prognostic marker and has the capability to predict the appropriate treatment strategy for sts patients with metastases. the modified gps systems demonstrated superior prognostic and predictive value compared with the traditional gps system. |
nasal tip surgery represents a challenge to rhinoplasty and requires the knowledge of essential anatomy and aesthetic points (figure 1). several techniques can be performed to improve nasal tip definition, such as cartilaginous resection, tip grafts, or suture placement. resection techniques have gradually been replaced by maneuvers that are effective in preserving more of the nasal tip support mechanisms1 2 3 4 5 6 7 8 9 10. education on suture techniques on the nasal tip through external access combined with surgeon sensibility has enabled great progress in the applicability of closed - access techniques. the surgical algorithm is fundamental to the comprehension, judgment, and performing of surgical maneuvers, being necessary to surgery systematization whether at the clinic or at educational institutions the objective to classify the approach to nasal tip refinement through septocolumellar and intercartilaginous bilateral incisions based on nasal tip type (classification proposed by the authors). this classification considers the interdomal distance (angle of domal divergence and intercrural distance), domal arch width, alar cartilage consistency, and skin type (figures 2 and 3). to develop a surgical algorithm based on the findings that describes the proposed maneuvers. skin inspection, lateral inferior lower lateral cartilage palpation, and measurement of angles and distances are fundamental to define the nasal tip type of the caucasian nose (table 1). based on the tip type (i, ii, or iii), it is possible to correlate the best surgical maneuver presented by the authors (table 2). lower lateral cartilage intermediate intercrural access to the tip is performed through incisions made according to the reduction rhinoplasty technique for the caucasian nose described by converse - diamond 1 (figures 4 and 5), which means making septocolumellar and intercartilaginous bilateral incisions. in cases that require weakening or cephalic removal of the lower lateral cartilage, the access is performed through an intercartilaginous incision with eversion of the lower lateral cartilage (mcindoe technique) (figure 6). this technique is carried out preferably with local anesthesia (1:100,000 xylocaine and epinephrine) and sedation. at the end of the surgery, a patch is placed over the nose with a mold (aquaplast) and removed after the seventh day. surgical technique (converse - diamond) steps : septocolumellar and intercartilaginous bilateral incisions, displacement of the soft tissues from the osteocartilaginous structure. separation of the upper lateral cartilage from the nasal septum junction, septoplasty with or without turbinate surgery, and reduction of the upper lateral and septal cartilage. nasal tip refinement based on the proposed surgery algorithm. bone hump reduction using cutting - type maury - parkes. osteotomies side by pecking performed with an osteotome converse 3 mm in women and 4 mm in men. displacement of the skin from the nasal tip is carried out (la garde maneuver).. its tightening must be gradual and progressive, permitting adjustments related to the definition of a more suitable tip. it must be carried out after septocolumellar and intercartilaginous suture using monocryl absorbable thread or colorless pds 4.0 thread with a straight, sharp needle. performing the interdomal suture at the end of the surgery prevents excessive manipulation of the nose tip and possible enlargement of the interdomal distance. subsequent to the lower lateral cartilage skin displacement using le garde 's maneuver, introduce the pds 4.0 thread to the inner portion of the dome. the thread must be introduced from right to left at the posterior border, returning from left to right to the anterior border, more cephalic. return the needle to the nostril in which the suture was initiated, tightening the suture gradually. exposure of the intralateral domal area through which the needle and thread will pass. step 3. returning the needle through the more interior portion of the domal area. subsequent to the lower lateral cartilage skin displacement using la garde 's maneuver, introduce the pds 4.0 thread to the inner portion of the dome. the thread must be introduced from right to left at the posterior border, returning from left to right to the anterior border, more cephalic. return the needle to the nostril in which the suture was initiated, tightening the suture gradually.. its tightening must be gradual and progressive, permitting adjustments related to the definition of a more suitable tip. it must be carried out after septocolumellar and intercartilaginous suture using monocryl absorbable thread or colorless pds 4.0 thread with a straight, sharp needle. performing the interdomal suture at the end of the surgery prevents excessive manipulation of the nose tip and possible enlargement of the interdomal distance. subsequent to the lower lateral cartilage skin displacement using le garde 's maneuver, introduce the pds 4.0 thread to the inner portion of the dome. the thread must be introduced from right to left at the posterior border, returning from left to right to the anterior border, more cephalic. return the needle to the nostril in which the suture was initiated, tightening the suture gradually. exposure of the intralateral domal area through which the needle and thread will pass. step 3. returning the needle through the more interior portion of the domal area. subsequent to the lower lateral cartilage skin displacement using la garde 's maneuver, introduce the pds 4.0 thread to the inner portion of the dome. the thread must be introduced from right to left at the posterior border, returning from left to right to the anterior border, more cephalic. return the needle to the nostril in which the suture was initiated, tightening the suture gradually. the substitution of cartilaginous resection techniques by suture techniques to improve nasal tip definition is a global trend5. the final result of the suture is influenced mainly by factors such as the cartilage intrinsic force, degree of suture tightening, and the limitation imposed by soft tissues (ligaments, subcutaneous tissue, and skin).. however, the interdomal suture may be ineffective in patients with thicker skin and excessive subcutaneous tissue, requiring a reduction of the angle of domal divergence and of the lateral intercrural width, domal arch width, and domal definition through other techniques such as transdomal suture (delivery), or alar lateral spanning - type suture, lateral intercrural suture6, as well as the goldman technique. some details concerning the interdomal suture must be observed : slight projection of the supratip region2.. a suture more to the anterior may result in slight pinching of the skin and caudal rotation. discrete pinching of the supra - alar region, especially in patients with very thin skin and weak supra - alar cartilage, when performed at a distance greater than 23 mm from the nasal dome. asymmetry of the tip can be observed if the sutures are not placed at corresponding levels. the suture can be held using long - term absorbable threads or with nonabsorbable threads considering the tensile strength and absorption levels of each material. the interdomal suture is an effective method to improve nasal tip definition on the caucasian nose and can be performed through endonasal rhinoplasty without delivery. such a technique has already been reported in the literature through open and closed access with delivery, but not through closed access with intercartilaginous and septocolumellar incisions for surgery on the caucasian nose. this suture is easy to perform, predictable, controlled, and extremely useful to the surgeon dedicated to nasal tip surgery. the main sutures applied on the nasal tip were reported by guyuron and behmand in 20036. our use of the interdomal suture has produced admirable results for more than 13 years. our records show that only one patient presented complications : recurrent cellulites of the tip, which resolved after suture removal. | summary introduction : refinement of the nose tip can be accomplished by a variety of techniques, but currently, the use of sutures in the nasal tip with conservative resection of the alar cartilage is the most frequently recommended approach. objective : to classify the nasal tip and to demonstrate the interdomal suture applied to nasal tip refinement in the caucasian nose, as well as to provide a simple and practical presentation of the surgical steps. method : development of surgical algorithm for nasal tip surgery : 1. interdomal suture (double binding suture), 2. interdomal suture with alar cartilage weakening (cross - hatching), 3. interdomal suture with cephalic removal of the alar cartilage (mcindoe technique) based on the nasal tip type classification. this classification assesses the interdomal distance (angle of domal divergence and intercrural distance), domal arch width, cartilage consistency, and skin type. interdomal suture is performed through endonasal rhinoplasty by basic technique without delivery (converse - diamond technique) under local anesthesia. conclusion : this classification is simple and facilitates the approach of surgical treatment of the nasal tip through interdomal suture, systematizing and standardizing surgical maneuvers for better refinement of the caucasian nose. |
the p21-activated kinases (paks) are downstream effectors of the ras - related rho - family gtpases rac and cdc42. as such, the paks are involved in the regulation of cell survival, proliferation and migration, making them attractive therapeutic targets for cancer. the biological roles of the paks under normal and pathological conditions such as cancer are covered by other reviews in this issue and we will therefore summarize the recent and ongoing research being conducted to develop inhibitors of the paks. there are two groups of mammalian p21-activated kinases (paks) : group i, which is comprised of pak1, pak2 and pak3, and group ii consisting of pak4, pak5 and pak6 (fig. while the groups carry out different functions and have distinct modes of regulation, both groups share a high degree of protein similarity within the kinase domains. the group i paks share 93 - 95% sequence identity within the kinase domain and group ii share 75% sequence identity within this domain. comparing group i to ii there is an overall 54% sequence identity within this domain. previous studies have indicated that the group i paks exist as homodimers that are regulated by an auto - inhibitory domain (aid) from one molecule in the dimer that interacts, in trans with the kinase domain of the other molecule. cdc42/rac interactive binding (crib) domain ] and upon binding of a gtp - bound form of rac or cdc42 the auto - inhibition is alleviated (fig. 1). recent work from multiple groups now indicates that all paks harbor an aid. the kung group has identified an auto - inhibitory fragment at the n - terminal of pak5 that can inhibit kinase activity in the absence of cdc42 binding. recently published data showed that pak5 and pak6 contain the same related crib / aid and that pak4 contains aid positioned similarly to the pak i aid (fig. 1). however, in spite of these similarities it appears the mechanisms underlying the activation of the group i and ii paks are different. (b) molecular structures of small molecule pak inhibitors. clearly, one of the major challenges has been subgroup - specific drug design / discovery given the similarity in structure of the pak - family proteins. one possibility would be to develop allosteric inhibitors, which would target regions outside the active site that are divergent between group i and ii kinases. an alternative approach would be to take advantage of the sequence differences that do exist between the two groups kinase domains to develop small molecule atp competitive inhibitors. such an approach requires a better understanding of the physical structure of the active site of each of the paks. the first report on the identification and characterization of a highly selective allosteric small molecule inhibitor that targets the auto - regulatory mechanism of group i paks was described by the peterson group. by developing a screen for allosteric inhibitors targeting pak1 activation they identified the inhibitor ipa-3 (p21-activated kinase inhibitor 3). it showed limited activity against the group ii paks and against a panel of kinases from which it significantly inhibited (> 50% inhibition at 10 m) only 9 from 214 kinases tested (4% total). further studies showed that ipa-3 binds covalently to the pak1 regulatory domain and thus prevents binding to the gtpase cdc42. the kinase inhibition by ipa-3 in vitro occurs in a temperature - dependent and irreversible manner. unfortunately, a structural isomer of ipa-3, pir-3.5, was found to have no inhibitory activity against pak1. the primary screen of 33,000 structurally diverse small molecules was performed based on catalytic activity of full - length pak1. a secondary screen was based on identification of compounds that are non - atp competitive. from 32 compounds shown to inhibit pak1 activity at 1 mm atp, ipa-3 is clearly an interesting lead compound and further work is required to overcome issues related to the in vivo stability and oral availability of the compound. in particular, the presence of a disulfide bond suggests that the compound might act through covalent redox modification of pak1. nonetheless, the approach taken to identify allosteric inhibitors of the paks will prove useful for the study of paks biology as well as identification of additional inhibitors. in addition to the small molecule allosteric inhibitors, a number of other inhibitors have been described. these include peptide inhibitors, such as the pak1 aid, which unfortunately exhibits pak1-independent effects and the cell - permeable pak1-inactivating peptide tat - pak18, which has been previously suggested to inhibit pak activity by disrupting the interaction between pak1 and pix and to block the growth of ovarian cancer cell lines. a potential new therapeutic approach relies on the use of rna interference, delivered by different approaches. the kissil group has previously shown that shrna - mediated depletion of pak1, 2 and 3 inhibits the proliferation and tumorigenicity of nih3t3/nf2 cells. these data suggest that using rnai - based inhibitors against the paks might be a potential tool in future applications for cancer treatment. nevertheless, questions regarding the delivery approaches for rnai based therapeutics as well as stability of gene silencing remain to be addressed. the challenge in the developing of atp - competitive inhibitors is the high degree of structural similarity between the atp binding pocket of the paks and other kinases. that is exemplified by compounds such as the natural product staurosporine, a broad - range kinase inhibitor, and its derivative k252a which shows potent, but not selective, inhibition of pak1. a subsequent synthetic derivative of k252a, cep-1347, proved to be a more potent inhibitor of pak1 but later was shown to be about 100-fold more selective toward mlk3 (mixed lineage kinase 3). another example of a kinase inhibitor exhibiting significant pak inhibitory capacity was provided by the heerding group. from a series of novel akt inhibitors containing 2,3,5-trisubstituted pyridines, one compound showed significant potency against pak1 (ic50 31 nm). however, it was also observed to be a potent inhibitor of many other kinases in the agc superfamily, such as p70s6k, pdk1, pka and rsk. osu-03012 unexpectedly reduced levels of phosphorylated pak with an ic50 value of ~1 m which is lower than required to block pdk1-mediated akt phosphorylation. an interesting approach was taken by the meggers group to combine organoruthenium chemistry, small - molecule screening and structure - based design to identify a pak1 inhibitor (fl172) with an ic50 value of about 100 nm. the screen of fl172 (at 3) against a panel of 264 kinases revealed that only 15 kinases (5.7% of total) were significantly inhibited by this compound. fl172 also showed isoform selectivity by exhibiting poor inhibitory potency against the group ii paks and displayed dose - dependent pak1 inhibitor activity when tested in mammalian cells. recently, the same group has reported the development of octahedral metal - based kinase inhibitors, including a highly specific pak1 inhibitor (os-2). this study demonstrated that octahedral metal complexes are sophisticated molecules providing versatile scaffolds for developing highly selective and potent kinase inhibitors. recent work from the pfizer oncology group has identified, through high - throughput screening and structure - based design, the compound pf-3758309, a potent (kd = 2.7 nm), reversible atp - competitive, pyrrolopyrazole inhibitor of pak4. pf-3758309 binds the atp binding site and makes multiple contacts with the hinge region through hydrogen - bond interactions with the pyrrolopyrazole core and the amine linker to the thienopyrimidine ring. pf-3758309 shows similar activity across the group i and ii paks and displays growth inhibitory activity against a broad range of tumor cell lines. in order to test selectivity, pf-3758309 was screened against a panel of 146 of the 518 known human kinases and was shown to have cellular activity against src family kinases ampk, rsk, chk2 and others. pf-3758309 has been also shown to be a potent anti - tumor agent in human xenograft tumor models, with plasma ec50 value of 0.4 nm. pf-3758309 demonstrated oral availability and additional steps have been taken to reduce the efflux of the inhibitor by medicinal chemistry strategies, specifically by lowering the molecular charge. these strategies ultimately lead to the discovery of a number of pyrroloaminopyrazoles as orally bio - available pak4 inhibitors. two compounds showed good anti- tumor growth activity (52 - 87%) through oral administration in a xenograft tumor model. recently a novel pak4 inhibitor, lch-7749944, was developed by the le group through structure - informed design. this atp - competitive inhibitor was shown to inhibit a number of pak4 signaling pathways including the pak4/limk1/cofilin and pak4/mek-1/erk1/2/mmp2 pathways as well as egfr activity. the first report on the identification and characterization of a highly selective allosteric small molecule inhibitor that targets the auto - regulatory mechanism of group i paks was described by the peterson group. by developing a screen for allosteric inhibitors targeting pak1 activation they identified the inhibitor ipa-3 (p21-activated kinase inhibitor 3). it showed limited activity against the group ii paks and against a panel of kinases from which it significantly inhibited (> 50% inhibition at 10 m) only 9 from 214 kinases tested (4% total). further studies showed that ipa-3 binds covalently to the pak1 regulatory domain and thus prevents binding to the gtpase cdc42. the kinase inhibition by ipa-3 in vitro occurs in a temperature - dependent and irreversible manner. unfortunately, a structural isomer of ipa-3, pir-3.5, was found to have no inhibitory activity against pak1. the primary screen of 33,000 structurally diverse small molecules was performed based on catalytic activity of full - length pak1. a secondary screen was based on identification of compounds that are non - atp competitive. from 32 compounds shown to inhibit pak1 activity at 1 mm atp, ipa-3 is clearly an interesting lead compound and further work is required to overcome issues related to the in vivo stability and oral availability of the compound. in particular, the presence of a disulfide bond suggests that the compound might act through covalent redox modification of pak1. nonetheless, the approach taken to identify allosteric inhibitors of the paks will prove useful for the study of paks biology as well as identification of additional inhibitors. in addition to the small molecule allosteric inhibitors, a number of other inhibitors have been described. these include peptide inhibitors, such as the pak1 aid, which unfortunately exhibits pak1-independent effects and the cell - permeable pak1-inactivating peptide tat - pak18, which has been previously suggested to inhibit pak activity by disrupting the interaction between pak1 and pix and to block the growth of ovarian cancer cell lines. a potential new therapeutic approach relies on the use of rna interference, delivered by different approaches. the kissil group has previously shown that shrna - mediated depletion of pak1, 2 and 3 inhibits the proliferation and tumorigenicity of nih3t3/nf2 cells. these data suggest that using rnai - based inhibitors against the paks might be a potential tool in future applications for cancer treatment. nevertheless, questions regarding the delivery approaches for rnai based therapeutics as well as stability of gene silencing remain to be addressed. the challenge in the developing of atp - competitive inhibitors is the high degree of structural similarity between the atp binding pocket of the paks and other kinases. that is exemplified by compounds such as the natural product staurosporine, a broad - range kinase inhibitor, and its derivative k252a which shows potent, but not selective, inhibition of pak1. a subsequent synthetic derivative of k252a, cep-1347, proved to be a more potent inhibitor of pak1 but later was shown to be about 100-fold more selective toward mlk3 (mixed lineage kinase 3). another example of a kinase inhibitor exhibiting significant pak inhibitory capacity was provided by the heerding group. from a series of novel akt inhibitors containing 2,3,5-trisubstituted pyridines however, it was also observed to be a potent inhibitor of many other kinases in the agc superfamily, such as p70s6k, pdk1, pka and rsk. osu-03012 unexpectedly reduced levels of phosphorylated pak with an ic50 value of ~1 m which is lower than required to block pdk1-mediated akt phosphorylation. an interesting approach was taken by the meggers group to combine organoruthenium chemistry, small - molecule screening and structure - based design to identify a pak1 inhibitor (fl172) with an ic50 value of about 100 nm. the screen of fl172 (at 3) against a panel of 264 kinases revealed that only 15 kinases (5.7% of total) were significantly inhibited by this compound. fl172 also showed isoform selectivity by exhibiting poor inhibitory potency against the group ii paks and displayed dose - dependent pak1 inhibitor activity when tested in mammalian cells. recently, the same group has reported the development of octahedral metal - based kinase inhibitors, including a highly specific pak1 inhibitor (os-2). this study demonstrated that octahedral metal complexes are sophisticated molecules providing versatile scaffolds for developing highly selective and potent kinase inhibitors. recent work from the pfizer oncology group has identified, through high - throughput screening and structure - based design, the compound pf-3758309, a potent (kd = 2.7 nm), reversible atp - competitive, pyrrolopyrazole inhibitor of pak4. pf-3758309 binds the atp binding site and makes multiple contacts with the hinge region through hydrogen - bond interactions with the pyrrolopyrazole core and the amine linker to the thienopyrimidine ring. pf-3758309 shows similar activity across the group i and ii paks and displays growth inhibitory activity against a broad range of tumor cell lines. in order to test selectivity, pf-3758309 was screened against a panel of 146 of the 518 known human kinases and was shown to have cellular activity against src family kinases ampk, rsk, chk2 and others. pf-3758309 has been also shown to be a potent anti - tumor agent in human xenograft tumor models, with plasma ec50 value of 0.4 nm. pf-3758309 demonstrated oral availability and additional steps have been taken to reduce the efflux of the inhibitor by medicinal chemistry strategies, specifically by lowering the molecular charge. these strategies ultimately lead to the discovery of a number of pyrroloaminopyrazoles as orally bio - available pak4 inhibitors. two compounds showed good anti- tumor growth activity (52 - 87%) through oral administration in a xenograft tumor model. recently a novel pak4 inhibitor, lch-7749944, was developed by the le group through structure - informed design. this atp - competitive inhibitor was shown to inhibit a number of pak4 signaling pathways including the pak4/limk1/cofilin and pak4/mek-1/erk1/2/mmp2 pathways as well as egfr activity. there is now ample evidence to implicate both pak families as targets in different types of cancers. however, a number of questions remain to be answered. first, is there a need for inhibitors that would specifically target the group i or ii pak independently ? second, how difficult would it be to attain this goal ? in regards to the first question, given the different tissue and cell type distributions and substrate specificities of the different paks, it is clear that the identification and development of inhibitors that can distinguish between the group i and ii paks will be instrumental to our understanding of the basic functions of these two groups of proteins, under normal physiological and disease conditions. moreover, in the context of diseases where evidence exists implicating aberrant activation of paks from either of the groups, a major open question is whether inhibiting both groups of paks has a confounding effect. in other words, if inhibiting pak from both groups results in undesired effects, such as reduced tolerability, then clearly inhibitors targeting one group or the other are likely to be beneficial. further studies using highly selective tool compounds and experimental approaches, such as rnai - based technologies to knockdown specific paks, will no doubt contribute to resolving these questions. in regards to the second question, the accumulated data on structure and regulation of the two groups of paks now provides tools toward the development of selective inhibitors. indeed such efforts have already resulted in competitive inhibitors that display specificity toward one group or the other, as illustrated by a number of examples discussed in this review. efforts over the next few years using structure - informed design and novel chemistries will likely lead to even more selective and potent inhibitors. in addition, approaches to identify allosteric inhibitors are still in early phases, but will likely prove fruitful, as illustrated by the identification of ipa-3. | the p21-activated kinases (paks) are downstream effectors of the small g - proteins of the rac and cdc42 family and have been implicated as essential for cell proliferation and survival. recent studies have also demonstrated the promise of paks as therapeutic targets in various types of cancers. the paks are divided into two major groups (group i and ii) based on sequence similarities. although the different roles the pak groups might play are not well understood, recent efforts have focused on the identification of kinase inhibitors that can discriminate between the two groups. in this review these efforts and newly identified inhibitors will be described and future directions discussed. |
spinal muscular atrophy (sma) is a neuromuscular disorder characterized by degeneration of the -motor neurons, leading to symmetrical muscle weakness and atrophy mainly of the lower limbs 1. here, we report on an infant with sma type 1 with a rare compound heterozygosity of the survival of motor neuron 1 gene (smn1) and characteristic fasciculations of the tongue. a 4-month old, caucasian boy with an unremarkable antenatal history (first child of nonconsanguineous, healthy parents, no polyhydramnios) was admitted to our hospital due to dyscataposia and mucus congestion with suspicion of a pulmonary infection. a mild muscular hypotonia was observed by the family 's pediatrician during standard examinations by age 1 month. our examination revealed a poor sucking reflex, failure to thrive, proximal and peripheral muscular weakness with a bell - shaped chest and arreflexia. lower limbs were more strongly affected than upper limbs. on inspection, fasciculations of the tongue were observed most prominently at the periphery of the tongue (video s1). magnetic resonance imaging (mri) of the brain showed a mega - cisterna magna (mcm, fig.1). during hospitalization, molecular genetic testing showed a rare compound heterozygosity for a point mutation c.815a > g (p.y272c) in exon 6 of the smn1 gene (omim 600354) on the paternal allele and a deletion of smn1 exon 7 and 8 on the maternal allele, in the presence of two copies of the smn2 gene (omim 601627) confirming the diagnosis of sma. at parental request spinal muscular atrophy is a heterogeneous neuromuscular disorder and the second most common lethal, autosomal recessive disease in caucasians after cystic fibrosis 2. three clinical subtypes depending on age of clinical onset and maximum motor function have been identified 1. type 1 (werdnig - hoffmann, omim 253300) the most severe and common type of sma has an early onset and progressive unrelenting course resulting in death due to respiratory insufficiency within the first 2 years 2. characteristic clinical features include profound hypotonia, symmetrical paralysis, little or no head control and areflexia 1,3. fasciculations and atrophy of the tongue affect roughly one - third to one - half of patients with sma, and may be noted during the first months of life, as in our patient 3. fasciculations of the tongue may also be seen in neonates with other medical conditions such as hypoxic - ischemic injury (hie), mobius syndrome, and storage disorders (pompe disease) 3. usually, these distinct clinical entities can be differentiated from sma by taking a detailed birth history (e.g., for hie) or taking into account other characteristic clinical findings (e.g., macroglossia in pompe disease). in addition to tongue involvement, fasciculation of the eyelids may also be seen in children with sma. several studies in infants have found an association between cerebrospinal fluid space abnormalities such as ventricular dilatation and congenital myotonic dystrophies 4 ; such abnormalities, like dilatation of the cisterna observed in our patient, may not result in serious complications and remain in most of the cases uninvestigated. mcm is a controversial entity which is generally thought to be an anatomic variant with no clinical significance but may constitute part of several malformation syndromes such as dandy walker complex (dwc). to the best of our knowledge, there is only one reported association between sma type i and blake 's pouch cyst, which along with mcm is regarded as a less severe malformation included in the dwc 5. the vast majority of patients display a homozygous absence of smn1 exons 7 or 8 or exon 7 only, whereas only a few (approximately 4%) show compound heterozygosity for a point mutation on one and a deletion on the other chromosome 2. unfortunately, in patients who do not present a homozygous disruption of the smn1 gene, adversities in terms of diagnosis, prognosis and genetic counseling still occur. c.815a > g (p.y272c), a missense mutation in a highly conserved region in exon 6 of smn1 68, accounts for approximately 20% of sma patients with compound heterozygosity for a point mutation 2,6. p.y272c is found to result in almost complete reduction in the self - oligomerization capacity 9, which suggests it to be a severe mutation8 and is in agreement with our case. in conclusion, muscular hypotonia is a common clinical sign in infants and may be associated with several conditions including neuromuscular disorders, connective tissue disorders, metabolic diseases, or even prematurity. the presence of tongue fasciculations, although not pathognomonic, and in combination with a thorough history, may be suggestive of sma diagnosis. additional supporting information may be found in the online version of this article : video s1. | key clinical messagemuscular hypotonia in infants may be associated with several conditions, such as spinal muscular atrophy (sma). we report on an infant with tongue fasciculations and a rare mutation of the smn1 gene. the presence of tongue fasciculations in combination with a thorough history may be suggestive of sma. |
zolpidem is a short - acting nonbenzodiazepine hypnotic of the imidazopyridine class used for short - term treatment of insomnia. zolpidem selectively activates only one of the benzodiazepine binding sites of the gabaa receptor (alpha-1) which may account for its selective sedative effects and relative lack of muscle relaxant and anticonvulsant effects compared to benzodiazepines. previously, some rare cases of zolpidem - induced hallucinations have been reported from developed countries. a 20-year - old female patient visited our psychiatric clinic with sadness of mood and irritable behavior for 4 months. now she would remain worried and preoccupied with thoughts of her marital life most of the time in a day with gradual loss of interest in pleasurable as well as routine activities. she had to push herself to go to office and would not take much interest in grooming herself now. her biological functions were disturbed and her risk assessment for suicide revealed a high score. she was diagnosed as a case of major depressive disorder and was put on paroxetine 12.5 mg twice a day and zolpidem 10 mg at bed time for sleep. next day, she reported with complaints of experiencing dream - like pleasurable state in which there was lilliputian like visual and elementary auditory hallucinations after half an hour of ingestion of zolpidem. the mood disturbances observed in the patient slowly over the next 3-month treatment were replaced by euthymia most of the time. the patient started doing her routine activities and no any hallucinations were reported by the patient in her follow - up. in the index case report, the patient developed hallucinations after intake of first dose of zolpidem. patient experienced lilliputian visual and elementary auditory hallucinations within half -an hour of intake of zolpidem. its onset of action is usually within 15 minutes and its half - life is 2.6 hours., the australian therapeutic goods administration attached a black box warning to zolpidem, stating, that zolpidem may be associated with potentially dangerous complex sleep - related behaviours that may include sleep walking, sleep driving, and other psychotic behaviours. keto and koga reported visual hallucinations in an 82-year - old woman with diagnosis of major depressive disorder after 1 and half month of administration of zolpidem. in contrast, our index patient reported the visual hallucination on the first night. there are reports of occupational hazards with zolpidem - induced hallucinations in a 54-year old driver and the continued use of zolpidem with fluoxetine resulted in nystagmus and gait disturbances. it was recommended that zolpidem be used for short periods of time using the lowest effective dose. zolpidem 10 mg is effective in treating insomnia when used intermittently no fewer than three and no more than five pills per week for a period of 12 weeks. tsai. postulated that visual hallucinations associated with zolpidem may be related to sudden withdrawal and restarting of zolpidem. further the author hypothesized the mechanism of this phenomena was associated with alteration in the gaba - a receptor. the author also suggested that zolpidem should never be used as needed basis and the dose should be lowest effective dose. zolpidem has not proven effective in maintaining sleep and is more used for sleep initiation problems. our case highlights that clinicians must be aware of the various pharmacological properties of zolpidem and this molecule can induce hallucinations as was observed in the indexed case. further, such cases suggest more research in this clinical area for the better wellbeing of the patients. | we are reporting a case of zolpidem - induced hallucinations in a 20-year - old patient. the duration of this phenomenon was brief, 15 - 20 minutes. our case suggests that clinicians must be aware of this phenomenon while prescribing zolpidem. |
mild - to - severe pruritus accompanies numerous inflammatory skin disorders including atopic dermatitis, eczema, psoriasis, or lichen planus. psoriasis is one of the most common chronic inflammatory skin diseases with a complex, multifactorial, and still not fully understood etiopathogenesis. the main factors contributing to the development of psoriatic lesions are genetic predispositions and immunological disturbances [1, 2 ]. however, the exacerbation of psoriasis can also be provoked by numerous exogenous factors including stress, smoking, infections, and some drugs. pruritus is observed in about 70 to 90% of patients with psoriasis [39 ], and many of them (at least 30%) had generalized itching [5, 6 ]. the mean intensity of this symptom assessed according to 10 point visual analogue scale ranged between 3.76.4 points [5,7, 1012 ]. this is less than the intensity of pruritus observed in atopic dermatitis or uremic pruritus [13, 14 ]. however, despite less intensive, pruritus was mentioned by many psoriatic patients as the most bothersome symptom of psoriasis and it was clearly documented that pruritus intensity significantly correlated in psoriatics with degree of quality of life impairment, level of stigmatization, as well as the presence and severity of depressive symptoms. it seems that patients with pruritus suffer from more severe psoriasis [4, 6, 8 ] although some authors did not find a significant relationship between pruritus intensity and psoriasis severity. the presence and intensity of itching were independent on age, gender, marital status, family history of psoriasis or atopy, type of psoriasis, alcohol or smoking habits, duration of the disease, as well as duration of the last outbreak of psoriasis [5, 6, 8 ]. despite the high frequency of this symptom, here, we reviewed the available literature data on this symptom in order to summarize our current knowledge of the origin of pruritus in psoriasis. histamine, one of the major mediators of pruritus, does not seem to be involved in its development in psoriasis. there was no correlation between pruritus intensity and histamine plasma level in psoriasis, as well as no difference was observed in histamine plasma levels between pruritics and nonpruritics patients with psoriasis. in addition, less than 20% of psoriatic subjects claimed that oral antihistaminics were effective in reducing pruritus. it seems that only sedating antihistaminics should be tried in pruritic psoriatics as they sometimes could be effective due to evoked sedation. it is generally accepted that the histamine blockade does not prevent pruritus in psoriasis. the most often discussed theory on pruritus in psoriasis mentioned the importance of impaired innervation and neuropeptides imbalance in psoriatic skin. interactions between nerves, neuropeptides, and mast cells, leading to neurogenic inflammation, have also been implicated in another chronic itchy immunodermatosis : atopic dermatitis [16, 17 ]. several studies demonstrated altered expression and/or distribution of several neuropeptides and their receptors within various layers of psoriatic skin, including substance p (sp), calcitonin gene - related peptide (cgrp), vasoactive intestinal peptide (vip), somatostatin, -endorphin, or pituitary adenylate cyclase activating polypeptide (pacap) [1726 ]. neuropeptides degranulate mastocytes, activate dendritic cells, lymphocytes, macrophages, and neutrophils, and produce vascular changes in the skin by inducing angiogenesis, dilatation of vessels, and stimulation of synthesis of nitric oxide. they also stimulate synthesis and release of many proinflammatory cytokines from mast cells, lymphocytes, dendritic cells, fibroblasts, and keratinocytes, induce expression of vascular adhesion molecules on endothelium, and exert hyperproliferative effect on keratinocytes. neuropeptides in the skin may be released from dermal nerve endings, but they can also be directly produced by several cell types, for example, mastocytes. nakamura. observed that pruritic psoriatic skin demonstrated significantly increased number of nerve growth factor- (ngf-) immunoreactive keratinocytes, elevated ngf content in the lesional skin, and enhanced expression of high - affinity receptor for ngf (trk - a) in the epidermis and dermal nerve fibres. moreover, pruritic skin showed increased number of protein gene product (pgp) 9.5-immunoreactive nerve fibers in the epidermis and in the upper dermal areas, increased number of sp - containing nerves in the perivascular areas, as well as decreased expression of neutral endopeptidase (nep) in the epidermal basal layer and in the endothelia of blood vessels. the pruritus intensity correlated with the number of pgp 9.5-immunoreactive intraepidermal nerve fibers, the number of ngf - immunoreactive keratinocytes and the expression level of trka in the epidermis. nakamura. also found an increased number of mast cells in the papillary dermis of pruritic psoriatic skin among the various cellular components examined, including resident cells and infiltrating cells in the skin lesions. ultrastructural examination showed that these mast cells possessed degranulating specific granules indicating that mast cells in pruritic psoriatic skin are activated. the particularly characteristic finding of mast cells in lesional skin from patients with pruritus was the presence of free mast cell granules in close apposition to the perineurium surrounding unmyelinated nerve fibers. did not find any differences between pruritic and nonpruritic psoriatics regarding the skin expression of brain - derived neurotrophic factor, neurotrophin-3, vip, neuropeptides y (npy), somatostatin, low - affinity receptor for ngf, and angiotensin - converting enzyme. in another study, a hyperproliferation of small cutaneous nerves was found in the lesional skin of pruritic psoriatic subjects compared to nonpruritic ones. keratinocytes in the psoriatic plaques of patients with pruritus also showed consistently increased expression of sp receptor, trka and cgrp receptor, but the immunoreactivity for sp, cgrp, vip, and pacap was independent on the occurrence of pruritus. the expression of ngf, neurotrophin-4, low - affinity receptor for ngf, pacap receptor expression, as well as nep activity did not differ between pruritus and nonpruritus group. interestingly, remrd. did not find any relationship between sp - positive fibers nor cells and the degree of pruritus, but the analyzed group of patients in this study was very small. in addition, the npy plasma level was significantly decreased in patients with pruritus compared to patients without pruritus. plasma levels of sp, cgrp, and vip did not differ significantly between pruritics and nonpruritics, however, a tendency to lower sp and vip plasma levels in patients with pruritus was noted. moreover, significant, negative correlations between pruritus severity and sp as well as vip plasma levels were found. it seems probable, that increased expression of neuropeptides in the pruritic skin might activate the neuropeptides degrading enzymes like nep or angiotensin - converting enzyme in a regulatory mechanism. this hypothesis could be supported by the observations that the proportion between chymase- and tryptase - positive mast cells was shown to be disturbed in lesional psoriatic skin as well as patients with psoriasis were characterized by higher serum activity of angiotensin - converting enzyme which was normalized after effective antipsoriatic treatment. in the study by our group it was noted that cgrp plasma level was significantly elevated in pruritic psoriatic patients compared to healthy subjects, a difference that was not found between nonpruritic psoriatics and healthy volunteers, and that cgrp plasma level correlated with itching intensity in some subgroups of psoriatics. the important role of altered innervations and neuropeptide imbalance in pruritus accompanying psoriasis may also be supported by the observations that topically applied capsaicin, a potent sp depletory, effectively treated pruritus in psoriatics [30, 31 ]. finally, it was documented that stress - exacerbated pruritus in psoriasis and neuropeptides seem to be good candidates for linking nervous system and skin. it could be hypothesized that increased innervations in the skin of psoriatic patients with pruritus may lead to a lower threshold for pruritic stimuli compared to patients without pruritus. additionally, pruritus might be evoked by the release of selected neuropeptides from dermal nerve endings and cells during stress, but this hypothesis still requires further investigations (table 1). concerning the role of cytokines in pruritus in psoriasis, nakamura. found an increased number of interleukin (il)-2 immunoreactive cells in pruritic versus nonpruritic lesions of psoriasis (table 1). there were no significant differences in the expression of other cytokines (interferon (inf)-, tumor necrosis factor (tnf)-, il-1, il-1, il-4, il-5, il-6, il-8, il-10, and il-12). recently, a novel cytokine, il-31, was suggested to play an important role in pruritus in atopic dermatitis, as il-31 caused the itch - associated scratching behavior in conventional nc / nga mice, an experimental animal model for atopic dermatitis. it seems that changes of dermal vasculature may be important in the pathogenesis of pruritus in psoriasis (table 1). a marked increase of the density of e - selectin - positive venules was found in psoriatic patients with pruritus compared to nonpruritic subjects. however, there was no statistical difference in the number of vessels immunoreactive for intercellular cell adhesion molecule (icam)-1, vascular cell adhesion molecule (vcam)-1, or platelet endothelial cell adhesion molecule (pecam)-1 in the upper dermis or in the expression of icam-1 in the epidermis. however, significant correlation was observed between the itching intensity and the density of e - selectin - immunoreactive vessels. in addition, madej. found an increased serum concentration of soluble vascular adhesion protein (vap)-1 in psoriatic subjects with pruritus compared to patient free of this symptom. despite the lack of solid laboratory data, other mediators may also play a role in the pathogenesis of pruritus in psoriasis (table 1). they were found to be important in several pruritic conditions, but have not been investigated in psoriasis yet. it could be speculated that neuropeptides in psoriatic skin may induce expression and/or activity of dermal proteases, and these enzymes acting via protease - activated receptors (par) might be responsible for prurtius. recent findings suggested that proteases are not only degrading enzymes, but rather represent a group of mediators communicating with nerves, and thereby modulating inflammation, pain, and pruritus [43, 44 ]. tryptase and microbial proteases induced itch by the par-2-mediated neurogenic mechanism [43, 45 ]. activation of par-2 evoked itching both in mice and in human [4346 ]. because par-2 is irreversibly activated by proteases it is believed that activation of -opioid receptors induces while activation of -opioid receptors alleviates pruritus. a significantly altered - and -opioid receptor expression was observed in the epidermis of patients with atopic dermatitis, showing mainly downregulation of -opioid system [35, 36 ]. puva treatment, a frequently applied and effective therapy of atopic dermatitis, was shown to reconstitute the altered opioid receptor distribution in epidermis of these patients. it was shown that intrathecal administration of morphine elicits pruritus and both naloxone and naltrexone, the potent -opioid receptor antagonists, reduces histamine - induced pruritus in atopic dermatitis subjects to greater extend than antihistaminic drugs [37, 38 ]. on the other hand, nalfurafine, a -opioid receptor agonist, led to significant reduction of itching in patients with uremic or cholestatic pruritus [39, 40 ]. prostanoids, mainly prostaglandin d2 [41, 42 ] and tromboxane a2 or serotonin, could be further candidates as mediators of pruritus in psoriasis. the importance of the latter one might be supported by the observations that mirtazapine, an antihistaminic drug acting also via noradrenergenic 2-receptors and 5ht2 and 5ht3 serotonin receptors, relieved psoriatic itch even in cases of severe pruritus associated with erythrodermic psoriasis. pruritus causes the desire to scratch the skin and is experienced as a sensation arising in the skin. however, like all other skin sensations, itch is a product of central nervous system activities. the itch - selective spinal neurons form a distinct pathway projecting from lamina i of the spinal cord to the ventrocaudal part of the nucleus medialis, which projects to the anterior cingulated and dorsal insular cortex. recent studies characterized the supraspinal processing of itch in humans by different imaging techniques. intradermal injection of histamine in healthy volunteers led to activation of anterior cingulate cortex, supplementary motor area, premotor area, and inferior parietal lobe [50, 51 ]. prolonged itch stimuli activated a superior frontal gyrus and the gyrus rectus in both hemispheres as well as in a small area of the left anterior cingulated gyrus. further activation was located in the left temporal pole and some parts of the left cerebellum. repetitive scratching induced bilateral activation of the secondary somatosensory cortex, insular cortex, inferior parietal lobe, and cerebellum while anterior and posterior cingulated cortices were deactivated. the main limitation of these studies is the observations of healthy subjects. as it was demonstrated by ishiuji., the brain processing of itch in chronic skin conditions like in atopic dermatitis is significantly different than in healthy individuals. therefore, further data are needed to identify the brain areas responsible for pruritus in patients with chronic itch, including those having psoriasis. summarizing, pruritus is an important symptom of psoriasis. despite the fact that several studies have been undertaken to investigate the pathogenesis of pruritus in psoriasis, therefore, the pathogenesis of this symptoms is far to be well understood and, as a consequence, the therapy of pruritic psoriatic patients still remains a big challange for clinicians. we hope that in the near future new studies will be conducted to better characterize and understand this symptom in psoriasis. we do believe that this progress may facilitate the development of new effective antipruritic treatment modalities. | the pathogenesis of pruritus in psoriasis remains unclear. many possible mediators were implicated to transmit or modulate this sensation in psoriasis, but none has been clearly proven to be a causative agent of itching. the most often discussed theory mentioned the importance of impaired innervations and neuropeptides imbalance in psoriatic skin. other possible causes of itching might be increased expression of interleukin 2 or vascular abnormalities. recent data indicated that pruritus could be also evoked by opioid system, prostanoids, interleukin 31, serotonin, or proteases. whether these mechanisms are also involved in pruritus accompanying psoriasis requires further investigation. limited knowledge of pruritus origin in psoriasis is responsible for the lack of the effective antipruritic treatments for psoriatics. here, we summarize the current knowledge about the pathogenesis of pruritus in psoriasis and point out possible directions of future studies aiming the pathogenesis of this symptom in psoriasis. |
progesterone (p4) exerts a broad spectrum of physiological actions in the cardiovascular and respiratory systems, kidney, adipose tissue, bone, testis and the brain (graham and clarke, 1997 ; gellersen., 2009 ; 2010). the primary target, however, is the female reproductive tract where p4 has facilitatory roles in modulating the contractile waves of the junctional myometrial zone, tubal transport, and cervical secretion. these processes are superseded by indispensible p4 functions in follicular growth, ovulation and luteinization, embryo implantation, decidualization and maintenance of pregnancy during gestation. we summarise here the actions of p4 in the gravid uterus, focusing on common mechanisms that operate in the myometrium and decidua that may be relevant to term and preterm labor. during the menstrual cycle, the luminal epithelium and underlying endometrial stroma undergo substantial transformation that renders the uterus receptive to embryo implantation (dey., this transformation is a highly coordinated and sequential response to the postovulatory rise in p4 levels, commencing with arrest of estrogen - dependent epithelial cell proliferation, followed by the secretory transformation of the glands, recruitment of various bone marrow - derived immune cells, and angiogenesis (brosens., 1999 ; the actions of p4 are primarily mediated by differentiating stromal cells (simon., 2009). decidualization, is characterized by a mesenchymal - epithelial transition that transforms endometrial stromal cells into specialized secretory decidual cells (dey., 2004 ; gellersen., 2007 ; once decidualized the endometrium relies on a constant supply of p4 to maintain the integrity of the tissue. in the absence of successful implantation, the corpus luteum involutes and declining p4 levels trigger a switch in the secretory phenotype of the decidualizing stroma. this change in phenotype entails release of pro - inflammatory cytokines, chemokines and matrix metalloproteinases, leading to breakdown of the superficial endometrial layer, focal bleeding and menstrual shedding (marbaix., 1995 ; kokorine., 1996 ; brosens and gellersen, 2006 ; brosens., 2009 ; brun., 2009 ; gaide chevronnay., 2009). in addition to the ability to undergo apoptosis upon p4 withdrawal, decidualized stromal cells display a number of unique properties commensurate with their function to safeguard the early conceptus, including resistance to oxidative stress induced cell death, the ability to regulate local immune responses and to coordinate trophoblast invasion (labied., 2006 ; gellersen., 2010 ; endometrial responses to p4 are primarily transduced through binding to, and activation of, the nuclear receptors pr - a and -b, members of the superfamily of ligand - activated transcription factors (misrahi., 1987). in addition to the primary genomic response, it is recognised that there are more rapid short - term actions of p4 that are independent of the transcriptional machinery. in contrast to genomic mechanisms the precise non - genomic actions of p4 are not well defined and are reviewed in detail elsewhere (gellersen., 2009). the nuclear receptors pr - a and pr - b are members of the nuclear steroid receptor family that share structure with the estrogen, androgen, glucocorticoid and mineralocorticoid receptors. this class of transcription factors has a modular structure of distinct functional domains that can be swapped experimentally without significant loss of function (kastner., pr - a and -b are transcribed from the same gene on chromosome 11 by alternative promoter usage (kastner., 1990). the resultant transcripts generate two proteins that differ in size with pr - b containing an additional 164 amino acids at the amino terminus. close analysis of the pr gene has revealed the potential for multiple protein products generated by alternative transcription, translation, or splicing. there has been speculation about the functional relevance of alternative transcripts such as pr - c, pr - m and pr - s, although the existence and physiological relevance of these isoforms remain controversial (samalecos and gellersen, 2008). while the dna and hormone binding affinities of pr - a and -b are indistinguishable, their transcriptional actions are remarkably divergent. early experiments on reporter constructs of simple or complex progesterone response elements (pres), suggested that pr - a displays very little intrinsic transcriptional activity and acts primarily as a dominant inhibitor of pr - b and other steroid hormone receptors (vegeto. it is now clear that pr - a and pr - b govern distinct networks of target genes in a cell - specific context (richer., 2002). unequivocal support for this notion came from selective gene knockout studies in mice, demonstrating that pr - a is indispensible for ovarian and uterine functions whilst pr - b is obligatory for mammary gland development (conneely., 2002 ; mulac - jericevic., thus pr - a is likely to be the dominant receptor isoform in both endometrium and myometrium. ligand binding is thought to occur at prs anchored in cytoplasmic multi - subunit protein complexes consisting of variable heat shock proteins (p23, hsp70, hsp40 and hsp90) and the immuophilins fkbp51 and fkb52 (kosano., 1998 ; tranguch., 2007) the assembly of pr with these macromolecular complexes plays a key role in both the dynamic trafficking of the receptor and the maintenance of an active pool of protein ready to receive freely diffusing p4 from the circulating plasma binding protein transcortin. once bound to p4, the subsequent conformational change in pr promotes dissociation from the chaperone scaffold, followed by homologous dimerization prior to translocation to the nucleus. in the nucleus, activated pr binds to specific nucleotide recognition sequences in promoters of target genes, leading to recruitment of chromatin - modifying co - repressor or -activator complexes, and finally transcriptional repression or activation, respectively (brosens., 2004). this classical model of action predicts that response to p4 signaling should be proportional to the cellular abundance of pr and associated binding proteins. evidence in endometrial cells during the decidualization process demonstrates that this is emphatically not the case. despite the presence of abundant pr in primary endometrial cells, exposure to p4 triggers the expression of few, if any, genes (aghajanova., 2011). although initially difficult to reconcile with the fact that decidualization is a p4 dependent process in vivo, subsequent experiments demonstrated that sustained activation of the protein kinase a (pka) pathway is required to sensitize endometrial cells to p4 (brosens., 1999 ; gellersen and brosens, 2003 ; jones., 2006). the endometrial response to rising p4 during the secretory phase of the cycle is therefore preceded by the rising intracellular camp levels and pka activation, which in turn induces a diverse array of transcription factors including c / ebp, stat5 and foxo1 (gellersen and brosens, 2003). it seems likely that the recruitment of these transcription factors into the pr dependent transcriptional complex is necessary for the classical p4 decidualisation response. importantly, because camp levels are under the control of exogenous factors such as prostaglandin e2, corticotropin releasing factor and relaxin, the p4 response in decidualizing endometrium is both cell and environment specific. as previously alluded to, nuclear receptors such as pr do not have the intrinsic ability to modify chromatin structure to allow access of the transcriptional machinery to dna (lonard and omalley, 2006 ; han. the required modifications are made by recruitment of co - regulators that possess histone and dna modifying activity. the number of known co - regulator proteins, which can either promote or inhibit transcription now exceeds 300 (onate., 1995 ; thakur and paramanik, 2009), and their defined roles in regulating pr transcription in the endometrium requires further investigation. once formed, assembled transcription factor complexes containing pr are capable of initiating gene transcription at other, sequence - specific, transcription factor sites. such cross - talk is critical to the camp dependent decidualisation response and is dependent on the binding partners p53, foxo1, hoxa10, hoxa11, stat5 and c / ebp (christian., 2002a ; 2002b ; 2002c ; mak., 2002 ; since pr - a is essential for decidualization it seems likely that it acts as a critical scaffold protein upon which transcription factor complexes are assembled to transcribe a cohort of decidua specific genes. such complexes are not limited to genes containing pr binding elements (pres) or to ligand bound pr. the pr has been demonstrated to modulate activator protein 1 (ap1), nuclear factor - kappab (nf - b) and specificity protein 1 (sp1) transcriptional activity thus expanding the transcriptional network beyond pre containing genes (bamberger., 1996 ; kalkhoven., 1996 ; owen., 1998) and independent of p4 (cloke., 2008). a final layer of complexity in pr signalling in endometrium comes from posttranslational modifications of the receptor. these modifications (phosphorylation, sumoylation, ubiquitination, and acetylation) are rapid and dynamic and provide a means to fine tune pr signalling in the context of complex environmental signals (brosens., 1999 ; lange., 2000 ; the consequences of modification are many - fold and can involve changes in sub - cellular localisation, protein stability, targeted degradation in the proteasome, altered interactions with co - factors and/or target gene expression. a well - defined example of a physiologically important modification in the reproductive tract is the suppressive effect of sumoylation on transcriptional activity of pr - a (jones., 2006). this sumoylation dependent suppression is potently stimulated by oxidative stress in endometrial cells, but is selectively disabled during the process of decidualisation, thus emphasising the context dependence of posttranslational modifications (leitao. much of the evidence of pr action in the decidua is provided from studies on decidualisation during the menstrual cycle, implantation and early support for pregnancy prior to establishing the placenta. relatively less well studied is the role of decidua during late pregnancy and in particular potential roles for the decidua in initiating parturition. there is abundant evidence from different species that the decidua is a major source of prostaglandins at term (keelan., 2003 ; recent genetic studies in mice demonstrated that increased decidual prostanoid production precipitates preterm labor in the absence of p4 withdrawal, a prerequisite for term parturition in this species. uterine - specific deletion of p53 was sufficient to induce decidual senescence, increased akt signalling, prostaglandin - endoperoxide synthase 2 (ptgs2), prostaglandin f synthase and consequently greater production of the uterotonin pgf2 (hirota., 2010). this effect is mediated by mammalian target of rapamycin complex 1 (mtorc1) and is reversed by low doses of the mtorc1 inhibitor rapamycin (hirota., 2011). the same phenotype (i.e. preterm birth in the absence of progesterone withdrawal) is induced in mice hypomorphic for the prostaglandin - degrading enzyme 15-hydroxyprostaglandin dehydrogenase (15-hpgd) (roizen., 2008). thus, increased prostanoid synthesis, or decreased degradation, in the decidua is sufficient to trigger preterm birth in a species normally reliant on progesterone withdrawal for parturition. this effect can be replicated upon administration of other agonists, such as oxytocin (ot), in either wild - type or ot - deficient mice where the threshold for stimulus is lower in ko mice (imamura., 2000). these observations suggest that a complex relationship between uterine sensitivity and the magnitude of stimulation determines the timing of labor. it is clear that p4 regulates uterine sensitivity to stimulation (see infra) but it is much less obvious if it also regulates the level of stimulation per se. the question that arises is how this change in sensitivity and stimulation is achieved in species such as humans that do not depend on falling p4 levels to initiate labor. there is a set of experimental observations that potentially shed light on the role of pr in modulating these thresholds in different species. there is a clear difference in the efficacy of pr antagonists to induce labor between species that normally deliver in the presence of high p4 and those that exhibit systemic p4 withdrawal. in the latter, administration of ru486 (mifepristone), a mixed pr / gr antagonist, this contrasts to species that normally deliver in the presence of high circulating p4 levels. administration of ru486 here leads to an increased uterine sensitivity and cervical ripening but induction of labor requires co - administration of oxytocics (e.g. oxytocin or prostaglandins). in contrast, the pure pr antagonist onapristone precipitates labor without the need for oxytocics, although only when administered during mid - to - late gestation but not earlier (elger., 1986 ; 1987). the effect of onapristone is pr - specific and reversible by co - administration of the pr agonists r5020 or gestodene (chwalisz., 1995). the reason for the discrepancy in the actions of onapristone and ru486 is not understood but may be related to the high camp / pka activity in the decidua, which converts ru486 into a partial pr agonist (nordeen., 1993). if so, these observations suggest that the gestation - dependent increase in oxytocic drive may emanate from the decidua / fetal membranes in species lacking systemic p4 withdrawal. for example, it is possible that decidual senescence, associated with increased prostaglandins production and/or loss of prostaglandin dehydrogenase activity, could be a predetermined process that is timed relative to the implantation process. the majority of gestation is characterized by a dominance of uterine quiescence, whereby the growing fetus develops in a safe uterine environment until a point sufficient for extra - uterine survival. it is generally accepted that prior to the onset of labor the myometrium undergoes a process of) whereby the muscle becomes more electrically excitable and susceptible to stimulation by pro - contractile hormones. this process is mediated by the increase in expression of certain contraction associated protein (cap) genes (e.g. oxytocin receptor (oxtr), prostaglandin endoperoxidase synthase 2 (ptgs2), connexin 43 (gja1) etc.), concomitant changes is resting membrane potential (parkington, tonta. 1999), and a decrease in camp / pka activity (dodge., 1999). cumulative evidence from different mammalian species indicates that only some labor - associated myometrial changes are mediated directly by p4. as mentioned, administration of ru486 or onapristone leads to increased myometrial responsiveness in all species tested so far, irrespective of the time in gestation (chwalisz and garfield, 1994). the observed increase in uterine responsiveness occurs for both ot and prostaglandins and is not mediated by an increase is receptor number (elger. the fact that increased uterine responsiveness prior to parturition is not accounted for by an increase in either ligand or receptor suggests that a more fundamental change in electrophysiological properties of the myometrium may underpin this phenomenon. the central process that governs uterine contractions is the generation of electrical activity in the form of complex action potentials that mediate voltage - gated calcium entry and hence contractions (blanks., 2007) the spread of electrical activity throughout the uterine smooth muscle is critically dependent on the formation of electrical synapses by gap junction proteins between cells (garfield., 1977 ; 1978 ; 1988). an increase in cell coupling would render the uterus much more sensitive to stimulation by dramatically increasing the efficacy of oxytocics to trigger membrane depolarization (blanks., 2007). this is certainly true for ot. while the uterus is sensitive to picomolar concentrations of ot in vivo, the binding affinity of ot for its receptor is much higher (1 nm) (blanks, 2003). thus, coupling intracellular calcium release to a tissue level action potential and voltage gated calcium entry enables ot to elicit a full agonist response with comparatively low receptor occupancy. consistent with this hypothesis, administration of anti - progestins in rats and guinea pigs dramatically increases gap junction proteins at the plasma membrane of uterine myocytes (garfield., 1987 ; chwalisz., 1991) in addition to gap junction proteins, pr regulates the expression of the main pore forming subunit of the voltage - gated l - type calcium channel, further intimating that p4 modulates uterine excitability (chwalisz., 1995). the observation that most mammalian species initiate parturition in response to falling circulating progesterone levels combined with the fact that pr antagonists universally increase myometrial responsiveness to uterotonics underpin the widely held view that local p4 withdrawal must trigger the onset of labor in humans. in fact, numerous mechanisms of local p4 withdrawal have been proposed, focusing either on modulation of pr function, p4 metabolism, and/or p4-dependent suppression of inflammation (mendelson, 2009 ; mesiano., 2011). none of these mechanisms are necessarily mutually exclusive and yet - in our view conclusive proof that local p4 withdrawal is obligatory for labor is as yet lacking. it is indeed striking that p4 therapy is effective in the prevention of preterm labor in some but not all women (da fonseca., 2003 ; meis., 2003 ; fonseca., 2007). a popular concept is that a change in the ratio of pr isoforms accounts for local p4 withdrawal in the myometrium. this is based on the observation that myometrial biopsies taken during labor express relatively more pr - a than pr - b when compared to samples obtained prior to labor (merlino., 2007). further, an increase in pr - a / pr - b ratio in an immortalized myometrial cell line has been shown to activate pro - inflammatory genes (tan., 2012). while attractive, the in vivo relevance of these observations remains difficult to test as a change in pr - a / b ratio can not underpin labor in mice (the usual in vivo model) as gestation and parturition are unperturbed upon pr - b silencing. the hypothesis is pertinent to those species that do not exhibit systemic p4 withdrawal, although the model fails to explain the requirement for oxytocics to induce labor upon treatment with pr antagonists. a related hypothesis is predicated on the observation that p4 and inflammatory signalling pathways are closely intertwined and converge on the reciprocal inhibitory interaction between pr and the nf - b transcription factor complex. for example, p4 has been shown to inhibit binding of the nf - b - p65 complex to response elements in the ptgs2 promoter (hardy., 2006), a process that may be mediated through physical interaction between p65 and the activated pr (kalkhoven., 1996). p4 also stimulates the expression of the binding protein ib responsible for maintaining nf - b in a transcriptionally inactive state in the cytosol (hardy., 2006). this model assumes that the inhibitory effects of p4 are overridden in response to increased nf - b activation, which in turn establishes a positive feedback mechanism by decreasing p4-mediated repression. in support of this notion, increased nf - b activity has been shown to decrease the expression of pr co - activators, thus diminishing receptor activity (condon.. however, a recent study using primary human myocytes indicated that nf - b activation interferes only with the ability of the pr to activate but not repress target genes (lee., 2012). further, and in contrast to observations in an immortalized cell line, p4 did not inhibit the inflammatory response in primary cultures. a final proposed pathway for p4 withdrawal, which may be complimentary to the mechanisms proposed for pr, is a local metabolism of p4. the onset of labor in mice is associated with striking non - labor phenotypes in knockouts of the p4 metabolizing enzyme 20-hydroxysteroid dehydrogenase (20-hsd) and 5-reductase type 1 (mahendroo., 1996 ; 1999 ; in a species that normally experiences systemic p4 withdrawal these interesting phenotypes suggest that p4 clearance from uterine tissues is also important. interestingly, recent evidence suggests that 20-hsd may be regulated in the uterus by stat5b, which itself is under the regulation of mi - r200a(williams., 2012). furthermore, mir-200a is up regulated at term in mice and humans and is also capable of regulating the e - box binding homeobox proteins zeb1 and zeb2 (renthal., 2010). these transcription factors also regulate the oxytocin receptor (oxtr) and connexin-43 (cx43) in a pr dependent manner. thus, p4 metabolism and pr transcriptional activity may be co - regulated to create a concerted alteration in the p4 response. it is clear that there is much work still to be done before we can establish exact mechanisms of p4 and pr action in the myometrium throughout gestation and prior to parturition. of particular importance is the need to reconcile data obtained from various in vitro systems and cell lines with in vivo observations, for example in response to pr antagonists. furthermore, the role of pr action in late gestation in the decidua requires greater focus as relatively little is known compared to our understanding of the role of this nuclear receptor during menstrual cycle. it seems highly probable that a focus on the juxtacrine interactions between uterine compartments may yield a better understanding of the role of p4 in both term and preterm labor. | progesterone is central to many reproductive processes and is critical in regulating the menstrual cycle and maintaining pregnancy. we discuss here similarities in the molecular mechanisms that regulate the process of decidualisation in endometrial stromal cells and uterine quiescence in myometrial smooth muscle cells. we discuss recent evidence that the decidua may be an important mediator of progesterone actions in the onset of labor in mammalian species lacking progesterone withdrawal. these observations have relevance to recent clinical observations of the effect of progesterone administration in the prevention of preterm labor. we suggest that further research is required to understand the role of progesterone in maintaining the decidua in late pregnancy and particular focus should be given to the mechanisms that increase prostaglandin production in the uterus at term. |
the advent of effective chemotherapy has made limb salvage an oncologically safe option for a large majority of bone tumors without compromising long term survival. the femur is the commonest site for primary bone tumors, and rarely, the extent of tumor may necessitate resection of the complete femur to achieve adequate oncologic clearance. options include total femoral prostheses (tfp), allografts, and rotationplasty.16 few reports in literature address the use of tfp exclusively for bone sarcomas. most of these describe the functional results with expensive internationally available prostheses.25 in a resource challenged population, the cost of prostheses can occasionally be a limiting factor to limb salvage, especially in cases of malignant tumors where the prognosis is guarded.7 we present our experience with indigenously manufactured low cost total femoral prosthesis (tfp) for oncologic outcomes and implant survival after resection and replacement of the total femur. nine patients were operated between december 2003 and june 2009 for resection and tfp implantation. endoprosthetic reconstruction after resection was achieved with a modular total femur prosthesis using an indigenously manufactured fixed hinge tfp (tmh - nice / restor, sushrut - adler mediequip pvt. ltd., devrukh, india www.sushrut.com/restor.htm) in eight cases and an expandable howmedica modular resection system (hmrs) prosthesis in one case. the eight cases in which the indigenously manufactured tfp was implanted were analyzed in this case series. there were four males and four females with a mean age of 32 years (range 1372 years). all except one were done as a limb salvage procedure for a primary or recurrent malignant bone tumor. case 7 was for revision of a broken proximal femur replacement, originally done for a malignant bone tumor [table 1 ]. the diagnosis included osteogenic sarcoma (n=5), ewing 's sarcoma (n=1), and chondrosarcoma (n=2). the primary goal of surgery was limb preservation with complete excision of the tumor while retaining adequate muscle for satisfactory residual function. surgery involved disarticulation of the hip and knee with enbloc excision of the entire femur and surrounding muscles involved with the tumor while preserving the neurovascular bundle. the indigenously manufactured modular total femur system comprises a trochanteric component, a central resection segment, and a distal femoral component, designed to lock into each other with a customized self - locking taper. the central resection segments are available in various sizes to match the length of the patient 's femur. restoration of length was based on preoperative radiological assessment and reconfirmed intraoperatively by measurement of the resected specimen [figure 1 ]. (a) preoperative t1-weighted mri showing involvement of the entire femur in a case of ewing 's sarcoma. (c) radiograph demonstrating restoration of limb length after total femur prosthesis to further reinforce hip stability and prevent dislocation, the residual hip capsule was sutured around the neck of the prosthesis. in cases where the capsule was deficient, it was reinforced with a polypropylene mesh (prolene johnson and johnson, ethicon division, aurangabad, india) that was anchored to the acetabulum and extended to form a sleeve around the proximal part of the prosthesis.8 only glutei were either sutured directly to the prosthesis and/or sutured to the polypropylene mesh. progressive mobilization using a walker or crutches was started within the first few postoperative days and patients gradually progressed to ambulation without the use of supports. patients were instructed to restrict hip flexion to 60 for the first 6 weeks while encouraging active quadriceps exercises to regain knee extension. patients were asked to followup every 3 months for the first 2 years and six monthly subsequently. functional status was assessed at the time of last followup using the musculoskeletal tumor society (msts) scoring system.9 this was based on the analysis of six factors (pain, use of supports for ambulation, walking ability, functional activities, gait, and emotional acceptance). for each of the six factors, values of 05 were assigned based on established criteria. the result was expressed as a sum total with a maximum score of 30 and as a percentage of the expected normal function for the patient. survival of the implant was analyzed with the starting point defined as the date of implantation of the endoprosthesis and the endpoint being removal (whatever the cause). patients were censored for statistical analysis (observation stopped before the event occurred) if removal had not occurred at the time the patient was last assessed. the mean followup was 33 months (972 months) for all patients (n=8) and 40 months (2472 months) in survivors (n= 5). 8 patients analysed, 1 lost to follow up, 2 dead, 5 alive and were available for followup. there was one case of infection which developed after 7 months in a patient who had the tfp implanted after excision of a recurrence around an earlier distal femoral prosthesis. repeated wound lavage failed to control the infection and a hip disarticulation was finally performed (case 1). it occurred in a 72-year - old male who had a large open biopsy elsewhere. his biopsy diagnosis was chondrosarcoma which was revised to chondroblastic osteosarcoma after evaluation of the excised femur. another patient (case 1) who had the tfp implanted after excision of a recurrence around an earlier distal femoral prosthesis eventually succumbed to distant metastasis. thus, five of the seven patients available for follow up are currently alive. the msts score for patients evaluated at their last followup ranged from 21 to 25 with a mean of 24 (80%). all the patients were independent ambulators and none of them required a support for walking. there was one case of infection which developed after 7 months in a patient who had the tfp implanted after excision of a recurrence around an earlier distal femoral prosthesis. repeated wound lavage failed to control the infection and a hip disarticulation was finally performed (case 1). it occurred in a 72-year - old male who had a large open biopsy elsewhere. his biopsy diagnosis was chondrosarcoma which was revised to chondroblastic osteosarcoma after evaluation of the excised femur. another patient (case 1) who had the tfp implanted after excision of a recurrence around an earlier distal femoral prosthesis eventually succumbed to distant metastasis. the msts score for patients evaluated at their last followup ranged from 21 to 25 with a mean of 24 (80%). all the patients were independent ambulators and none of them required a support for walking. reconstructing large defects after resection of a tumor has always been challenging. in the past, most patients with extended sarcomas of the femur had an amputation with poor function.3 the advent of better imaging modalities, effective chemotherapy, better understanding of anatomy with continuous refinement in surgical techniques, and advances in prosthesis design and materials have all played a part in increasing the incidence of limb salvage surgery. limb salvage should not compromise patient survival while ensuring that patients are able to resume near - normal function as soon as possible.5 the prostheses used must also demonstrate good long term survival. when evaluating a reconstruction technique, various factors need to be considered including its complications, functional outcome, and durability. in resource - challenged populations, cost too often plays a role in the decision making.10 in most cases of tumors involving the femur, adequate resection may be possible with retention of enough bone either proximally or distally to seat an intramedullary stem for a distal femoral or proximal femoral prosthesis. occasionally, for oncologic reasons, entire femoral resection may be necessary or the residual bone stump after proximal / distal femoral resection may be too short to adequately seat a stable intramedullary stem. hip dislocation is a common complication reported after total femur replacement.24611 a combination of various factors can contribute to this. these include lack of functional abductor musculature, resection of the joint capsule, acetabular resurfacing, and use of a rotating hinge design at the knee since rotation at the knee can lead to hip dislocation.25 in our series, we did not have any dislocation. this can be attributed to the use of a fixed hinge prosthesis, using a bipolar head in all cases, and reinforcing the residual hip capsule with a polypropylene mesh in cases where the capsule was deficient. the incidence of local recurrence is a reflection of adequate oncologic clearance and the effectiveness of chemotherapy.12 we had one local recurrence occurring in a case of osteosarcoma, who had an open biopsy at a non oncology center and received no chemotherapy. having identified definite adverse contributing factors in this case, we believe that limb salvage with a tfp is an oncologically sound procedure in appropriately selected cases. local control rates are similar to those described for limb salvage procedures at other sites.7 the survival of the implant was 88%, with removal as the endpoint. the average functional score (80%) in our study as evaluated by the msts scoring system is encouraging when compared with other series describing the use of tfp [table 2].246 one case (case 8) currently has 3 cm shortening and is managing with a shoe raise. patients reported very good psychological acceptance after limb salvage with the tfp despite having a poor gait which is primarily due to the lack of good abductor muscle attachment. this is significant as the prostheses used for limb salvage were low - cost, locally manufactured devices costing approximately us $ 2000 (international prosthesis costs approximately us $ 18,000). thus, due to cost constraints, we are unable to offer conventional limb salvage to some young children requiring total femur excision and do a rotationplasty instead.13 our study is limited by its small numbers, but the uncommon use of tfp for bone sarcomas would necessitate results from various centers being pooled to reach meaningful conclusions.4 though late local recurrence is uncommon, the incidence of implant related complications can be expected to increase with a longer followup.12 in spite of these shortcomings, we believe that this study does add to the existing literature on use of massive implants in limb salvage, especially the use of tfp in appropriately indicated patients with malignant bone tumors. the successful use of locally manufactured megaprostheses demonstrates that cost constraints need no longer be an insurmountable barrier for the use of limb salvage implants in resource - challenged populations. | background : the extent of tumor may necessitate resection of the complete femur rarely to achieve adequate oncologic clearance in bone sarcomas. we present our experience with reconstruction in such cases using an indigenously manufactured, low - cost, total femoral prosthesis (tfp). we assessed the complications of the procedure, the oncologic and functional outcomes, and implant survival.materials and methods : eight patients (four males and four females) with a mean age of 32 years, operated between december 2003 and june 2009, had a tfp implanted. the diagnosis included osteogenic sarcoma (5), ewing 's sarcoma (1), and chondrosarcoma (2). mean followup was 33 months (972 months) for all and 40 months (2472 months) in survivors. they were evaluated by musculoskeletal tumor society score, implant survival as well as patient survival.results:there was one local recurrence and five of seven patients are currently alive at the time of last followup. the musculoskeletal tumor society score for patients ranged from 21 to 25 with a mean of 24 (80%). the implant survival was 88% at 5 years with only one tfp needing removal because of infection.conclusions:a tfp in appropriately indicated patients with malignant bone tumors is oncologically safe. a locally manufactured, cost - effective implant provided consistent and predictable results after excision of the total femur with good functional outcomes. |
pre - crystallization lcp - frap assays are optional ; however, they can significantly accelerate the process of searching for initial crystallization conditions, especially in the case of difficult membrane proteins. purify a membrane protein of interest in a detergent solution and concentrate the protein / detergent complexes to ~10 - 20 mg / ml, taking care not to over - concentrate the detergent. transfer ~25 mg of an lcp host lipid (typically monoolein) or a lipid mixture into a 1.5 ml plastic tube and incubate at 40 c for few minutes until the lipid melts. load another 100 l syringe with the protein solution at a protein solution - to - lipid ratio 2/3 v / v. push the syringe plungers alternately to move the lipid and protein through the inner needle of the coupler, back and forth, until the lipid mesophase becomes homogeneous. lcp forms spontaneously upon mechanical mixing, and the protein becomes reconstituted in the lipid bilayer of lcp. formation of lcp can be verified by its transparent and gel - like consistency and by the absence of birefringency when viewed under a microscope equipped with cross - polarizers, or, if possible, by using small - angle x - ray diffraction. lcp - frap assays are designed to measure the diffusion properties of membrane proteins reconstituted in lcp at a variety of screening conditions. the long - range diffusion of membrane proteins in lcp is essential for successful crystallization ; however, the microstructure of lcp constrains diffusion of large proteins or oligomeric protein aggregates. a common reason for failure of an in meso crystallization experiment is a fast protein aggregation leading to a loss of diffusion. it has been shown that the aggregation behavior of a protein depends on the particular protein construct, the host lipid and the composition of the screening solution. label the protein with a fluorescent dye (cy3 or similar) at a protein / dye ratio of ~100/1, remove the unreacted dye and concentrate the protein to ~ 1 mg / ml. when labeling free amines, use ph between 7 and 7.5 to predominately label the free n - terminus. be aware that amino labeling can also label lipids co - purified with the protein. reconstitute the labeled protein in lcp as described in section 1). set up assay plates as described in section 3) using lcp - frap screening solutions instead of crystallization screens. incubate the plates at 20 c in the dark for at least 12 hours to achieve an equilibrium state. place one of the plates on the lcp - frap station and focus on the first well using a 10x objective. trigger the laser. the laser power and number of pulses should be adjusted to bleach ~30 - 70% of the labeled protein in the middle of the bleached spot. immediately after triggering the laser, start recording a fast post - bleaching sequence of ~200 images at the fastest possible rate. follow with recording of a slow post - bleach sequence of ~50 images, selecting the delay between images as 1 - 20 s, depending on the diffusion rate of the protein. integrate the intensity inside the bleach spot in all frames and correct it for bleaching and light intensity fluctuations during the acquisition by dividing the intensity inside the bleached spot by the averaged intensity of a reference spot outside of the laser bleached area. normalize the corrected intensity to make the pre - bleached intensity equal to 1 and the initial bleached intensity equal to 0. fit the curve of the normalized intensity vs. time, f(t), using the following equation : f(t) = m x exp(-2t / t) x (i0(2t / t) + i1(2t / t)), (eq.1) where m is the mobile fraction of diffusing molecules, t is the characteristic diffusion time, t is the real time of each recorded frame, i0 and i1 are the 0 and 1 order modified bessel functions. calculate the diffusion coefficient, d, as : d = r/4 t, (eq.2) where r is the radius of the bleached spot. design new crystallization screens based on the components that facilitated protein diffusion and excluding conditions for which protein diffusion was not observed. if the protein did not diffuse in any of the screened conditions, consider broadening the screening space or trying a new protein construct. transfer the protein - laden lcp into a 10 l gas - tight syringe attached to a repetitive syringe dispenser. attach a short removable needle (gauge 26, 10 mm length) to the 10 l syringe. dispense 200 nl boluses of lcp on the surface of four adjacent wells forming a 2x2 square. repeat steps 3.4)-3.6) with the next set of 4 wells until the whole plate is filled. incubate the plate at a constant temperature, periodically checking for crystal formation and growth. place a plate with protein crystals under a stereo microscope with variable zoom, equipped with a linear rotating polarizer and analyzer. focus on the well of interest using a low power zoom so that the whole well is placed within the field of view. score the coverslip glass in four strokes making a square inside the well boundaries using a sharp corner of a ceramic capillary cutting stone. press around the scored perimeter with strong sharp - point tweezers to propagate the scratches through the thickness of the coverslip glass. inject few l of precipitant solution through one of the holes to reduce dehydration during the subsequent steps. using an angled sharp needle probe break up the glass along one or two sides to free the cut - out square. if the bolus is stuck to the coverslip, then flip the glass square over and place on the bottom of the well. add an extra few l of precipitant solution, supplemented with a cryo - protectant, if necessary, on top of the exposed cubic phase bolus in the well. adjust the angle between the polarizer and the analyzer to increase the contrast between the birefringent crystal and the background, while keeping enough light to see the harvesting loop. select a mitegen micromount with a diameter matching the crystal size and then harvest the crystal directly from the lcp by scooping it into the micromount. flash freeze the micromount with the harvested crystal in liquid nitrogen, and ship it to a synchrotron source beamline for x - ray data collection. an engineered human beta 2 adrenergic g protein - coupled receptor (2ar - t4l) was expressed in baculovirus infected sf9 insect cells and purified in dodecylmaltoside (ddm)/ cholesteryl hemisuccinate (chs) detergent solution bound to a partial inverse agonist carazolol. the protein was labeled with cy3 nhs ester and used in lcp - frap pre - crystallization assays (figure 2). coarse grid screens based on several conditions selected from the results of lcp - frap assays produced initial crystal - like hits (figure 3). a) results of an lcp - frap assay performed in an automatic high - throughput mode, in which each sample of a 96-well plate is bleached sequentially and fluorescence recovery is measured after a 30 min incubation. the obtained fluorescence recoveries, which represent the mobile fraction in each sample, are plotted for all 96 samples. the screening solutions contain 0.1 m tris ph 8, 30 % v / v peg 400 combined with 48 different salts at two different concentrations. 1.the mobile fractions and the diffusion coefficients are determined using eqs. 1 and 2. fast recovery of less than 10% in the sample containing na chloride is due to fluorescently labeled lipids co - purified with the protein. initial crystal hits of 2ar - t4l / carazolol obtained by a coarse grid screening around most promising conditions identified by lcp - frap, containing na sulfate (panel a) and na formate (panel b). the protein is labeled with cy3 nhs ester and the fluorescent images are taken using excitation at 543 nm and emission at 605 nm. the images of crystals grown in the presence of na sulfate (panels a and b) and k formate (panels c and d) are taken in the brightfield mode (panels a and c) and using cross - polarizers (panels b and d). purify a membrane protein of interest in a detergent solution and concentrate the protein / detergent complexes to ~10 - 20 mg / ml, taking care not to over - concentrate the detergent. transfer ~25 mg of an lcp host lipid (typically monoolein) or a lipid mixture into a 1.5 ml plastic tube and incubate at 40 c for few minutes until the lipid melts. load another 100 l syringe with the protein solution at a protein solution - to - lipid ratio 2/3 v / v. push the syringe plungers alternately to move the lipid and protein through the inner needle of the coupler, back and forth, until the lipid mesophase becomes homogeneous. lcp forms spontaneously upon mechanical mixing, and the protein becomes reconstituted in the lipid bilayer of lcp. formation of lcp can be verified by its transparent and gel - like consistency and by the absence of birefringency when viewed under a microscope equipped with cross - polarizers, or, if possible, by using small - angle x - ray diffraction. lcp - frap assays are designed to measure the diffusion properties of membrane proteins reconstituted in lcp at a variety of screening conditions. the long - range diffusion of membrane proteins in lcp is essential for successful crystallization ; however, the microstructure of lcp constrains diffusion of large proteins or oligomeric protein aggregates. a common reason for failure of an in meso crystallization experiment is a fast protein aggregation leading to a loss of diffusion. it has been shown that the aggregation behavior of a protein depends on the particular protein construct, the host lipid and the composition of the screening solution. label the protein with a fluorescent dye (cy3 or similar) at a protein / dye ratio of ~100/1, remove the unreacted dye and concentrate the protein to ~ 1 mg / ml. when labeling free amines, use ph between 7 and 7.5 to predominately label the free n - terminus. be aware that amino labeling can also label lipids co - purified with the protein. reconstitute the labeled protein in lcp as described in section 1). set up assay plates as described in section 3) using lcp - frap screening solutions instead of crystallization screens. incubate the plates at 20 c in the dark for at least 12 hours to achieve an equilibrium state. place one of the plates on the lcp - frap station and focus on the first well using a 10x objective. trigger the laser. the laser power and number of pulses should be adjusted to bleach ~30 - 70% of the labeled protein in the middle of the bleached spot. immediately after triggering the laser, start recording a fast post - bleaching sequence of ~200 images at the fastest possible rate. follow with recording of a slow post - bleach sequence of ~50 images, selecting the delay between images as 1 - 20 s, depending on the diffusion rate of the protein. integrate the intensity inside the bleach spot in all frames and correct it for bleaching and light intensity fluctuations during the acquisition by dividing the intensity inside the bleached spot by the averaged intensity of a reference spot outside of the laser bleached area. normalize the corrected intensity to make the pre - bleached intensity equal to 1 and the initial bleached intensity equal to 0. fit the curve of the normalized intensity vs. time, f(t), using the following equation : f(t) = m x exp(-2t / t) x (i0(2t / t) + i1(2t / t)), (eq.1) where m is the mobile fraction of diffusing molecules, t is the characteristic diffusion time, t is the real time of each recorded frame, i0 and i1 are the 0 and 1 order modified bessel functions. calculate the diffusion coefficient, d, as : d = r/4 t, (eq.2) where r is the radius of the bleached spot. design new crystallization screens based on the components that facilitated protein diffusion and excluding conditions for which protein diffusion was not observed. if the protein did not diffuse in any of the screened conditions, consider broadening the screening space or trying a new protein construct. reconstitute the protein in lcp as described in section 1). transfer the protein - laden lcp into a 10 l gas - tight syringe attached to a repetitive syringe dispenser. attach a short removable needle (gauge 26, 10 mm length) to the 10 l syringe. dispense 200 nl boluses of lcp on the surface of four adjacent wells forming a 2x2 square. apply a gentle pressure on the coverslip to seal the wells. repeat steps 3.4)-3.6) with the next set of 4 wells until the whole plate is filled. incubate the plate at a constant temperature, periodically checking for crystal formation and growth. place a plate with protein crystals under a stereo microscope with variable zoom, equipped with a linear rotating polarizer and analyzer. focus on the well of interest using a low power zoom so that the whole well is placed within the field of view. score the coverslip glass in four strokes making a square inside the well boundaries using a sharp corner of a ceramic capillary cutting stone. press around the scored perimeter with strong sharp - point tweezers to propagate the scratches through the thickness of the coverslip glass. punch two small holes at opposite corners of the scored square. inject few l of precipitant solution through one of the holes to reduce dehydration during the subsequent steps. using an angled sharp needle probe break up the glass along one or two sides to free the cut - out square. if the bolus is stuck to the coverslip, then flip the glass square over and place on the bottom of the well. add an extra few l of precipitant solution, supplemented with a cryo - protectant, if necessary, on top of the exposed cubic phase bolus in the well. adjust the angle between the polarizer and the analyzer to increase the contrast between the birefringent crystal and the background, while keeping enough light to see the harvesting loop. select a mitegen micromount with a diameter matching the crystal size and then harvest the crystal directly from the lcp by scooping it into the micromount. flash freeze the micromount with the harvested crystal in liquid nitrogen, and ship it to a synchrotron source beamline for x - ray data collection. an engineered human beta 2 adrenergic g protein - coupled receptor (2ar - t4l) was expressed in baculovirus infected sf9 insect cells and purified in dodecylmaltoside (ddm)/ cholesteryl hemisuccinate (chs) detergent solution bound to a partial inverse agonist carazolol. the protein was labeled with cy3 nhs ester and used in lcp - frap pre - crystallization assays (figure 2). coarse grid screens based on several conditions selected from the results of lcp - frap assays produced initial crystal - like hits (figure 3). a) results of an lcp - frap assay performed in an automatic high - throughput mode, in which each sample of a 96-well plate is bleached sequentially and fluorescence recovery is measured after a 30 min incubation. the obtained fluorescence recoveries, which represent the mobile fraction in each sample, are plotted for all 96 samples. the screening solutions contain 0.1 m tris ph 8, 30 % v / v peg 400 combined with 48 different salts at two different concentrations. 1.the mobile fractions and the diffusion coefficients are determined using eqs. 1 and 2. fast recovery of less than 10% in the sample containing na chloride is due to fluorescently labeled lipids co - purified with the protein. initial crystal hits of 2ar - t4l / carazolol obtained by a coarse grid screening around most promising conditions identified by lcp - frap, containing na sulfate (panel a) and na formate (panel b). the protein is labeled with cy3 nhs ester and the fluorescent images are taken using excitation at 543 nm and emission at 605 nm. the images of crystals grown in the presence of na sulfate (panels a and b) and k formate (panels c and d) are taken in the brightfield mode (panels a and c) and using cross - polarizers (panels b and d). the protocols provide a basic visual guidance for the main steps involved in performing in meso crystallization experiments. more in - depth details related to these protocols, emphasizing possible pitfalls, shortcomings or alternative routes are available elsewhere. optional lcp - frap assays can help at the earlier stages to select the most promising protein construct, lcp host lipid and lipid additives, as well as limit the range of possible precipitants and buffer conditions. once an initial crystallization hit is found, it should be optimized to obtain better quality crystals. optimization of in meso crystallization conditions is essentially similar to optimizing conditions for soluble proteins with the addition of extra parameters associated with the composition of lcp. membrane protein crystals grown in lipidic mesophase are typically smaller in size than crystals obtained in detergent solution, but more ordered, thus, benefitting strongly from using microfocus beamlines available at modern synchrotron sources. many of the procedures related to in meso crystallization, including setting up crystallization or assay plates, conducting lcp - frap assays and detecting crystals, have been semi- or fully automated, allowing screening of a large range of conditions while consuming small amounts of protein and lipid. on the other hand, protein reconstitutions in lcp and crystal harvesting remain manual and more tedious operations and, thus, have a need for improvement. | membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are implicated in a multitude of malfunctions and diseases. however, a structural and functional understanding of membrane proteins is strongly lagging behind that of their soluble partners, mainly, due to difficulties associated with their solubilization and generation of diffraction quality crystals. crystallization in lipidic mesophases (also known as in meso or lcp crystallization) is a promising technique which was successfully applied to obtain high resolution structures of microbial rhodopsins, photosynthetic proteins, outer membrane beta barrels and g protein - coupled receptors. in meso crystallization takes advantage of a native - like membrane environment and typically produces crystals with lower solvent content and better ordering as compared to traditional crystallization from detergent solutions. the method is not difficult, but requires an understanding of lipid phase behavior and practice in handling viscous mesophase materials. here we demonstrate a simple and efficient way of making lcp and reconstituting a membrane protein in the lipid bilayer of lcp using a syringe mixer, followed by dispensing nanoliter portions of lcp into an assay or crystallization plate, conducting pre - crystallization assays and harvesting crystals from the lcp matrix. these protocols provide a basic guide for approaching in meso crystallization trials ; however, as with any crystallization experiment, extensive screening and optimization are required, and a successful outcome is not necessarily guaranteed. |
jaundice is one of the most prevalent clinical problems during neonatal period which requires interventional treatment. it affects 60% of healthy term neonates during the first week of life. despite the fact that most icteric neonates have not any other disorders, physicians are worried about the toxic effects of bilirubin on neural cells and the subsequent appearance of kernicterus which is a chronic, degenerative and debilitating disease. due to socioeconomic reasons, there is a global agreement on the early discharge of healthy neonates from hospitals and if neonatal hyperbilirubinemia was not predicted at this stage, the early discharge of these neonates will be leading to an increase in the incidence of kernicterus. different methods of measuring the serum levels of bilirubin are currently used which can be categorized into three groups of cutaneous (a non - invasive method of measuring bilirubin precipitated in the layers of skin by relevant apparatus, capillary (bilirubin measurement using a spectrophotometer) and laboratory (the typical method of serum bilirubin measurement using chemical reactions). there are only a few studies investigating the correlations between the popular and the new laboratory methods as alternative methods for bilirubin measurement. the apparatus for cutaneous and capillary measurements are currently presented in iran. by achieving this study, the correlation rate of these apparatuses has been clearly defined and there is a chance to test the jm103 (draeger medical system, inc, telford, pa) setting and to clarify the functions of the capillary and cutaneous methods of bilirubin measurement in different levels for the first time, in iran. in addition, this study suggests replacing the ordinary laboratory systems with the capillary and cutaneous methods as a practical and reliable method to minimize the pain and discomfort during blood sampling in neonates. this prospective non - blind randomized clinical trial study was performed in 3 steps and in 3 different locations. infants with rh or abo isoimmunisation, major congenital malformations, hemoglobinopathies, evidence of liver disease or receiving phototherapy 12 hours before study were excluded from the study (fig. 1). randomized controlled diagram (rct) the first step : 120 term healthy infants without sepsis or hemolysis were enrolled to determine the correlation and measurement accuracy of bilirubin serum level by the 2 methods laboratory and capillary : all the icteric neonates who were brought into the emergency department of afzalipour hospital of kerman university of medical sciences and all the icteric neonates admitted to the neonatal ward of this hospital entered this study. to determine the serum bilirubin level after several minutes, 2 capillary samples were taken in capillary tubes from the baby 's heel and bilirubin measured by a bilirubinometer apparatus after centrifugation. the average value of the 2 samples was recorded as the capillary bilirubin level. the second step : 160 infants involved in evaluating the accuracy and correlation rates of the bilirubin level measured by cutaneous measurement apparatuses : healthy neonates brought into the clinic to be checked for any possible jaundice were investigated by 2 cutaneous measurement apparatuses, simultaneously over chest skin, to measure the level of bilirubin. the third step : 148 infants enrolled in determining the correlation and measurement accuracy of bilirubin level by the 2 methods cutaneous and capillary : this step of the study was performed at the imam hassan mojtaba health center. a blood sample, from the samples taken for thyroid screening purposes, was prepared in a capillary tube and was immediately centrifuged and analyzed by a bilirubinometer. the bilirubin level was then measured over the thorax of the neonates by cutaneous measurement apparatus. the apparatuses were correlated at this stage in each working shift according to the manufacturer 's guidelines. the research was approved by ethics committee of kerman university of medical sciences. considering a maximum of 5% as the first type error, 20% as the second type error and 4% as the minimum valuable correlation, the sample size was estimated to be 65 for the present study. by comparing the three methods, the estimated figure was multiplied by the square root of 2, according to which, a sample size of 90 was estimated for calculating simple correlations. for eliminating the effects of at least 2 confounding variables such as skin color, 20% was added to the estimated sample size and the final figure of 108 was estimated as the study sample size. the correlation rate of the results of the two other methods and the method of measurement byvenous sample (standardized chemical analysis) was then calculated using the linear regression method. in this study the correlation coefficient of higher than 0.8 and p value of less than 0.05 was considered statistically significant. there was no statistically significant difference between the three groups of study regarding the age and sex at entrance. a total of 73 neonates were excluded from the study because of the hemolysis of their capillary blood samples. the range of bilirubin level measured in the first, second and the third stage of the study was 1 - 30 mg / dl, 1 - 30 mg / dl and 1 - 18 mg / dl, respectively. 2 illustrates that the david icterometer apparatus has measured the bilirubin concentration averagely 2.36 mg / dl more than that measured by the jm103 apparatus. the correlation coefficient between the concentrations reported by these two tools is 0.91 which is also statistically significant. the figure also shows that the difference in the concentration of bilirubin measured by these two apparatuses is seen in almost all ranges of bilirubin concentration. comparing the measured bilirubin level by two cutaneous apparatuses fig. 3 shows that the bilirubin concentration in capillary blood sample is averagely 0.91 mg / dl lower than that measured in venous sample. the correlation coefficient between the concentrations reported by these two tools is obtained as 0.96 which is also statistically significant. in addition, this figure shows that the difference in the concentration of bilirubin by the two tools is almost seen in all the ranges of bilirubin concentration reported. comparing the measured bilirubin levels by the capillary and laboratory methods fig. 4 indicates that david icterometer apparatus has measured the bilirubin concentration 3.1 mg / dl more than the capillary samples on average. this difference is more obvious at lower concentrations of bilirubin whilst it is not detected above the bilirubin concentration of 9 mg / dl, and at this level of bilirubin shows a better correlation with the capillary method. comparing the measured bilirubin level by the capillary and david icterometer fig. 5 indicates also that the jm103 apparatus has measured the bilirubin level averagely 0.57 mg / dl more than the capillary samples. the correlation coefficient between the concentrations reported by these two tools is 0.89 but the difference pattern among different concentrations of bilirubin is almost constant. comparing the measured bilirubin level by the capillary and jm 103 apparatuses because of the early discharge of neonates from hospitals, a worldwide concern appeared on the uncontrolled neonatal hyperbilirubinemia and its sequels. in july 2004, the american academy of pediatrics proposed a practical protocol to all neonatal surveillance centers as to examine any possible jaundice in the neonates above 35 weeks of age. it also notified that all neonatal wards must possess a practical instruction as to examine and pursue jaundice in neonates. although this academy did not define any methods of bilirubin examination or measurement, measuring the serum bilirubin level is one of the most common laboratory tests carried out in neonates. this test is painful and can put the neonate at increased risk of sepsis. on the other hand, this test is stressful for both the neonate and parents and requires a lot of time and money. therefore, a reduction in repeated blood sampling is very important which might be achieved by utilizing the new set of apparatuses, available on market, for cutaneous and capillary measurement of bilirubin concentration. different studies have evaluated the sensitivity and accuracy of these tools, according to which different results have been obtained. according to the literature review concerning the role of cutaneous measurement apparatuses, it has been shown that the jm103 tool has a good correlation with serum bilirubin level in neonates ; however, because of its different error factors, it can not predict the precise serum bilirubin concentration and therefore, the cutaneous bilirubin test, on its own, can not be a reliable criterion for exchange transfusion or phototherapy in neonates. in another study jm103 and bilicheck in measuring the serum bilirubin concentration. the accuracy of these two tools on estimating different levels of bilirubin was assessed and it was shown that a significant correlation and similarity existed between the serum and the cutaneous bilirubin concentration measured by these tools. the difference between the level of serum bilirubin and the cutaneous bilirubin measured by jm103 was 0.7 mg / dl which was also 0.7 mg / dl for the bilicheck apparatus. another study compared the correlation between the bilirubin levels measured by 9 different methods (3 cutaneous measuring apparatuses, 3 photometric apparatuses, 3 chemical and laboratory analyses) and concluded that photometric apparatuses had good correlations with chemical and laboratory methods ; however, when the serum level of bilirubin is high, the cutaneous measurement apparatuses show a lower bilirubin concentration. the present study showed that the jm103 has a significant correlation with the laboratory and capillary methods and that the bilirubin concentration measured by this apparatus is averagely 0.57 mg / dl higher than the capillary method and 0.34 mg / dl lower than that measured by laboratory method (correlation coefficient 0.89). jm103 was constant among all the different levels of bilirubin and even on high concentrations of bilirubin, it is possible to decide reliably, based on the bilirubin level measured by this apparatus, on the procedures for pursuing the icteric neonates. jm103 can, with a high sensitivity, replace the ordinary laboratory methods of neonatal bilirubin measurements, even though the manufacture setting of this apparatus is set in a way that no more than 20 mg / dl of bilirubin concentration can be measured. this study showed that the david icterometer apparatus measured the bilirubin level averagely 3.1 mg / dl higher than the capillary method and about 2.2 mg / dl higher than the serum level in venous samples. this difference is statistically significant (p<0.001) and is more obvious in lower bilirubin levels. at bilirubin levels higher than 9 mg / dl, however, the results obtained showed a slight difference and had a better correlation with capillary method. although the statistical results are indicative of a correlation between bilirubin levels measured by david icterometer and capillary (correlation coefficient 0.85), however, during evaluation of neonatal hyperbilirubinemia, particularly at levels which need interventional treatment, this amount of difference in serum bilirubin level is not accepted and can lead to incorrect decisions on presuming jaundice in neonates. this study showed that the capillary method has a significant correlation with the laboratory method and that the bilirubin level measured is averagely 0.9 mg / dl less than the serum level (correlation coefficient 0.96). this difference is constant among the different levels of bilirubin and is independent of its concentration. this study also showed that the capillary method can be used not only as a tool for pursuing jaundiced neonates, is also as a suitable alternative for laboratory methods. the capillary method, however, is influenced by many variables which, in turn, limit its application as a preliminary option in measuring neonatal bilirubin as well as its potential in becoming a suitable alternative for ordinary laboratory methods. this method, compared to venous blood sampling, is less invasive, causes less pain in the neonate and requires less talent and experience. to avoid frequent and repetitive blood sampling, the capillary and jm103 methods can be used, with a high confidence, to measure the neonatal bilirubin level. the david icterometer method, however, measures the bilirubin level averagely 2.2 mg / dl higher than the serum level which, from a clinical point of view, is a significant difference and, at higher bilirubin levels, can lead to wrong decisions in pursuing jaundiced neonates. second, we excluded neonates with prematurity, sepsis, hemolysis or liver disease. therefore, our study could not be used for sick or preterm newborns. the jm103 apparatus has a significant correlation with the serum bilirubin level without a notable difference between the apparatus - measured and the actual serum bilirubin level. therefore, at bilirubin concentrations less than 20 mg / dl, this apparatus can be used both as a monitoring tool and as an alternative for routine laboratory methods of bilirubin measurement. the jm103 technique can be used as the primary method in assessing jaundice in neonates ; however, at bilirubin levels higher than the limits of this apparatus or levels requiring interventional treatments such as phototherapy or exchange transfusion, the capillary method could, instead, be used as the first option in investigating jaundice in neonates. considering the fact that healthy neonates with normal peripheral perfusion were investigated in this study to assess the accuracy and sensitivity of cutaneous bilirubin measuring methods, it seems that the results of this study can not be extrapolated to sick or premature neonates. | objectivethere are different methods for measuring bilirubin concentration ; however, it is quite important for practitioners to know which method should be used in certain clinical situations. the present prospective study aimed to compare three different methods for measuring neonatal bilirubin concentrations.methodsall full term neonates who were either brought into emergency departments or admitted to the neonatal wards in kerman city in 2011 were recruited (n = 428). the correlation coefficients were estimated for the routine ways of bilirubin concentrations including capillary, cutaneous and laboratory methods.findingsof 428 recruited neonates, 178 were female. mean age sd was 17871 hours. the correlation coefficient for david icterometer vs jm103 was 0.91, while the corresponding coefficient for david icterometer vs capillary was 0.96. it was also equivalent to 0.85 for correlation between jm103 and capillary methods. the david icterometer measured an average of 2.36 mg / dl levels of bilirubin concentration compared to the jm103 method. the capillary method showed a lower bilirubin level than the venous concentration (0.91 mg / dl on average). compared with the capillary, the jm103 measured a slightly higher level of bilirubin with an average 0.57 mg / dl.conclusiondue to low difference (less than 1 mg / dl) between jm103 and the capillary methods for measurement of neonatal bilirubin concentration, these two methods could alternatively be used instead of usual laboratory method. |
recently, helicobacter pylori (h. pylori) infection has been increasingly reported to be inversely associated with allergic diseases, although some study groups proposed discrepancies in the data, i.e. improvement or no improvement in allergic symptoms after the eradication. we by chance checked nonspecific serum immunoglobulin e (ige) and found a marked rise in its titers following h. pylori eradication therapy. a 49-year - old japanese man, ex - smoker without allergic disorders, visited kamifukuoka - kyodo clinic checking for low serum pepsinogen (pg) levels : pg i 40.8 ng / ml, pg ii 15.7 ng / ml, and pg i : pg ii ratio 2.60, suggesting possible gastric cancer. gastrofiberscopic examination did not reveal gastric cancer, but showed atrophic gastritis with h. pylori infection, i.e. positive results for rapid urease test and pathologic findings. the patient wanted to eradicate h. pylori, and a triple drug regimen using lansoprazole 60 mg / day, amoxicillin 1,500 mg / day, and clarithromycin 400 mg / day for 7 days was initiated in february 2008. pylori antibody titers and immunohematologic parameters, complete blood counts, leukocyte differentials, and serum levels of immunoglobulin (igg, iga, igm, and ige) were serially measured. eight weeks later, a second endoscopic examination confirmed successful eradication : negative results for both rapid urease test and pathologic findings. at that time, the titer of anti - h. pylori igg antibody, neutrophil counts, and monocyte counts had decreased. on the contrary, peripheral counts of lymphocytes, eosinophils, basophils, and platelets in addition, serum levels of igg, iga, igm, and ige had all risen. 10 more weeks later, the level of serum ige increased to four times its pretreatment level : 306 485 1,325 u / ml (normal < 170). along with these increases, the number of peripheral eosinophils also increased to triple : 99 139 298 per l. in addition, we pathologically evaluated ige - containing cells and mast cells in the gastric mucosa (fig. 1, fig. the density of mast cells apparently increased in the antrum, although that of ige - containing cells did not show any difference on the posttreatment specimen. the patient neither began to keep animals nor did he receive any additional medications, and was free from any signs of allergic disorders by december 2008. infiltration of both neutrophils and lymphocytes into the gastric mucosa is a well - known phenomenon among h. pylori - infected patients. in addition, monocytes, eosinophils and basophils also have been reported to infiltrate into the gastric mucosa in h. pylori - infected persons. these observations suggest that h. pylori infection can upregulate the peripheral numbers of such leukocytes in infected patients. we therefore presume that eradication of h. pylori decreases the numbers of these leukocytes and of immunoglobulins in the peripheral blood. the precise mechanism of this phenomenon is unclear, but the following is able to explain the phenomenon : (1) skewed differentiation of helper t cell seen in h. pylori - infected patients, (2) hygiene hypothesis, and (3) the influence of lansoprazole on serum immunoglobulins. h. pylori infection is now accepted to skew t helper differentiation toward type 1 (th1) property (production of interleukin-2, interferon-, and tumor necrosis factor - a) that counteracts the th2-dependent process. actually some cell contents of h. pylori have been reported to downregulate th2 inflammation in experimental models. as a result of successful eradication, the liberation of th1 property could enhance humoral immunity to upregulate immunoglobulin production, and this regain may finally exert upregulation of serum ige and eosinophilic inflammation induced by the th2-dependent process. in recent decades, the decreased prevalence in infectious diseases had led to a rapid increase in the prevalence of allergic diseases especially in western societies, and such observation has been explained with the hygiene hypothesis. exposure to some pathogens, including bacteria and viruses, is proven to induce th1 skew that could be preventive for the induction of allergic diseases through counteraction against th2 cells. we stained ige - containing cells and mast cells in the pretreatment and posttreatment gastric mucosa. as a result, mast cells have been reported to involve gastric mucosal damage in patients with gastritis, especially those infected with h. pylori. we thus presumed the decreased density of mast cells in the posttreatment specimen, we however could not confirm such decrease microscopically. since mast cells involve allergic response in humans, this observation may represent h. pylori eradication - induced allergy. concerning serum immunoglobulins, we recently reported increases in serum igg and igm in h. pylori - infected patients treated with lansoprazole, suggesting the altering ability on th1 shift. we however confirmed continuous increases in lymphocytes, eosinophils, basophils, igg and ige in the peripheral blood at follow - up. since the patient was free from lansoprazole on follow - up, the continuous increases in both ige and eosinophil should be an aftereffect induced by h. pylori eradication. this case presentation suggests that some patients are able to develop allergic disorders through h. pylori eradication. since a sole case presentation can not be applied for an exact explanation for the phenomenon, further evaluations using sufficient numbers of patients should be performed. | helicobacter pylori infection has been reported to be inversely associated with allergic disorders. we by chance experienced a patient with atrophic gastritis who presented marked elevations of both nonspecific serum immunoglobulin e and eosinophil counts after h. pylori eradication. a 49-year - old japanese man received eradication of h. pylori using lansoprazole 60 mg / day, amoxicillin 1,500 mg / day, and clarithromycin 400 mg / day for 7 days. serum immunoglobulin e increased to more than four times its pretreatment level, 306 485 1,325 u / ml, and peripheral eosinophil counts increased to more than three times, 99 139 298 per l. deducing from the current case, h. pylori eradication might develop allergic disorders in some patients. |
in orthodontics, several studies have showed that bracket and wire materials, sections, surface conditions, type and force of ligation, use of self - ligating brackets, saliva, and other oral functions influence friction (f) at the archwire - slot interface.110 self - ligating bracket1112 is a family of ligatureless brackets characterized by a metal device to close off the edgewise slot2 that demonstrates a significant decrease in f compared to conventional brackets.7,10,1318 recently, new low - friction systems and self - ligating brackets were introduced in trading, with differentiated performances according to their manufacturers. among them, for example, there are low - friction ligatures (slide ; leone, firenze, italy), which are similar to conventional elastic ligatures, but have an anterior part that is more rigid and similar to the cap of self - ligating brackets ; they are recommended when low f is desired, but they can be replaced when more f is needed. then, there are also the recently introduced vision lp brackets (american orthodontics, sheboygan, wisconsin, usa), which seem to generate low f even when thick rectangular archwires are employed. on the contrary, there are other recently introduced brackets, called time3 brackets (american orthodontics), which guarantee high f with thick rectangular archwires in order to better control dental torque. according to the proper manufacturers, each of these brackets has different clinical indications and can be helpful in various situations in which different levels of f are needed. thus, the aim of this in vitro study is to compare the f generated by these recently introduced self - ligating brackets, low - friction ligatures, and conventional stainless steel (ss) brackets. the null hypothesis for this investigation was that there are no significant differences among the various self - ligating brackets and between the low - friction system and traditional system, in terms of f generated, when these brackets are coupled with the same type of archwire. in this investigation, we included passive, interactive, and conventional brackets, according to the classification introduced in literature by voudouris.19 the brackets tested were : damon mx brackets (sds ormco, glendora, california, usa) (passive),time3 brackets (american orthodontics) (interactive),victory series brackets (3 m unitek, monrovia, california, usa) ligated with ligature ringlet elastic modules (rmo, denver, colorado, usa) (traditional),victory series brackets (3 m unitek) ligated with slide low - friction ligatures (leone) (conventional), andvision lp brackets (american orthodontics) (passive). damon mx brackets (sds ormco, glendora, california, usa) (passive), time3 brackets (american orthodontics) (interactive), victory series brackets (3 m unitek, monrovia, california, usa) ligated with ligature ringlet elastic modules (rmo, denver, colorado, usa) (traditional), victory series brackets (3 m unitek) ligated with slide low - friction ligatures (leone) (conventional), and vision lp brackets (american orthodontics) (passive). the testing model (fabricated by myrmex laboratory, foggia, italy) was described in a previous investigation.9 it was composed of a metal bar, approximately 10 cm long, 3.5 cm wide, and 1 cm thick. on one of the larger surfaces of this metal bar, 10 brackets (to represent the upper right to the upper left second bicuspid) were bonded by the same technician, mr. ten brackets of the same group were mounted in alignment on the metal bar using a cyanoacrylate adhesive (loctite 416 ; loctite corp. the number of procedures was calculated using a test for the calculation of sample numerosity on the basis of value fixed at 0.05, considering the minimum detectable difference and the number of groups. alignment of the brackets was obtained through preliminary insertion of a 0.0210.028-inch ss archwire into the slots of the brackets, without ligation. however, as minor misalignments of the brackets or non - linearity of the wire could not be controlled to estimate the extent to which the f could be attributed to misalignment rather than ligation, a preliminary confirmatory check was performed by measuring f for each bracket - archwire combination with only the terminal brackets ligated. the models with only the terminal brackets ligated were compared with each other, and then, with the models with all the brackets ligated. the comparison among models with only the terminal brackets ligated is useful because if the comparison was among different types of brackets (with the same slot size) coupled with the same archwire but without any ligation except for the terminal brackets (ligated in order to obtain only the stabilization of the archwire along the slots), no differences would be observed among the brackets in terms of f, except if the brackets (of 1 type, with respect to another) were positioned in misalignments. in this case, the expectation is that the 10 misaligned brackets could generate a higher f with respect to the 10 brackets positioned in alignment. in total, 25 testing models were constructed, i.e., 5 models for each group of brackets (victory series, time3, damon mx, slide ligatures, and vision lp). for each group, a single model was used 10 times to test the same bracket - archwire combination with all the brackets ligated and 10 times to test the same bracket - archwire combination with only the terminal brackets ligated. the archwires tested were 0.014-inch, 0.016-inch, 0.0140.025-inch, 0.0180.025-inch, and 0.0190.025-inch nickel - titanium (niti) ; 0.0170.025-inch beta - titanium (tma) ; and 0.0190.025-inch ss (table 1). the tests were run in dry state at an ambient temperature of 34c maintained through the use of an air conditioner. for frictional evaluation, a mechanical testing machine (model lloyd 30k ; lloyd instruments ltd., segensworth, uk) with a 10-lb tension load cell, set at a range of 1 lb and calibrated from 0 to 1000 g, was employed. the archwires moved through all the 10 brackets at a crosshead speed of 0.5 mm / min (each run lasted approximately 5 min). f was calculated as the mean of all the values recorded as the wire was drawn through the brackets. the load cell registered the force levels needed to move the wire along the 10 aligned brackets, and these levels were transmitted to a computer. due to skewed data, nonparametric tests were used to investigate statistically significant differences in f among the groups. the data were analyzed as differences in f observed in the groups of archwires among the 5 groups of brackets with all the brackets ligated through the kruskal - wallis test ; if the results of the kruskal - wallis test were significant, a mann - whitney test was performed as post hoc analysis to evaluate the significance of the differences among the groups. in order to estimate the extent to which f could be attributed to the misalignment of brackets rather than to the type of ligation, a statistical comparison (kruskal - wallis test) was also performed on all the groups of brackets with only the terminal brackets ligated. finally, a mann - whitney test was performed on the data for all ligated brackets and the data for those with only the terminal brackets ligated ; comparisons were made for each bracket - archwire combination in order to verify the effect of ligation on f. for each statistical test, the statistical significance was set at =0.05. in this investigation, we included passive, interactive, and conventional brackets, according to the classification introduced in literature by voudouris.19 the brackets tested were : damon mx brackets (sds ormco, glendora, california, usa) (passive),time3 brackets (american orthodontics) (interactive),victory series brackets (3 m unitek, monrovia, california, usa) ligated with ligature ringlet elastic modules (rmo, denver, colorado, usa) (traditional),victory series brackets (3 m unitek) ligated with slide low - friction ligatures (leone) (conventional), andvision lp brackets (american orthodontics) (passive). damon mx brackets (sds ormco, glendora, california, usa) (passive), time3 brackets (american orthodontics) (interactive), victory series brackets (3 m unitek, monrovia, california, usa) ligated with ligature ringlet elastic modules (rmo, denver, colorado, usa) (traditional), victory series brackets (3 m unitek) ligated with slide low - friction ligatures (leone) (conventional), and vision lp brackets (american orthodontics) (passive). the testing model (fabricated by myrmex laboratory, foggia, italy) was described in a previous investigation.9 it was composed of a metal bar, approximately 10 cm long, 3.5 cm wide, and 1 cm thick. on one of the larger surfaces of this metal bar, 10 brackets (to represent the upper right to the upper left second bicuspid) were bonded by the same technician, mr. ten brackets of the same group were mounted in alignment on the metal bar using a cyanoacrylate adhesive (loctite 416 ; loctite corp. the number of procedures was calculated using a test for the calculation of sample numerosity on the basis of value fixed at 0.05, considering the minimum detectable difference and the number of groups. alignment of the brackets was obtained through preliminary insertion of a 0.0210.028-inch ss archwire into the slots of the brackets, without ligation. however, as minor misalignments of the brackets or non - linearity of the wire could not be controlled to estimate the extent to which the f could be attributed to misalignment rather than ligation, a preliminary confirmatory check was performed by measuring f for each bracket - archwire combination with only the terminal brackets ligated. the models with only the terminal brackets ligated were compared with each other, and then, with the models with all the brackets ligated. the comparison among models with only the terminal brackets ligated is useful because if the comparison was among different types of brackets (with the same slot size) coupled with the same archwire but without any ligation except for the terminal brackets (ligated in order to obtain only the stabilization of the archwire along the slots), no differences would be observed among the brackets in terms of f, except if the brackets (of 1 type, with respect to another) were positioned in misalignments. in this case, the expectation is that the 10 misaligned brackets could generate a higher f with respect to the 10 brackets positioned in alignment. in total, 25 testing models were constructed, i.e., 5 models for each group of brackets (victory series, time3, damon mx, slide ligatures, and vision lp). for each group, a single model was used 10 times to test the same bracket - archwire combination with all the brackets ligated and 10 times to test the same bracket - archwire combination with only the terminal brackets ligated. the archwires tested were 0.014-inch, 0.016-inch, 0.0140.025-inch, 0.0180.025-inch, and 0.0190.025-inch nickel - titanium (niti) ; 0.0170.025-inch beta - titanium (tma) ; and 0.0190.025-inch ss (table 1). the tests were run in dry state at an ambient temperature of 34c maintained through the use of an air conditioner. for frictional evaluation, a mechanical testing machine (model lloyd 30k ; lloyd instruments ltd., segensworth, uk) with a 10-lb tension load cell, set at a range of 1 lb and calibrated from 0 to 1000 g, was employed. the archwires moved through all the 10 brackets at a crosshead speed of 0.5 mm / min (each run lasted approximately 5 min). f was calculated as the mean of all the values recorded as the wire was drawn through the brackets. the load cell registered the force levels needed to move the wire along the 10 aligned brackets, and these levels were transmitted to a computer. descriptive statistics were calculated for each bracket - archwire combination. due to skewed data, the data were analyzed as differences in f observed in the groups of archwires among the 5 groups of brackets with all the brackets ligated through the kruskal - wallis test ; if the results of the kruskal - wallis test were significant, a mann - whitney test was performed as post hoc analysis to evaluate the significance of the differences among the groups. in order to estimate the extent to which f could be attributed to the misalignment of brackets rather than to the type of ligation, a statistical comparison (kruskal - wallis test) was also performed on all the groups of brackets with only the terminal brackets ligated. finally, a mann - whitney test was performed on the data for all ligated brackets and the data for those with only the terminal brackets ligated ; comparisons were made for each bracket - archwire combination in order to verify the effect of ligation on f. for each statistical test, the statistical significance was set at =0.05. for majority of the bracket - archwire combinations, f values obtained with all the 10 brackets ligated were significantly higher than those obtained with only the terminal brackets ligated ; no significant differences were observed in only 4 cases, i.e., when the damon mx brackets and vision lp brackets were engaged with round archwires (table 2). the f recorded with all the brackets ligated and their significant differences are shown in table 2 and figure 1. coupled with round archwires, damon mx and vision lp brackets generated significantly lower f than victory series, time3, and slide ligatures brackets (p<.05). victory series brackets generated significantly higher f than the other groups (p<.05). coupled with rectangular archwires, victory series, slide ligatures, and vision lp self - ligating brackets generated significantly lower f than time3 and damon mx brackets (p<.05). in this investigation, we made a comparison of the effects of different self - ligating and conventional brackets on f, but we did not include a group ligated with ss ligature wire to avoid unexpected results associated to the force spent by the operator during ligation. in addition, because of its design, this in vitro study can not add data on the clinical indications of these brackets. however, the results will be useful to indicate the proper clinical study that must be conducted to obtain such data. then, clinical conclusions are not possible due to the limitations of the experimental conditions, as, for example, it did not take into account the canine bracket tipping during retraction or the undesirable rotation during retraction. as reported in table 2, for majority of bracket - archwire combinations, the f values obtained with all 10 brackets ligated were significantly higher than those recorded with only the terminal brackets ligated (p<.05), confirming that ligation plays an important role in generating f. this is in accordance with previous literature.14,20 there were no significant differences in only 4 cases, i.e., when damon mx and vision lp brackets were engaged with round archwires, probably due to the shapes of their rigid caps. as reported, the archwires were moved at a crosshead speed of 0.5 mm / min (each run lasted approximately 5 min) to avoid failure of the brackets from the metal bar. this low crosshead speed is generally lower than in other investigations, such as, for example, khambay,7 who used a crosshead speed of 5 mm / min (each test run lasted for 4 min) but included only 1 bracket in their model. in this study, 3 different self - ligating brackets showed different trends when used with the various archwires (table 2 and figure 1). coupled with round archwires (0.014-inch and 0.016-inch niti archwires), time3 self - ligating brackets generated significantly higher f than the other 3 self - ligating brackets (damon mx and vision lp brackets), while no significant differences were observed between slide ligatures and time3 brackets. on the contrary, victory series generated significantly higher f than all the other groups (table 2 and figure 1). a consistent agreement was found among the reviewed studies that self - ligating brackets produce lower f compared to conventional brackets when coupled with small round archwires, as pointed by a recent systematic review.21 the differences in f observed among the self - ligating brackets could be explained by the differences in the shapes of their little caps. damon mx and vision lp brackets show a little cap that keeps the bracket closed without pressing the archwire against the slot, while the time3 bracket is characterized by a bell - shaped cap that can squeeze the archwire into the slot, probably increasing f at the archwire - slot interface. as noted, damon mx and vision lp brackets showed a lower level of f with round archwires compared to the other groups, suggesting that among the archwire - bracket combinations considered, they could guarantee the lowest f during the alignment phase of orthodontic treatment. in addition, it must be noted that when coupled with all the rectangular archwires, damon mx brackets showed significantly higher f than majority of the other brackets (table 2 and figure 1), except for time3 brackets, for which no significant differences were observed between the 2 systems (table 2 and figure 1). this finding suggests that damon mx bracket results in low f only when engaged with round archwires but not with rectangular archwires, probably because of the shape of its sliding little cap, which when engaged with rectangular archwires, provides a large contact surface between the archwire and slot walls, thus increasing the level of f. similarly, when coupled with rectangular archwires, time3 bracket could probably allow for an increase in f because of its bell - shaped cap, which when closed, can compress the archwire against the slot walls. vision lp brackets exhibited a different behavior with respect to the other 2 self - ligating brackets, showing a low f when matched with either round or rectangular archwires (table 2 and figure 1). coupled with round archwires, vision lp brackets showed significantly lower f than victory series, slide ligatures, and time3 brackets, but no significant differences with damon mx brackets ; as previously mentioned, this finding could be explained through the little cap of this bracket, which could prevent the compression of the archwire against the slot, thus reducing the f generated, as is also assumed for damon mx brackets. however, coupled with rectangular archwires, vision lp brackets showed significantly lower f than both, damon mx and time3 brackets (table 2 and figure 1), probably because the particular design of closing mechanism of this the little cap to slide along a rail to maintain space between the archwires and slot walls, thus reducing the f produced. in addition, the little cap of vision lp brackets is not exactly on a parallel plane to the base of the slot, but is lightly inclined to leave a small trilateral free space between the archwire and the little cap. consequently, this little cap seems to allow for a decrease in f at the archwire - slot interface. taken together, these findings suggest the importance of the closing mechanism and cap design in determining the f generated at the bracket - archwire interface. differences in the structural design and material composition of the bracket slot and cap can influence the level of f generated at the bracket - archwire interface when coupled with rectangular or round archwires. we must state that the particular design of this study allowed a possible lack of alignment of the 10 brackets, which presents as a limitation of the study. in passive systems, such as damon mx and vision lp brackets, any rigid lack of alignment will lead to high levels of binding with rectangular wires, while in active systems and conventional ligated systems, the give in the ligation method will allow for a lack of alignment. in this regard, our findings are also a function of the experimental setup and do not actually reflect the situation in clinical use wherein the periodontal ligament provides give. this clarification is fundamental as this concept limits the possibility of making any clinical extrapolation from this study. this point has enormous importance from a clinical point of view, because the clinician must know the different in vitro behaviors of the different self - ligating brackets. from a clinical point of view, for example, the low f observed with slide ligatures or vision lp brackets, compared to time3 and damon mx brackets when coupled with rectangular archwires (table 2 and figure 1), could be considered either as an advantage or a disadvantage in different situations ; during anterior tooth retraction, low f is desired in the lateral segment of dental arches, while in the final phase of stabilization, high f is desired in all slots. for slide ligatures, however, the primary advantage seems to be that they can be used only when low f is necessary or vice versa. from a clinical point of view, the observations in this study altogether indicate that the clinician must select the type of self - ligating system to be employed depending on the type of malocclusion to treat. with regard to the literature, our findings about damon mx and time3 brackets are in accord with those of khambay,7 who employed a model with only 1 damon sl ii bracket and 1 conventional straight - wire bracket, and tested (for 10 times, in the presence of human saliva) various archwires with 4 types of elastomeric modules and preformed 0.09-inch ss ligatures. they found that the damon sl ii (sds ormco, glendora, california, usa) self - ligating bracket and unligated conventional ss bracket produced negligible f with any of the round wires tested, but coupled with 0.0170.025-inch and 0.0190.025-inch ss and 0.0190.025-inch tma archwires, the damon sl ii bracket produced the highest f (as in this study), while the ss ligatures produced the lowest. as recently reported in a systematic review, there is not enough evidence to claim that with large rectangular wires, in the presence of tipping and/or torque and in arches with considerable malocclusion, sl brackets produce lower f compared to conventional brackets.21 the primary finding of this study is that self - ligating brackets with different slot designs show different behaviors in terms of f generated at the bracket - archwire interface when coupled with rectangular or round archwires. in this study, slide ligatures showed a similar behavior as the conventional ligatures when coupled with all the rectangular archwires (table 2 and figure 1). when coupled with round archwires, they showed significantly lower f than victory series and significantly higher f than damon mx and vision lp brackets, but similar f as the time3 brackets. when coupled with rectangular archwires, they showed significantly lower f than damon mx and time3 brackets, but similar f as the vision lp brackets. this behavior is probably associated with their design ; their elastic properties decrease when coupled with thicker archwires, resulting in low f. in contrast, self - ligating brackets have a built - in metal device (to close off the edgewise slot) that is rigid and rather stiff, compared to the soft and elastic surface of low - friction ligatures. our findings are not in agreement with the general statement that self - ligating brackets generate lower f than conventional ss brackets,2,9,1415,1718,22 probably because a variety of different self - ligating brackets were tested, each with different mechanical characteristics:19 passive or interactive slots. the differences among the passive and interactive sl brackets could be explained by the flexibility of the spring clip of active sl brackets that can actively engage the wire also in the presence of tipping.21 in addition, in this study, no significant differences were observed between the tma, ss, and niti archwires in terms of f, although it was reported that tma generates higher f than both ss and niti for all bracket - archwire combinations.2,2330 this variability may be due to the differences in the experimental setup, number of brackets, or bracket angulations.31 therefore, a direct comparison of various published studies on this topic is complex. one limitation of this study is that it was carried out under ideal conditions, in a passive configuration with no misalignment of brackets, as shown by previous reports.2,9,17,20,21,2425,2930 self - ligating brackets appear to be a family of very different brackets. for majority of the bracket - archwire combinations, the f values obtained with all 10 brackets ligated were significantly higher than those obtained with only the terminal brackets ligated ; no significant differences were observed in only 4 cases, i.e., when damon mx and vision lp brackets were engaged with round archwires. coupled with round archwires, damon mx and vision lp brackets generated significantly lower f than victory series, time3, and slide ligature brackets, while victory series brackets generated significantly higher f than all the other groups. coupled with rectangular archwires, victory series brackets, slide ligatures, and vision lp self - ligating brackets generated significantly lower f than time3 and damon mx brackets. however, as these findings do not actually reflect the situation in clinical use wherein the periodontal ligament provides give, the possibility of making any clinical extrapolation from this study is limited. | objectives : this in vitro study evaluated the friction (f) generated by aligned stainless steel (ss) conventional brackets, self - ligating damon mx brackets (sds ormco, glendora, california, usa), time3 brackets (american orthodontics, sheboygan, wisconsin, usa), vision lp brackets (american orthodontics), and low - friction slide ligatures (leone, firenze, italy) coupled with various ss, nickel - titanium (niti), and beta - titanium (tma) archwires.methods:all brackets had a 0.022-inch slot, and the orthodontic archwires were 0.014-inch, 0.016-inch, 0.0140.025-inch, 0.0180.025-inch, and 0.0190.025-inch niti ; 0.0170.025-inch tma ; and 0.0190.025-inch ss. each bracket - archwire combination was tested 10 times. in the test, 10 brackets of the same group were mounted in alignment on a metal bar. the archwires moved through all the 10 brackets at a crosshead speed of 0.5 mm / min (each run lasted approximately 5 min). the differences among 5 groups of brackets were analyzed through the kruskal - wallis test, and a mann - whitney test was calculated as post hoc analysis. the p value was set at 0.05.results:coupled with 0.014-inch niti and 0.016-inch niti, victory series brackets generated the greatest f, while damon mx and vision lp brackets generated the lowest (p<.05) ; no significant differences were observed between time3 brackets and slide ligatures. coupled with all the rectangular archwires, victory series brackets, slide ligatures, and vision lp self - ligating brackets generated significantly lower f than did time3 and damon mx self - ligating brackets (p<.05).conclusions : these findings suggest that self - ligating brackets are a family of brackets that, in vitro, can generate different levels of f when coupled with thin or thick, rectangular, or round archwires. clinical conclusions based on our results are not possible due to the limitations of the experimental conditions. |
although ischemic stroke occurs mainly in elderly individuals aged 65 years or older, nowadays, there are growing evidences of an increasing trend of childhood ischemic stroke (1, 2). childhood ischemic stroke is generally considered as a rare and benign occurrence, with an incidence of at least 3.3/100,000 (3). however, childhood ischemic stroke is increasingly recognized as an important cause of disability and lifelong morbidity and it is among the top 10 causes of death in children (2). reperfusion following ischemia causes a wide range of pathophysiological process that leads to further damage, and the process may be defined as ischemia / reperfusion injury (4, 5). limited oxygen availability is associated with impaired endothelial cell barrier function (6) and a concomitant increase in vascular permeability and leakage (7). in addition, ischemia / reperfusion leads to the activation of cell death programs, including apoptosis, autophagy - associated cell death and necrosis (8). some studies using ischemic animal models have shown that young animals are resistant to ischemia / reperfusion injury (9, 10). recently, we also compared neuronal damage in the ischemic ca1 region between the young and adult gerbils after 5 min of transient cerebral ischemia / reperfusion and showed that the neuronal death in the hippocampal ca1 region of the young was more delayed and less than that in the adult (11 - 13). however, the precise mechanisms of the more delayed neuronal death in the young remain unclear. glucose is a major energy substrate of energy metabolism for the central nervous system (cns). neurons in adult brains have a higher energy demand a continuous supply of glucose from blood (14). glucose transporters (gluts) play critical roles in regulating glucose transportation and controlling the level of glucose in the brain (15, 16). many studies have investigated that glut-1 is highly enriched in the endothelial cells of the blood brain barrier (bbb) (17 - 19). a disruption of glucose uptake and utilization in the brain is anticipated to cause negative effects on the function and survival of the brain cells. it has been reported that the alteration of glut-1 is closely related with the pathogenesis of cerebral edema (20). glut-1 expression increases in response to focal and global ischemia in the adult rat brain (21, 22). however, few studies regarding glut-1 changes in ischemic brains between the young and adult have been demonstrated. therefore, in the present study, we compared chronological change of glut-1 immunoreactivity in the hippocampus of the young gerbil with that in the adult following 5 min of transient cerebral ischemia reperfusion. we used male mongolian gerbils (meriones unguiculatus) obtained from the experimental animal center, kangwon national university, chunchon, south korea. gerbils were used at 1 (bw 25 - 30 g) and 6 months (bw 65 - 75 g) of age for the young and adult group. experimental protocols were approved by the institutional animal care and use committee (iacuc) at kangwon university and adhered to guidelines that are in compliance with the current international laws and policies (guide for the care and use of laboratory animals, the national academies press, 8th ed., 2011). the animals were divided into four groups : adult sham - operated - group, young sham - operated - group, adult ischemia - operated - group, and young ischemia - operated - group (n = 7 at each point in time in each group). transient cerebral ischemia was developed according to a previously published method by us (13). in brief, the animals were anesthetized with a mixture of 2.5% isoflurane in 33% oxygen and 67% nitrous oxide. the body (rectal) temperature under free - regulating or normothermic (370.5 c) conditions was monitored with a rectal temperature probe (tr-100 ; fine science tools, foster city, ca) and maintained using a thermometric blanket before, during and after the surgery until the animals completely recovered from anesthesia. sham - operated animals were subjected to the same surgical procedures except that the common carotid arteries were not occluded. as described previously method (13), in brief, gerbils (n= 7 at each point in time) in each group were sacrificed under anesthesia at designated times (1, 2, 4, 7 and 15 days after reperfusion) and perfused transcardially with 0.1 m phosphate - buffered saline (pbs, ph 7.4) followed by 4% paraformaldehyde in 0.1 m phosphate - buffer (pb, ph 7.4). the brains were serially sectioned into 30 m coronal sections on a cryostat (leica, wetzlar, germany). to investigate neuronal death in the hippocampus after ischemia - reperfusion, cv staining for normal cells and f - j b histofluorescence for dying or dead cells were performed. as described (13), in brief, the sections were stained with 1.0% (w / v) cresyl violet acetate (sigma aldrich, st. louis, mo, usa) and dehydrated by immersing in serial ethanol bath. for f - j b histofluorescence, the sections were immersed in a 0.0004% f - j b (histochem, jefferson, ar, usa) staining solution and examined using an epifluorescent microscope (carl zeiss, gttingen, germany) with blue (450 - 490 nm) excitation light and a barrier filter. immunohistochemistry was carried out as described (13). in brief, the brain sections were blocked with 10% normal goat serum in 0.05 m pbs followed by staining with primary mouse anti - neun (a neuron - specific soluble nuclear antigen) (diluted 1:1,000, chemicon international, temecula, ca, usa) and rabbit anti - glut-1 (diluted 1:200, santa cruz biotechnology, santa cruz, ca, usa) overnight at 4 c. burlingame, ca, usa) and were developed using vectastain abc (vector laboratories inc.). and they were visualized with 3,3-diaminobenzidine in 0.1 m tris - hcl buffer. in order to establish the specificity of the immunostaining, a negative control test was carried out with pre - immune serum instead of primary antibody. as applied (13), the sections were selected according to anatomical landmarks corresponding to ap from 1.4 to 1.8 mm of gerbil brain atlas. the number of neun - immunoreactive and f - j b - positive cells was counted in a 250250 m square, applied approximately at the center of the stratum pyramidale (sp) of the hippocampal ca1 region. cell counts were obtained by averaging the total cell numbers from each animal per group : a ratio of the count was calibrated as %. in order to quantitatively analyze glut-1 immunoreactivity, as described (23), briefly, the density of glut-1-immunoreactive structures was evaluated on the basis of a relative optical density (rod), which was obtained after the transformation of the mean gray level using the formula : rod = log (256/mean gray level). a ratio of the rod was calibrated as %, with the sham - group designated as 100 %. the data were evaluated by a tukey test for post - hoc multiple comparisons following one - way anova. we used male mongolian gerbils (meriones unguiculatus) obtained from the experimental animal center, kangwon national university, chunchon, south korea. gerbils were used at 1 (bw 25 - 30 g) and 6 months (bw 65 - 75 g) of age for the young and adult group. experimental protocols were approved by the institutional animal care and use committee (iacuc) at kangwon university and adhered to guidelines that are in compliance with the current international laws and policies (guide for the care and use of laboratory animals, the national academies press, 8th ed., 2011). the animals were divided into four groups : adult sham - operated - group, young sham - operated - group, adult ischemia - operated - group, and young ischemia - operated - group (n = 7 at each point in time in each group). transient cerebral ischemia was developed according to a previously published method by us (13). in brief, the animals were anesthetized with a mixture of 2.5% isoflurane in 33% oxygen and 67% nitrous oxide. the body (rectal) temperature under free - regulating or normothermic (370.5 c) conditions was monitored with a rectal temperature probe (tr-100 ; fine science tools, foster city, ca) and maintained using a thermometric blanket before, during and after the surgery until the animals completely recovered from anesthesia. sham - operated animals were subjected to the same surgical procedures except that the common carotid arteries were not occluded. as described previously method (13), in brief, gerbils (n= 7 at each point in time) in each group were sacrificed under anesthesia at designated times (1, 2, 4, 7 and 15 days after reperfusion) and perfused transcardially with 0.1 m phosphate - buffered saline (pbs, ph 7.4) followed by 4% paraformaldehyde in 0.1 m phosphate - buffer (pb, ph 7.4). the brains were serially sectioned into 30 m coronal sections on a cryostat (leica, wetzlar, germany). to investigate neuronal death in the hippocampus after ischemia - reperfusion, cv staining for normal cells and f - j b histofluorescence for dying or dead cells were performed. as described (13), in brief, the sections were stained with 1.0% (w / v) cresyl violet acetate (sigma aldrich, st. louis, mo, usa) and dehydrated by immersing in serial ethanol bath. for f - j b histofluorescence, the sections were immersed in a 0.0004% f - j b (histochem, jefferson, ar, usa) staining solution and examined using an epifluorescent microscope (carl zeiss, gttingen, germany) with blue (450 - 490 nm) excitation light and a barrier filter. immunohistochemistry was carried out as described (13). in brief, the brain sections were blocked with 10% normal goat serum in 0.05 m pbs followed by staining with primary mouse anti - neun (a neuron - specific soluble nuclear antigen) (diluted 1:1,000, chemicon international, temecula, ca, usa) and rabbit anti - glut-1 (diluted 1:200, santa cruz biotechnology, santa cruz, ca, usa) overnight at 4 c. burlingame, ca, usa) and were developed using vectastain abc (vector laboratories inc.). and they were visualized with 3,3-diaminobenzidine in 0.1 m tris - hcl buffer. in order to establish the specificity of the immunostaining, a negative control test was carried out with pre - immune serum instead of primary antibody. as applied (13), the sections were selected according to anatomical landmarks corresponding to ap from 1.4 to 1.8 mm of gerbil brain atlas. the number of neun - immunoreactive and f - j b - positive cells was counted in a 250250 m square, applied approximately at the center of the stratum pyramidale (sp) of the hippocampal ca1 region. cell counts were obtained by averaging the total cell numbers from each animal per group : a ratio of the count was calibrated as %. in order to quantitatively analyze glut-1 immunoreactivity, as described (23), briefly, the density of glut-1-immunoreactive structures was evaluated on the basis of a relative optical density (rod), which was obtained after the transformation of the mean gray level using the formula : rod = log (256/mean gray level). a ratio of the rod was calibrated as %, with the sham - group designated as 100 %. the data were evaluated by a tukey test for post - hoc multiple comparisons following one - way anova. in the adult and young sham - operated - groups, cv+ cells were well distributed in the hippocampus (figures 1a, 1a, 1b and 1b). in the adult ischemia - operated - groups, a significant loss of cv+ cells was observed in the stratum pyramidale (sp) of the ca1 region, not the other subregions, 4 days after ischemia / reperfusion (figures 1c and 1c). thereafter, the distribution pattern of cv+ cells in the sp of the ca1 region was similar to that at 4 days post - ischemia (figures 1e, 1e, 1 g and 1 g). cv staining in the hippocampus of the adult (left two columns) and young (right two columns) sham- (a, a, b and b) and ischemia - operated- (c h and c h) groups. in the adult ischemia - operated - groups, cv cells are damaegd in the stratum pyramidale (sp) from 4 days after ischemia / reperfusion (asterisk). however, in the young ischemia - operated - groups, cv cells are damaged from 7 days post - ischemia (asterisk). scale bar = 800 (a h) and 50 (a h) m in the young ischemia - operated - groups, the distribution pattern of cv+ cells in the sp of the ca1 region was not changed 4 days after ischemia / reperfusion (figures 1d and 1d). however, at 7 days post - ischemia, the morphological damage of cv+ cells was shown in the sp of the ca1 region (figures 1f and 1f), thereafter, similar damage was found in the sp (figures 1h and 1h). in the adult sham - group, pyramidal neurons in the ca1 region were well immuno - stained with neun and no f - j b+ pyramidal neurons were found (table 1, figures 2a and 2a). in the young sham - group, the distribution pattern of neun+ and f - j b+ neurons in the ca1 region was similar to that in the adult sham - group (table 1, figures 2b and 2b). changes in the mean average number of pyramidal neurons of the ischemic hippocampal ca1 region in the adult and young gerbils the mean number of neun - immunoreactive neurons and f - j b - positive cells was counted in a 250 x 250 m square of the stratum pyramidale of the ca1 region after ischemia - reperfusion (i - r) (n=7 per group ; p<0.05, significantly different from the corresponding sham - group ; p<0.05, significantly different from the respective pre - time point group ; p<0.05, significantly different from the corresponding adult - group) neun immunohistochemistry and f - j b histofluorescence staining in the ca1 region of the adult (left two columns) and young (right two columns) sham- (a, a, b, b) and ischemia - operated- (c h and c h) groups. in the adult ischemia - operated - groups, a few neun and many f - j b cells (asterisk) are shown in the stratum pyramidale (sp) from 4 days post - ischemia ; however, neun cells are significantly decreased and f - j b cells (asterisk) are observed in the young ischemia - operated - groups from 7 days post - ischemia. so, stratum oriens ; sr, stratum radiatum. scale bar = 50 m in the adult ischemia - operated - groups, a signi - ficant loss of neun+ neurons (about 90% of the adult sham - group) was observed in the sp of the ca1 region at 4 days post - ischemia (table 1, figure 2c), and, at this point in time, many f - j b+ cells were observed in the sp of the ca1 region (table 1, figure 2c). thereafter, the distribution pattern of neun+ and f - j b+ pyramidal neurons in the ischemic ca1 region was similar to that at 4 days post - ischemia (table 1, figures 2e, 2e, 2 g and 2 g). in the young ischemia - operated - groups, neun+ pyramidal neurons in the ca1 region at 4 days post - ischemia were similar to those in the sham - group (table 1, figure 2d), and no f - j b+ cells were detected in the sp (table 1, fig. however, at 7 days post - ischemia, neun+ neurons were significantly decreased (about 52 % of the sham - group) in the sp of the ca1 region (table 1, figure 2f), and, at this point in time, many f - j b+ cells were found in the sp (table 1, figure 2f). thereafter, distribution patterns of neun+ and f - j b+ pyramidal neurons were similar to those at 7 days post - ischemia (table 1, figures 2h and 2h). ca1 region : glut-1-immunoreactive micro - vessels in the adult sham - group were easily observed in all layers of the ca1 region (figure 3a). immunohistochemistry for glut-1 in the ca1 region of the adult (upper column) and young (lower column) of the sham- (a and g) and ischemia - operated- (b - f, h - l) groups. glut-1-immunoreactive microvessels (arrows) in the young sham - group are higher in density than to those in the adult sham - group. in the ischemia - operated - groups, the density of glut-1-immunoreactive microvessels is distinctively decreased at 1 and 4 days post - ischemia, respectively, in the adult and young, and, thereafter, the density in both groups is gradually increased. so, stratum oriens ; sp, stratum pyramidale ; sr, stratum radiatum. scale bar= 50 m. m : rod as % of glut-1-immunoreactive structures in the adult and young groups (n= 7 per group ; p<0.05, significantly different from the young sham - group, p<0.05, significantly different from the respective pre - time point group ; p<0.05, significantly different from the corresponding adult ischemia - group). the bars indicate the means sem in the young sham - group, the relative optical density (rod) of glut-1-immunoreactive microvessels in the ca1 region was significantly higher (about 56% of the adult sham - group) than that in the adult sham - group (figures 3b and 3 m). in the adult ischemia - operated - groups, the rod of glut-1-immunoreactive microvessels in the ca1 region was significantly decreased (about 35% of the adult sham - group) 1 day after ischemia / reperfusion compared with that in the adult sham - group (figures 3b and 3 m). at 2 days post - ischemia, the rod of glut-1-immunoreactive microvessels was recovered to the rod of the sham - group, thereafter, the rod of glut-1-immunoreactive microvessels was gradually increased with time after ischemia / reperfusion (figures 3c3f and 3 m). in the young ischemia - operated - groups, the rod of glut-1-immunoreactive microvessels in the ca1 region was not changed until 2 days post - ischemia (figures 3h, 3i and 3 m). four days after ischemia - reperfusion, the rod of glut-1-immunoreactive microvessels was decreased (about 20% of the young sham - group) (figures 3j and 3 m), thereafter, the rod of glut-1-immunoreactive microvessels in the ca1 region was increased with time (figures 3k, 3l and 3 m). ca2/3 region : the rod of glut-1-immuno - reactive microvessels in the ca2/3 region of the adult sham - group was similar to that in the ca1 region (figure 4a). in the young sham - group, the rod of glut-1-immunoreactive microvessels was also higher (about 24% of the adult sham - group) than that in the adult sham - group (figures 4 g and 4 m). in the adult ischemia - groups, the rod of glut-1-immunoreactive microvessels in the ca2/3 region as not changed after ischemia / reperfusion (figures 4b4f and 4 m). in addition, the pattern of glut-1 immunoreactivity in the young - ischemia groups was not changed after ischemia / reperfusion (figures 4h4l and 4 m). immunohistochemistry for glut-1 in the ca2/3 region of the adult (upper column) and young (lower column) groups of the sham- (a and g) and ischemia - operated- (b - f, h - l) groups. glut-1-immunoreactive microvessels (arrows) in the young sham - group are is higher in density than to those in the adult sham - group. in both ischemia - operated - groups, distribution patterns of glut-1-immunoreactive microvessels in the ca2/3 region so, stratum oriens ; sp, stratum pyramidale ; sr, stratum radiatum. scale bar = 50 m. m : rod as % of glut-1-immunoreactive structures in the adult and young groups (n= 7 per group ; p < 0.05, significantly different from the young sham - group). in the adult and young sham - operated - groups, cv+ cells were well distributed in the hippocampus (figures 1a, 1a, 1b and 1b). in the adult ischemia - operated - groups, a significant loss of cv+ cells was observed in the stratum pyramidale (sp) of the ca1 region, not the other subregions, 4 days after ischemia / reperfusion (figures 1c and 1c). thereafter, the distribution pattern of cv+ cells in the sp of the ca1 region was similar to that at 4 days post - ischemia (figures 1e, 1e, 1 g and 1 g). cv staining in the hippocampus of the adult (left two columns) and young (right two columns) sham- (a, a, b and b) and ischemia - operated- (c h and c h) groups. in the adult ischemia - operated - groups, cv cells are damaegd in the stratum pyramidale (sp) from 4 days after ischemia / reperfusion (asterisk). however, in the young ischemia - operated - groups, cv cells are damaged from 7 days post - ischemia (asterisk). scale bar = 800 (a h) and 50 (a h) m in the young ischemia - operated - groups, the distribution pattern of cv+ cells in the sp of the ca1 region was not changed 4 days after ischemia / reperfusion (figures 1d and 1d). however, at 7 days post - ischemia, the morphological damage of cv+ cells was shown in the sp of the ca1 region (figures 1f and 1f), thereafter, similar damage was found in the sp (figures 1h and 1h). in the adult sham - group, pyramidal neurons in the ca1 region were well immuno - stained with neun and no f - j b+ pyramidal neurons were found (table 1, figures 2a and 2a). in the young sham - group, the distribution pattern of neun+ and f - j b+ neurons in the ca1 region was similar to that in the adult sham - group (table 1, figures 2b and 2b). changes in the mean average number of pyramidal neurons of the ischemic hippocampal ca1 region in the adult and young gerbils the mean number of neun - immunoreactive neurons and f - j b - positive cells was counted in a 250 x 250 m square of the stratum pyramidale of the ca1 region after ischemia - reperfusion (i - r) (n=7 per group ; p<0.05, significantly different from the corresponding sham - group ; p<0.05, significantly different from the respective pre - time point group ; p<0.05, significantly different from the corresponding adult - group) neun immunohistochemistry and f - j b histofluorescence staining in the ca1 region of the adult (left two columns) and young (right two columns) sham- (a, a, b, b) and ischemia - operated- (c h and c h) groups. in the adult ischemia - operated - groups, a few neun and many f - j b cells (asterisk) are shown in the stratum pyramidale (sp) from 4 days post - ischemia ; however, neun cells are significantly decreased and f - j b cells (asterisk) are observed in the young ischemia - operated - groups from 7 days post - ischemia. so, stratum oriens ; sr, stratum radiatum. scale bar = 50 m in the adult ischemia - operated - groups, a signi - ficant loss of neun+ neurons (about 90% of the adult sham - group) was observed in the sp of the ca1 region at 4 days post - ischemia (table 1, figure 2c), and, at this point in time, many f - j b+ cells were observed in the sp of the ca1 region (table 1, figure 2c). thereafter, the distribution pattern of neun+ and f - j b+ pyramidal neurons in the ischemic ca1 region was similar to that at 4 days post - ischemia (table 1, figures 2e, 2e, 2 g and 2 g). in the young ischemia - operated - groups, neun+ pyramidal neurons in the ca1 region at 4 days post - ischemia were similar to those in the sham - group (table 1, figure 2d), and no f - j b+ cells were detected in the sp (table 1, fig. however, at 7 days post - ischemia, neun+ neurons were significantly decreased (about 52 % of the sham - group) in the sp of the ca1 region (table 1, figure 2f), and, at this point in time, many f - j b+ cells were found in the sp (table 1, figure 2f). thereafter, distribution patterns of neun+ and f - j b+ pyramidal neurons were similar to those at 7 days post - ischemia (table 1, figures 2h and 2h). ca1 region : glut-1-immunoreactive micro - vessels in the adult sham - group were easily observed in all layers of the ca1 region (figure 3a). immunohistochemistry for glut-1 in the ca1 region of the adult (upper column) and young (lower column) of the sham- (a and g) and ischemia - operated- (b - f, h - l) groups. glut-1-immunoreactive microvessels (arrows) in the young sham - group are higher in density than to those in the adult sham - group. in the ischemia - operated - groups, the density of glut-1-immunoreactive microvessels is distinctively decreased at 1 and 4 days post - ischemia, respectively, in the adult and young, and, thereafter, the density in both groups is gradually increased. so, stratum oriens ; sp, stratum pyramidale ; sr, stratum radiatum. scale bar= 50 m. m : rod as % of glut-1-immunoreactive structures in the adult and young groups (n= 7 per group ; p<0.05, significantly different from the young sham - group, p<0.05, significantly different from the respective pre - time point group ; p<0.05, significantly different from the corresponding adult ischemia - group). the bars indicate the means sem in the young sham - group, the relative optical density (rod) of glut-1-immunoreactive microvessels in the ca1 region was significantly higher (about 56% of the adult sham - group) than that in the adult sham - group (figures 3b and 3 m). in the adult ischemia - operated - groups, the rod of glut-1-immunoreactive microvessels in the ca1 region was significantly decreased (about 35% of the adult sham - group) 1 day after ischemia / reperfusion compared with that in the adult sham - group (figures 3b and 3 m). at 2 days post - ischemia, the rod of glut-1-immunoreactive microvessels was recovered to the rod of the sham - group, thereafter, the rod of glut-1-immunoreactive microvessels was gradually increased with time after ischemia / reperfusion (figures 3c3f and 3 m). in the young ischemia - operated - groups, the rod of glut-1-immunoreactive microvessels in the ca1 region was not changed until 2 days post - ischemia (figures 3h, 3i and 3 m). four days after ischemia - reperfusion, the rod of glut-1-immunoreactive microvessels was decreased (about 20% of the young sham - group) (figures 3j and 3 m), thereafter, the rod of glut-1-immunoreactive microvessels in the ca1 region was increased with time (figures 3k, 3l and 3 m). ca2/3 region : the rod of glut-1-immuno - reactive microvessels in the ca2/3 region of the adult sham - group was similar to that in the ca1 region (figure 4a). in the young sham - group, the rod of glut-1-immunoreactive microvessels was also higher (about 24% of the adult sham - group) than that in the adult sham - group (figures 4 g and 4 m). in the adult ischemia - groups, the rod of glut-1-immunoreactive microvessels in the ca2/3 region as not changed after ischemia / reperfusion (figures 4b4f and 4 m). in addition, the pattern of glut-1 immunoreactivity in the young - ischemia groups was not changed after ischemia / reperfusion (figures 4h4l and 4 m). immunohistochemistry for glut-1 in the ca2/3 region of the adult (upper column) and young (lower column) groups of the sham- (a and g) and ischemia - operated- (b - f, h - l) groups. glut-1-immunoreactive microvessels (arrows) in the young sham - group are is higher in density than to those in the adult sham - group. in both ischemia - operated - groups, distribution patterns of glut-1-immunoreactive microvessels in the ca2/3 region are not significantly changed after ischemia - reperfusion. so, stratum oriens ; sp, stratum pyramidale ; sr, stratum radiatum. scale bar = 50 m. m : rod as % of glut-1-immunoreactive structures in the adult and young groups (n= 7 per group ; p < 0.05, significantly different from the young sham - group). the mongolian gerbil has been commonly used for making a good animal model to investigate mechanisms of neuronal death following transient global cerebral ischemia / reperfusion (24, 25), because about 90% of the gerbils lack the communicating arteries between the carotid and vertebral arteries. thus, the bilateral occlusion of the common carotid arteries essentially and completely eliminates blood flow to the telencephalon while completely sparing the vegetative centers of the brain stem (26). among the brain regions, pyramidal neurons of the hippocampal ca1 region are the most vulnerable to transient cerebral ischemic insult (27), and this neuronal death is called delayed neuronal death since the neuronal death occurs very slowly after ischemia / reperfusion (28 - 30). therefore, we have chosen the gerbil as an animal model of transient cerebral ischemia to study this subject. among risk factors for ischemic stroke, age is an important in determining the outcome of cerebral ischemic injury. until now, age - related studies have been focused on neuronal death using adult gerbils (31, 32), and some reports have demonstrated that neuronal death induced by transient cerebral ischemia occurs much later in the aged than in the adult (31 - 33). on the other hand, childhood ischemic stroke is increasingly recognized as an important cause of disability and lifelong morbidity, although cerebral ischemia occurs mainly in the older (2). a previous study showed a greater resistance to various periods of transient cerebral ischemia using from 2 week - old to 12 week - old gerbils (10). some researchers have also reported that young animals are less vulnerable to brain ischemic insult (9, 34), and we recently reported that resistance to cerebral ischemic insults was different according to age ; young and aged gerbils are more resistant to cerebral ischemia than the adult under the same condition (11, 32, 35). in the present study, using cv staining, neun immunohistochemistry and f - j b histofluorescence staining, we observed the neuronal death in the young gerbil was much more delayed and less severe than that in the adult. this result is similar to previous studies that showed that the young gerbil was resistant to ischemic damage (9, 10). in addition, we recently reported that endogenous anti - oxidants and anti - inflammatory cytokines were markedly increased and they might be related with much more delayed and lesser neuronal death in the young gerbil hippocampus following transient cerebral ischemia / reperfusion (13, 36, 37). it is well known that transient cerebral ischemia leads in oxygen - glucose deprivation and energy failure, which is associated with the development of neuronal cell damage / death in the hippocampal ca1 region (38). because glucose is a major source of energy metabolism for the cns, it is essential to keep adequate glucose supply to the brain (14). glut-1 plays a critical role in regulating glucose transportation and controlling the level of glucose in the brain (17, 39), and its expression is modulated in concert with metabolic demand and regional rates of cerebral glucose utilization (16). glut-1 is specifically localized to capillary endothelial cells of the brain (18, 19). several studies have examined effects of global or focal ischemia on brain glut-1 expression in adult animal models (21, 40 - 42). a recent research demonstrated that the expression of glut-1 was increased in the hippocampus and cerebral cortex after ischemia / reperfusion injury in diabetic rats (43). on the other hand, li (44) reporetd that the accumulation of glut-1 induced by progesterone treatment showed neuroprotective effects against cerebral ischemic insults. in the present study, we found that the density of glut-1-immunoreactive microvessels was significantly higher in the young than that in the adult and that, in the ischemia - operated - groups, the density of glut-1-immunoreactive microvessels in the young were changed later and significantly higher than that in the adult. this finding is supported by previous studies that showed that cerebral hypoxia - ischemia significantly increased the expression of glut-1 in the immature rat brain (45, 46). based on these findings, high glut-1 expression seems to supply more glucose for ischemic brain, which may be an effective protection against cerebral ischemic insults. although it was recently reported that hypoxia stimulated glut-1 expression in endothelial cells in vitro and in vivo (47, 48) and that glut-1 contributed to maintaining the integrity of bbb (49), it needs to study the precise mechanism of the ischemia - induced change of glut-1 expression in brain microvessels. our present findings demonstrate that glut-1-immunoreactive microvessels in the hippocampal ca1 region of the young were more than those in the adult and that the density of the microvessels in the young following ischemic insult was significantly increased much later than that in the adult. we suggest that the ischemia - mediated increase of glut-1-immunoreactive microvessels in the young may contribute to less and more delayed neuronal death in the young gerbil. | objective(s):the alteration of glucose transporters is closely related with the pathogenesis of brain edema. we compared neuronal damage / death in the hippocampus between adult and young gerbils following transient cerebral ischemia / reperfusion and changes of glucose transporter-1(glut-1)-immunoreactive microvessels in their ischemic hippocampal ca1 region.materials and methods : transient cerebral ischemia was developed by 5-min occlusion of both common carotid arteries. neuronal damage was examined by cresyl violet staining, neun immunohistochemistry and fluoro - jade b histofluorescence staining and changes in glut-1 expression was carried out by immunohistochemistry.results:about 90% of pyramidal neurons only in the adult ca1 region were damaged after ischemia / reperfusion ; in the young, about 53 % of pyramidal neurons were damaged from 7 days after ischemia / reperfusion. the density of glut-1-immunoreactive microvessels was significantly higher in the young sham - group than that in the adult sham - group. in the ischemia - operated - groups, the density of glut-1-immunoreactive microvessels was significantly decreased in the adult and young at 1 and 4 days post - ischemia, respectively, thereafter, the density of glut-1-immunoreactive microvessels was gradually increased in both groups after ischemia / reperfusion.conclusion : ca1 pyramidal neurons of the young gerbil were damaged much later than that in the adult and that glut-1-immunoreactive microvessels were significantly decreased later in the young. these data indicate that glut-1 might differently contribute to neuronal damage according to age after ischemic insults. |
the incidence rate of community - acquired pneumonia (cap) is estimated to be 5.2 to 6.1 cases per 1000 adults, and the mortality rate may reach 23%. several independent risk factors for pneumonia have been identified, such as increasing age, comorbidities, swallowing dysfunction, and nutritional status. host genetic susceptibility has also been reported to be an important risk factor for pneumonia. toll - like receptors (tlrs) are transmembrane proteins that recognize infection with various pathogens and damaged host cells, which lead to the subsequent inflammation responses. tlr4 can distinguish between lipopolysaccharide and gram - negative bacteria, and can initiate intracellular signal cascades. standiford. showed that tlr4 played a protective role in lung epithelium during gram - negative bacterial pneumonia. tang. found that decreased tlr4 expression occurred in deceased patients compared with survivors. furthermore, tanaka. found that tlr4 agonistic monoclonal antibody ut12 could improve the prognosis of secondary pneumococcal pneumonia. previous studies have found 2 single - nucleotide polymorphisms (snps) of tlr4 : tlr4 a299 g and tlr4 t399i. figueroa. suggested that tlr4 a299 g polymorphism impaired lps - induced phosphorylation of p38 and tank - binding kinase 1, activation of nf-b and ifn regulatory factor 3, and induction of il-8 and ifn- mrna. in addition, long. found that the tlr4 a299 g variant, but not the t399i variant, was responsible for impaired responsiveness of tlr4 to lps and corresponding activation of nf-b. recently, studies assessed the association between tlr4 a299 g polymorphism and the risk of pneumonia [1219 ], but the results were inconclusive and mixed. we thus conducted this meta - analysis to investigate the association between tlr4 a299 g polymorphism and the risk of pneumonia. relevant studies were systematically searched for by using the ncbi, medline, web of science, and embase databases (he last retrieval date was august 29, 2014, using the search terms : pneumonia and toll like receptor 4 or all discovered studies were retrieved and only published studies with full - text articles were included. for publications with duplicates, the inclusion criteria was : (1) the diagnosis of pneumonia was confirmed by x - ray or ct ; (2) the research was a case - control study or a cohort study ; (3) the study focused on the association between the tlr4 a299 g polymorphism and the risk of pneumonia ; and (4) the tlr4 a299 g genotype of individual groups were provided, the exclusion criteria were : (1) pneumonia was not confirmed by x - ray or ct ; (2) animal studies ; and (3) reviews or abstracts. based on the selection criteria, 2 reviewers (xingjun cai and yihui fu) extracted and sorted the data independently (including author, year, ethnicity, age, type, and sample size). authors were contacted by email if further study details are needed. statistical analysis was conducted using stata software 11.0 (statacorp, college station, texas, usa). odds ratio (or) with a 95% confidence interval (ci) was presented for dichotomous data, and significant level was 0.05. q statistic and i statistic were used to measure statistical heterogeneity and significant level was 0.10. a fixed - effects model was selected when no significant heterogeneity, otherwise we used a random - effects model. we performed a cumulative meta - analysis by sequential random - effects pooling, starting with the earliest studies. results were presented as a series of mini meta - analyses, which were ordered chronologically in a forest plot to show the consequence of adding studies on the effect size. to evaluate the effect of individual studies on overall risk of pneumonia, we conducted sensitivity analyses by excluding each study individually and recalculating the ors and 95% ci. funnel plots were performed to estimate potential publication bias, with an asymmetrical plot suggesting possible publication bias. the asymmetry was assessed using the egger s linear regression test and p<0.05 was considered to represent statistically significant publication bias. relevant studies were systematically searched for by using the ncbi, medline, web of science, and embase databases (he last retrieval date was august 29, 2014, using the search terms : pneumonia and toll like receptor 4 or all discovered studies were retrieved and only published studies with full - text articles were included. for publications with duplicates, the inclusion criteria was : (1) the diagnosis of pneumonia was confirmed by x - ray or ct ; (2) the research was a case - control study or a cohort study ; (3) the study focused on the association between the tlr4 a299 g polymorphism and the risk of pneumonia ; and (4) the tlr4 a299 g genotype of individual groups were provided, the exclusion criteria were : (1) pneumonia was not confirmed by x - ray or ct ; (2) animal studies ; and (3) reviews or abstracts. based on the selection criteria, 2 reviewers (xingjun cai and yihui fu) extracted and sorted the data independently (including author, year, ethnicity, age, type, and sample size) statistical analysis was conducted using stata software 11.0 (statacorp, college station, texas, usa). hwe test in the healthy control group was conducted using test. odds ratio (or) with a 95% confidence interval (ci) was presented for dichotomous data, and significant level was 0.05. q statistic and i statistic were used to measure statistical heterogeneity and significant level was 0.10. a fixed - effects model was selected when no significant heterogeneity, otherwise we used a random - effects model. we performed a cumulative meta - analysis by sequential random - effects pooling, starting with the earliest studies. results were presented as a series of mini meta - analyses, which were ordered chronologically in a forest plot to show the consequence of adding studies on the effect size. to evaluate the effect of individual studies on overall risk of pneumonia, we conducted sensitivity analyses by excluding each study individually and recalculating the ors and 95% ci. funnel plots were performed to estimate potential publication bias, with an asymmetrical plot suggesting possible publication bias. the asymmetry was assessed using the egger s linear regression test and p<0.05 was considered to represent statistically significant publication bias. only 1 study used pediatric patients, while 7 studies used adult patients. only 1 study assessed ventilator - associated pneumonia (vap) patients, while other studies used cap patients. a total of 658 patients and 1862 controls were included in our meta - analysis. we investigated the association between tlr4 a299 g polymorphism and pneumonia risk in the recessive models. result of this meta - analysis showed that tlr4 a299 g polymorphism was significantly associated with pneumonia risk (or=1.74 ; 95% ci 1.192.53 ; p=0.004 ; figure 2). when we deleted the pediatric study, the result was not changed (or=1.81 ; 95% ci 1.222.69 ; p=0.003). when the vap study was deleted, the result was also not changed (or=1.67 ; 95% ci 1.062.62 ; p=0.03). to determine the stability of the result, we performed sensitivity analysis by omitting 1 study at a time. we found that single study did not impact the pooled or, indicating that the results of our research are statistically robust (figure 3). cumulative meta - analysis was also conducted by the assortment of studies by publication time (figure 4). there was no publication bias in this meta - analysis, because no funnel plot asymmetry was detected (figure 5). only 1 study used pediatric patients, while 7 studies used adult patients. only 1 study assessed ventilator - associated pneumonia (vap) patients, while other studies used cap patients. a total of 658 patients and 1862 controls were included in our meta - analysis. we investigated the association between tlr4 a299 g polymorphism and pneumonia risk in the recessive models. result of this meta - analysis showed that tlr4 a299 g polymorphism was significantly associated with pneumonia risk (or=1.74 ; 95% ci 1.192.53 ; p=0.004 ; figure 2). when we deleted the pediatric study, the result was not changed (or=1.81 ; 95% ci 1.222.69 ; p=0.003). when the vap study was deleted, the result was also not changed (or=1.67 ; 95% ci 1.062.62 ; p=0.03). to determine the stability of the result, we performed sensitivity analysis by omitting 1 study at a time. we found that single study did not impact the pooled or, indicating that the results of our research are statistically robust (figure 3). cumulative meta - analysis was also conducted by the assortment of studies by publication time (figure 4). there was no publication bias in this meta - analysis, because no funnel plot asymmetry was detected (figure 5). this meta - analysis of 8 case - control studies evaluated the association between tlr4 a299 g polymorphism and the risk of pneumonia. we found that tlr4 a299 g polymorphism might be a risk factor for developing pneumonia. this result suggests that tlr4 299aa genotype carriers might have increased pneumonia risk compared to the ag or gg carriers. in addition, we also found that tlr4 a299 g polymorphism was associated cap risk. since there was only 1 study each with pediatric patients and vap patients, more studies in these populations are needed to validate the result of this meta - analysis. did a meta - analysis and showed that tlr4 a299 g polymorphism increased risk for all parasitic infections, malaria, brucellosis, cutaneous leishmaniasis, neurocysticercosis, streptococcus pyogenes tonsillar disease, typhoid fever, and adult urinary tract infections. however, no study evaluated the association between tlr4 a299 g polymorphism and pneumonia. thus, to the best of our knowledge, this is the first meta - analysis of the association between tlr4 a299 g polymorphism and pneumonia risk. a recent study suggested that deficient recruitment of signaling adapters myd88 and trif to tlr4 was a mechanistic basis for a299g - mediated impairment of tlr4-elicited, lps - induced activation of myd88-dependent il-8 and tnf- genes, deficient trif - dependent phosphorylation of tbk1 and irf3, transactivation of irf3, and expression of ifn- mrna. thus, it is important to pay more attention to the subjects with tlr4 a299 g polymorphism. however, results from cumulative meta - analysis and sensitivity analysis suggest that our results are reliable and robust. secondly, pneumonia is a complex process modulated by a series of genetic factors beyond tlr4. however, our analysis only tried to explore the effect of tlr4 a299 g polymorphism on pneumonia, and did not link other gene variants that may be involved in pathophysiological pathways. therefore, larger clinical studies are required to validate the hypothesis and findings obtained in this study. once validated, they can be very helpful evidence in developing tailored therapeutics for individual patients. the results of this meta - analysis suggest that susceptibility to pneumonia is associated with tlr4 a299 g polymorphism. | backgroundseveral genetic studies have evaluated the association between toll - like receptor 4 (tlr4) a299 g polymorphism and the risk of pneumonia. however, the results were not consistent. we thus did this meta-analysis.material/methodsrelevant studies were systematically searched by using the ncbi, medline, web of science, and embase databases. data were extracted independently by 2 investigators. odds ratios (ors) and corresponding 95% confidence intervals (cis) were estimated.resultseight case - control studies with 658 patients and 1862 controls were included in this meta - analysis. tlr4 a299 g polymorphism was significantly associated with pneumonia risk (or=1.74 ; 95% ci 1.192.53 ; p=0.004). the result was significant in adults. in addition, tlr4 a299 g polymorphism was also associated with community - acquired pneumonia (cap) risk. results from cumulative meta - analysis and sensitivity analysis suggested that the results are reliable and robust.conclusionsthe results of this meta - analysis suggest that susceptibility to pneumonia was associated with tlr4 a299 g polymorphism. |
the world health organization (who) estimates that 21.4 million people suffer from active trachoma of which 7.2 million have blinding trichiasis while 1.2 million people are actually blind [1, 2 ]. the disease is still endemic in egypt, among 53 countries, as reported by the who. this disease is characterized by recurrent attacks of chronic follicular conjunctivitis, progressive conjunctival scarring with subsequent misdirected lashes that, on rubbing against the cornea, is called trichiasis. trachomatous trichiasis (tt) could also be secondary to metaplastic lashes or cicatricial entropion. in addition to being a source of chronic irritation, tt usually causes a threat to the cornea in the form of recurrent ulceration, corneal opacities, and secondary infection that my progress to corneal melting, perforation with loss of the globe. in 1997, the alliance for the global elimination of blinding trachoma by 2020 (get 2020) was founded. it is the strategy of surgical treatment, antibiotic therapy for acute infection, face cleanliness, and environmental changes to improve sanitation. conservative observation is appropriate only for few patients who have trichiasis in the far ends of the eyelid where the lashes are not threatening the cornea. bilamellar tarsal rotation (bltr) and posterior lamellar tarsal rotation (pltr) were the recommended procedures by the who. however, they were not recommended for treating distichiatic or metaplastic lashes as well as cicatricial entropion cases with defective lid closure due to tarsus shortening, irregular lid margin as well as lids with previous entropion surgery. anterior lamellar reposition (alr) with or without mucous membrane grafting was first described by welsh. to correct cicatricial entropion associated with distichiatic cilia yet its use expanded to treating trichiasis and cases of entropion with short or thin tarsus [7, 8 ]. other surgical options include direct excision of the lash bearing area and anterior lamella nearby, grey line splitting, and direct follicle ablation in addition to the less invasive options as electrolysis, epilation, cryotherapy, and laser ablation. although alr is an established treatment modality, reports about its use in tt especially in cases that are not secondary to cicatricial entropion are far less than reports of the other two commonly used procedures. in this work, this is a prospective noncomparative study that took place from february 2009 to november 2013. patients suffering from trachomatous trichiasis or entropion with short or thin tarsus were recruited from kasr al aini and el nour hospital clinics. all affected lids were examined by the slit lamp without distraction to evaluate the lid margin and lash position. conjunctiva and cornea were thoroughly examined for signs of trachomatous affection and fluorescein test was done. patients with 5 or less rubbing lashes or entropion with firm, thick, and long tarsus as well as patients who underwent previous anterior lamellar reposition were excluded. we also excluded patients with nontrachomatous conjunctival scarring, for example, ocular cicatricial pemphigoid or chemical injuries. all cases were requested to stop systemic antiplatelets or anticoagulants, after their physician consultation whenever applicable, prior to surgery. patients who had recently epilated the rubbing lashes were deferred till lashes regrew (at least two weeks). the study and data collection conformed to all local laws and complied with the principles of the declaration of helsinki. the addressed lid was then infiltrated in the submuscular space by mixture of lidocaine hcl 2% and epinephrine 1 : 200.000, and only lidocaine hcl 2% in cardiac or hypertensive patients. a scratch incision was done along the entire lid margin just behind the abnormal lashes using number 15 bard parker scalpel blade (figure 1(a)). undervision dissection was facilitated by asking the assistant to hold the posterior lamella and exert mild traction on the lid while keeping hemostasis. dissection was then continued in the created plane by westcott scissors to reach the proper submuscular space in order to separate the anterior and posterior lamellae as far as the peripheral tarsal edge (figure 1(b)). in cases of dysplastic lashes that replaced the meibomian orifices in the lid margin, the initial incision had to go through the tarsus. dissection was then carried out till the routes were exposed then the plane was changed to separate the two lamellae as other cases. the anterior lamella was recessed (figure 1(c)) as far as possible and three 4/0 silk transverse mattress sutures were taken. each suture passed through the lash line to the highest possible point above the tarsus and through the conjunctiva back to the lash line of the anterior lamella to be tied in square knot. the extra - lash bearing skin at the lid margins as well as visible routes embedded in the tarsus was excised. the bare area of the posterior lamella was left to granulate (figure 1(d)). if both upper and lower lids of the same side were affected, they were operated upon in separate sessions to avoid the possibility of induced ankyloblepharon. all patients received tobramycin / dexamethasone ointment on the lid and intraocular lubricating gel twice per day for ten days. patients completed at least 6 months of follow - up and were evaluated under the slit lamp by 1 week, 3 weeks, 3 months, and 6 months for lid margin position and abnormalities like notching, necrosis, madarosis, or thickening as well as the condition of conjunctiva and cornea. recurrence was evaluated as regards the site and the number of the recurrent lashes. for symptomatic patients, postoperatively, complete success was defined as no rubbing lashes were detected, even if electrolysis was required once or twice during the follow - up period. partial success was defined in 5 lashes or less that did not require further surgical intervention yet needed more than two sessions of electrolysis or laser ablation. failure was considered when recurrent rubbing lashes were 6 or more and required another surgical intervention. data was collected and analyzed where descriptive statistics were calculated and the numerical data were summarized as mean and standard deviation (sd), while categorical data were summarized in tables and percentages (%). this study included 752 eye lids of 445 patients (58.4% females, 41.6% males) with mean age 53.2 6.9 years. all of the included patients reported repeated epilation of lashes while 163 (36.7%) patients reported previous electrolysis. associated entropion with thin tarsus was detected in 179 lids (25.1%) and 48.5% of lids had underwent previous surgery (bltr). all patients had signs of trachoma in the form of pannus siccus, subconjunctival fibrosis, and/or ptds. corneal affection was found in 287 patients (64.5%) in the form of nebula or leucoma either localized or diffuse ; 89% of these patients (56.8% of the total sample) had bilateral corneal affection. the distribution of number of affected lids per included patients is demonstrated in figure 2. all lids showed marginal thickening by the 3rd week (figure 3(a)) that softened over time and disappeared by the 3rd month (figure 3(b)). the lid had an abnormal appearance in all patients that was present till the time of suture removal by the 2nd week. the lash line migrated back to normal position by the 4th week ; however, this abnormal lid appearance persisted in 2.66% of lids (figure 3(c)). none of the operated lids showed marginal ischemia or additional corneal lesions in the follow - up period yet localized madarosis developed in 5.72% (figure 3(d)). complete success was recorded in 66.09% (95% confidence interval ci = 0.620.69) while partial success was recorded in 19.01% (95% ci = 0.160.21) making the overall success 85.1% (ci = 0.830.88). an overall success rate of 83.8% was also obtained in cases which had previous lid surgery. the flow chart in figure 4 summarizes the outcome numbers throughout the follow - up period as well as any required interventions while the percentages are further illustrated in figure 5. by the 3rd week, 270 eyelids required electrolysis with 68.1% cure rate. however, by the 3rd month 110 lids required electrolysis of which 80 lids received their first electrolysis during the follow - up period with 68.8% total cure. reappearance of lashes in preoperative sites was found in 45% of lids yet the remaining 55% had lashes that appeared in previously lash - free areas. the numbers of lids that received electrolysis along the follow - up period as well as the cure rates are shown in table 1. by the end of the follow - up period, 143 lids (19.01% of the whole sample) had recurrence of 5 lashes and were considered partial success as none of these lids was enrolled for further surgeries. sixty - four lids (45.5%) of these recurrences received electrolysis while the rest of cases had the recurrence in the far ends of the lid with no corneal threatening and patients preferred to frequently epilate the recurrent lashes. recurrence of 6 lashes was reported in 112 lids (14.9%) (ci : 0.130.17) and was considered to be failure being in need for another surgery. alr was repeated in 87 lids (77.7% of 112 lids) while 10 lids (8.9%) required another surgical procedure mainly grey line splitting with excision of lash bearing area. ten patients (15 lids, i.e., 13.4% of failed cases) deferred surgical intervention. trichiasis is a painful irritating disease that is usually secondary to conjunctival cicatrizing disorder with subsequent threatening to both vision and globe integrity. surgical treatment of tt is a key component of the safe strategy supported by the who for combating trachoma with direct relation to reducing blindness. bltr and ptlr are the recommended procedures for treating cases associated with cicatricial entropion via horizontal tarsotomy and everting sutures to rotate the distal end of the lid yet this concept is not effective in cases of trichiasis or distichiasis without entropion. grey line splitting with anterior lamellar repositioning is an established procedure for treating cicatricial entropion with reported success rate between 75 and 97% [11, 12 ]. according to the preset definitions in the current study, complete success was achieved in 66.09% of the operated cases while the overall success rate by the end of the follow - up period was 85.1% including complete and partial success. reported the anatomic success rate of 62.5% and functional rate of 75% in their series of 24 lids (ci : 0.440.82). elder and collin reported anatomical success of 71% in their series of alr in 16 lids (ci : 0.490.93) with ocular cicatricial pemphigoid (ocp) and complete success of 61%. koreen. reported 77% success rate for primary repair in their sample of 35 eyelids (ci : 0.630.91) ; however, their sample included various causes of conjunctival cicatrization. sodhi. reported success rate of 88.4% in their study of 84 eyelids (ci : 0.820.95). on the other hand, some studies reported higher success rates. kemp and collin reported an overall success rate of 90.7% in their 183 lids series (ci : 0.880.95). a similar success rate was also reported by choi. in their series that included 30 lower lids with cicatricial entropion. the rate rose to 97% in hintschich 's study of 34 eyelids (ci : 0.911.03). the large sample included in this series compared to the published studies, the additional excision of extra - lash bearing area, the margin of defining success, and the different follow - up periods could explain these different outcomes. the intersurgeon variability was not considered in the current study as both authors received similar surgical training and they adopted the same surgical technique. however, intersurgeon variability was believed to be an important factor for the outcome by both emerson. and rajak.. hence, it could be presumed to be a contributing factor in explaining the different outcomes in the current study compared to other studies. additionally, most studies were concerned about cicatricial entropion in comparison to the current work where cicatricial entropion constituted less than quarter of the included cases. trachoma is also a unique disease and many of the above mentioned studies included cases of cicatricial entropion due to other causes. high success rate (83.8%) was reported in cases with history of previous bltr. similar results were reported by sodhi. who reported a success rate of 97% of the lids (ss : 66) as a secondary procedure after failed tarsus rotation. the underlying etiology of cicatricial entropion was found to be the major risk factor that significantly influences the surgical outcome of alr and higher failure rates were associated with infective causes. the severity of the preoperative trichiasis was also found to be a major risk factor for recurrence contrary to presence of preoperative entropion that was considered an independent risk factor. studied the 4-year overall recurrence of trichiasis and reported 41% recurrence ; three/4 of which occurred in the first 6 months. they referred its causes to disease severity, surgical factors, and the wound healing course. in the current study, 69.14% had residual rubbing lashes by the 3rd week of follow - up mostly 1 - 2 lashes (61.03%). although using the surgical microscope is of utmost importance in this procedure for better visualization, early recurring lashes were actually missed in the primary surgery either because they were fine and nonpigmented, hence were overlooked, or because the roots of metaplastic lashes embedded in the tarsus were not completely excised. some patients had epilated irritating lashes prior to surgery, even if instructed not to, thus contributing to postoperative lash regrowth. the residual lashes < 6 reduced over the follow - up period due to the adjuvant electrolysis. however, by the end of the 6 months, the actual rate of recurrence was 14.9% with lashes either in the original or in the new places and they were candidates for another surgical intervention mainly repeating alr. a similar recurrence rate (11%) was reported in koreen. study. this recurrence rate is also comparable to bltr procedure (7.4 to 63%) [10, 19 ] and ptlr (12% to 55%). in their retrospective study, barr. found that the recurrence rates for tt treated by both bltr and alr showed no statistically significant difference. however, they found that in cases that had equal follow - up periods for both procedures, recurrence in alr group is less. madarosis, persistent abnormal lid appearance, and recurrence are the common complications associated with alr while overcorrection, granuloma formation, and notching ischemia of the lid margin and defective lid closure have also been more reported with tarsotomy in bltr and pltr [18, 21 ]. although this work is a short term follow - up yet it should be noted that late recurrence is suggested to be due to an ongoing scarring process. west el al. reported 7.6% recurrence rate in one year compared to 2.3% at 6 weeks in their study in southern ethiopia. this late recurrence is linked to inflammatory mediators il-1b and genetic susceptibility bacterial reinfection while tumor necrosis factor tnf was linked to scarring. grey line splitting with anterior lamella repositioning has various modifications with tarsus fracture, wedge resection, putting a tarsus substitute, and use of everting sutures as well as use of mucous membrane to cover the bare tarsus. anterior lamellar reposition can also be carried out via skin crease incision in the upper lid. we believe that splitting starting from the lid margin provides more controlled placement of the incision behind the aberrant lash line before separating the two lamellae. leaving the bare posterior lamella to granulate, though it gave the patient an odd appearance, provided time for the granulation tissue to cover the lash bearing area before the anterior lamella migrated back to its original place,. persistence of the abnormal lid appearance was minimal and was due to delayed suture removal for these patients who were from border areas. in conclusion, anterior lamellar recession without mucous membrane graft is a good option for treating trachomatous trichiasis especially in the absence of associated cicatricial entropion with good functional and cosmetic outcomes. proper placement of the incision behind the aberrant lashes, visualizing their roots with excision of extra - lash bearing area, is believed to be of utmost importance in preventing recurrence. however, at least one adjunctive lash electrolysis or laser ablation session is usually required postoperatively. further studies are required to establish the factors affecting the outcome and to validate the value of excision of lash bearing area as well as comparing the outcome according to the surgical approach whether via skin crease or starting at the lid margin. studies with longer follow - up periods are also recommended to evaluate the long lasting effect of this procedure. | purpose. to evaluate the outcome of anterior lamellar reposition (alr) in treating trachomatous trichiasis. methods. patients with trachomatous trichiasis or entropion with short tarsus were treated by alr between february 2009 and november 2013. this included splitting of the lid margin behind the aberrant lash line to separate the lid lamellae. the anterior lamella was recessed and fixated using 4/0 silk sutures. the extra lashes and their routes were excised. sutures were removed by the 3rd week and patients completed 6 months of follow - up. recurrence of 5 lashes was treated by electrolysis. results. the study included 752 eyelids (445 patients ; 58.4% females, 41.6% males), mean age 53.2 6.9 y. 179 (25.1%) lids had entropion while 287 (64.5%) patients had corneal affection. by the third week, 2.66% lid had trichiasis while 30.8% had no rubbing lashes. by the 6th month, 14.9% of lids showed recurrence while 66.1% were completely cured (ci = 0.630.69) and 19% had partial success (ci = 0.160.21). abnormal lid appearance persisted in 2.66% and 12.9% required another surgery. conclusion. alr is a good option for treating trachomatous trichiasis especially without cicatricial entropion. excision of dysplastic lashes is thought to augment the surgical outcome. |
deoxycytidine kinase (dck) is a deoxyribonucleoside kinase capable of phosphorylating deoxycytidine, deoxyadenosine, and deoxyguanosine to their monophosphate forms using either atp or utp as phosphoryl donors. phosphorylation by dck is responsible for converting salvaged deoxycytidine into deoxycytidine monophosphate (dcmp), a precursor for both dctp and dttp pools. apart from the physiological role of generating dntps, dck plays a crucial role in activating multiple nucleoside analog prodrugs that are widely used in anticancer and antiviral therapy. recently, we and others identified a requirement for dck in hematopoiesis in lymphoid and erythroid progenitors. the kinase has also been implicated in regulating the g2/m transition in response to dna damage in cancer cells. more recently, we have shown that partial inhibition of dck activity, combined with perturbations of nucleotide de novo synthesis pathways, was synthetically lethal to acute lymphoblastic leukemia cells but not to normal hematopoietic cells. these aspects of dck s biology, and its potential role as a new therapeutic target in cancer, prompted us to develop small molecule inhibitors of its enzymatic activity. in earlier publications we reported the discovery of hit compounds from a high throughput screen and subsequent optimization of the molecules to lead compounds 1 and 2 (numbered 36 and 37, respectively, in ref (8)). lead compounds 1 and 2 can be divided into four distinct structural parts (figure 1a). part a is the pyrimidine ring, which is connected by a linker (part b) to a 5-subsituted - thiazole ring (part c), which in turn is connected to a phenyl ring (part d). conceptually, each of these parts can be modified to attain desired druglike properties. in previous work, we focused on the thiazole portion of the inhibitor. the crystal structure of dck with one of the early compounds suggested that the ring 5-position could accommodate hydrophobic substituents, which led to the discovery that a propyl group at the 5-position is strongly favored over a methyl group. part a indicates the pyrimidine ring, and part b is the linker connecting to a 5-substituted - thiazole ring (part c), which is followed by a phenyl ring (part d). compounds 1 and 2 differ at the substituent present at the phenyl meta position (rm). (b) in vitro (ic50 and ki) and cell (ic50) properties for 1 and 2. to guide and rationalize the medicinal chemistry efforts in other parts of the molecule, we solved the crystal structures of human dck with several of the inhibitors we developed. the crystal structures illuminate the relationship between the enzyme structure, the small molecule structure, and its inhibition potency. in the first part of this manuscript we report the in vitro binding affinities (ic50 and ki), cellular ic50 values, and crystal structures of dck in complex with compounds that differ in the pyrimidine and phenyl rings. unfortunately, despite nanomolar affinity for dck, when tested in a liver microsomal assay, these compounds exhibited low metabolic stability (data not shown). this shortcoming was recapitulated by pharmacokinetic studies in mice. to identify inhibitors with improved in vivo properties, we set out to explore additional chemical modifications, specifically, those that maintain the low nanomolar binding affinity of the lead compounds. in the second part of the manuscript, we report novel chiral derivatives of our inhibitors. crystal structures of these chiral compounds bound to dck played a key role in elucidating the chirality of the active form of the inhibitor. by combining organic chemistry intuition with detailed structural information on the target inhibitor complex, we have identified a lead compound that retains the nanomolar affinity for dck but has gained significant in vivo metabolic stability. this compound could play a vital role in any therapeutic strategy based on induction of dna replication stress overload by perturbing a cancer cell s dntp pools. the pyrimidine ring (part a of the molecules, figure 1a) was predicted to be the part of the molecule most difficult to improve. this is because, as observed in the crystal structures of dck in complex with lead compounds 1 and 2 (pdb codes 4l5b and 4kcg, respectively), the inhibitor s pyrimidine ring binds to dck at a position nearly identical to that adopted by the pyrimidine ring of the physiological substrate dc, making several hydrogen bonds, hydrophobic, and stacking interactions (supporting information figure s1). this binding mode suggested an already quite optimized enzyme pyrimidine ring interaction. for compounds 1 and 2, both pyrimidine ring exocyclic amino groups formed hydrogen - bonding interactions with side chains of glu53, gln97, and asp133. hence, not surprisingly, simultaneous removal of both amino groups resulted in complete loss of dck inhibition. in contrast, removal of a single amino group to generate compound 3 (figure 2a), which is identical to 1 except for having a single exocyclic amino group in the pyrimidine ring (figure 1a), resulted in similarly tight binding affinity as measured for 2 (figures 1b and 2b). to explain how the affinity of 3 for dck is maintained with only a single exocyclic amino group, we sought the crystal structure of the complex, but unfortunately, we were unable to obtain diffraction quality crystals. we speculate that the sole exocyclic amino group present in compound 3 is oriented in the dck active site such that it maintains its interaction with asp133, since only in that orientation can the neighboring pyrimidine ring n atom maintain its interaction with the side chain of gln97 (supporting information figure s1). the conclusion here is that the interaction with glu53 made by an exocyclic amino group, when present, provides only moderate additional binding energy. while a single exocyclic pyrimidine ring amino group is sufficient for a tight interaction with dck, in our cem cell - based assay compound 3 exhibited a much - increased ic50 value (21.8 nm, figure 2b) relative to compound 2 (4.9 nm, figure 1b). this result showcases the importance of evaluating the interaction between an inhibitor and its target in using both an enzymatic in vitro assay and a cell - based assay. because of the reduced inhibition of dck activity of 3 in the cell - based assay, all future compounds contained the two exocyclic amino groups. (a) schematic representation of compound 3 that has a single exocyclic amino group and of compound 4 that has a ring nitrogen atom between the two exocyclic amino groups. (b) in vitro (ic50 and ki) and cell (ic50) properties for 3 and 4. (c) overlay of the dck4 (orange, pdb code 4q18) and dck-1 (green, pdb code 4l5b) structures with a focus on the pyrimidine ring. note the 0.4 shifted position of 4 relative to 1 that is due to the presence of a water molecule (orange sphere). binding of this water molecule next, we assessed the importance of the position of the pyrimidine ring n atoms by synthesizing compound 4 (figure 2a). this compound was measured to bind with 50-fold higher ic50 relative to the very similar lead compound 1 (figure 1a), which only differs in the position of one pyrimidine ring nitrogen atom. we solved the 2.0 resolution crystal structure of the dck4 complex to understand how this subtle change so drastically impacted the interaction with the enzyme (see table 1 for the data collection and refinement statistics). all of the examined compounds bind to the open state of the enzyme, which is also the catalytically incompetent state (for a discussion about the open and closed states of dck, see refs (10) and (11)). inhibitors bind within a deep cavity, with the pyrimidine ring of the inhibitors positioned deepest and occupying the same position occupied by the pyrimidine ring of the nucleoside substrate. while preventing the binding of the nucleoside substrate, our inhibitors do not interfere with binding of nucleotide to the phosphoryl donor - binding site. in fact, all crystal structures of dck in complex with inhibitors also contained udp at the donor site. despite significantly different ic50 values between compound 1 (14.5 nm) and compound 4 (754 nm), the pyrimidine ring of these related molecules interacts with the enzyme via very similar hydrophobic and polar interactions. however, the entire molecule 4 is displaced about 0.4 away from the floor of the binding cavity relative to compound 1. the crystal structure suggests that the factor responsible for this shift is the recruitment of a water molecule (orange sphere, figure 2c) by the pyrimidine ring n present in compound 4. in contrast, for compound 1 the ch group in this position eliminates the potential for a hydrogen bond. this water molecule is also held in place through interactions with arg104 and asp133. hence, despite formation of this additional water - mediated interaction with the enzyme, the displacement away from the enzyme caused by allowing the water molecule to bind at that position ultimately reduces the binding affinity of 4. on the basis of these results, we decided to maintain the original structure of the pyrimidine ring and to focus on the other parts of the molecule as potential modification sites. we next examined the effect of various substituents at different phenyl group positions (part d of the molecule, figure 1a). previously, we reported that a compound with no phenyl ring substituents, but otherwise identical to compound 1, showed very modest potency in our cem cell based assay (ic50 = 37 nm). adding a hydroxyl group at the meta position decreased the ic50 in that assay by about half (compound 5, previously compound 31, figure 3). the effect of adding the longer hydroxyethoxy group at that position (compound 6, previously compound 32(3)) was more impressive, yielding an ic50 of 1 nm (figure 3). we are aware that primary hydroxyls as in 6 are prone to oxidation or glucuronidation, but these studies do inform us as to the importance of the type of substituent at the phenyl meta position. (a) schematic representation of compounds 5 and 6 that differ by the nature of the meta position substituent. (b) in vitro (ic50 and ki) and cell (ic50) properties for 5 and 6. (c) overlay of the dck5 (magenta, pdb code 4q19) and dck6 (pale green, pdb code 4q1a) structures with a focus on the phenyl ring meta position. the tighter binding of 6 relative to 5 can be rationalized by the interaction of the longer meta substituent (position highlighted with a gray background) with s144/s146 of dck. to understand the difference in affinities to dck between compounds 5 and 6, we determined the structures of dck in complex with these molecules, solved at 2.09 and 1.9 resolution, respectively (table 1). the structure of dck in complex with compound 5 reveals that the hydroxyl group at the phenyl group meta position does not make any inhibitor in contrast, the structure of dck in complex with compound 6 shows that the hydroxyethoxy at this position is able to interact with the side chains of ser144 and ser146 (figure 3c and supporting information figure s3). we attribute this added interaction to the superior binding of compound 6 versus compound 5. in terms of the importance of substituents at the phenyl meta position, it is clear that having none or a short one such as a hydroxyl (compound 5) diminishes the interaction with dck. on the other hand, the binding affinity measured by both the in vitro kinetic assay and by the cell - based cem assay of larger substituents (as present in compounds 1, 2, and 6) are comparable. previous crystal structures of dck in complex with compound 1 (pdb code 4l5b) and 2 (pdb code 4kcg) also show an interaction between the substituent at the phenyl meta position and the enzyme, this time to ser144. additional side chains such as 2-fluoroethoxy poly(ethylene glycol) (n = 2) (peg)2 (s16, s17, s19), 2-hydroxyethyl (peg)2 (s11), 2-methoxyethyl (peg)2 (s20, s22, s23, s25s29), and 2-(4,6-diaminopyrimidine-2-thio)ethyl (peg)2 (s10) substituents were well tolerated at the meta position (data not shown and supporting information table s1). we conclude that the precise nature of the substituent at the phenyl meta position is not critical as long as it contains a polar group that can extend to the proximity of ser144/ser146. to determine the importance of substituent at the phenyl group para position, we prepared compound 7 (previously compound 28(3)), which only differs from compound 2 by lacking a para position substituent (figure 4a). the in vitro measured binding affinity values (ic50 ; ki) of compound 7 are nearly identical to that of 2 (figure 4b), indicating that substituents at the para position are not required for tight binding. this is explained by the crystal structures of dck in complex with compounds 7 and 8 (previously compound 30(3)), which show a nearly identical binding mode, very similar to that observed for compound 2 (figure 4c and supporting information figure s4). this conclusion is supported by the properties of compound 8, which in contrast to the methoxy group in compounds 1 and 2 has the longer hydroxyethoxy group but similar binding affinity. hence, the in vitro binding affinities are largely unchanged between having no substituent at the phenyl group para position, having a methoxy, or the longer hydroxyethoxy. however, we did notice a 10-fold difference between compounds 7 and 8 in the cem cell - based assay, with compound 7 being less potent. furthermore, substituents at the phenyl ring s para position such as 2-fluoroethoxy (s4, s14, s18), fluoro (s5, s6), methoxymethyl terminated (peg)2 (s21, s24), and n - substituted methanesulfonamide (s29, s30) were relatively well tolerated (data not shown and supporting information table s1). groups attached to the thiazole like 4-pyridinyl (s7), meta monosubstituted phenyl (s17), and 3,5-disubstituted phenyl ring (s31) substituents were also tolerated (data not shown and supporting information table s1). therefore, while not directly important for the binding affinity, having even a small substituent at the phenyl group para position improves the relevant cell - based measurements. as a result, most subsequent compounds contained the methoxy group at that position. (a) schematic representation of compounds 7 and 8 that differ by the nature of the para position substituent. (b) in vitro (ic50 and ki) and cell (ic50) properties for 7 and 8. (c) overlay of the dck7 (teal, pdb code 4q1b) and dck8 (beige, pdb code 4q1c) structures with a focus on the phenyl ring para position. the inhibitors bind very similarly ; the meta position substituents make a direct interaction with the enzyme, but the para substituent does not. the very similar ic50 and ki values of 7 and 8 are explained by the lack of direct interactions to the enzyme via the para position. in contrast, the presence of a para position substituent lowers the cell - based determined ic50 value. in previous work we demonstrated that the nature of the substituent at the thiazole ring 5-position (part c of the molecule, figure 1a) plays a crucial role in binding affinity. in short, we compared having no substituent at that position to having a methyl, ethyl, or propyl. we found that propyl dramatically improved the binding affinity, and as a result, compounds with a propyl at the 5-position became our lead compounds (i.e., compounds 1 and 2, figure 1). interestingly, compounds with a small / no substituent at the thiazole 5-position were observed to bind two inhibitor molecules per dck active site, to binding sites that we refer to as position 1 and position 2. in contrast, the tighter binding propyl - containing molecules were observed to bind with a single inhibitor molecule, at position 1, per dck active site. this revealed that binding of two molecules is not required for high affinity. in our previous report, we analyzed the implication of single versus double binding of inhibitor molecules to dck and concluded that inhibition of dck is primarily caused by the binding of the inhibitor at position 1, whereas the molecule bound at position 2 does not appreciably enhance the inhibition. however, when tested for metabolic stability, we discovered that the propyl - group - containing compounds 1 and 2 are less stable relative to those having the shorter methyl group, e.g., compound 15a as reported by murphy. we also explored the activity of cyclopropyl and phenyl groups at the thiazolyl 5-position (supporting information table s1 and data not shown). the cyclopropyl analog (s27) had a good ic50 value, but it failed in the pet l - fac assay. hence we were forced to revert to the methylthiazole ring substituent despite a weaker interaction with dck. to compensate for the loss of affinity provided by the thiazole propyl group, we searched for a compensating modification that would restore the in vitro binding affinity while maintaining acceptable metabolic stability. for that purpose, we decided to explore modifications on the linker moiety (part b of the compounds, figure 1a). test concentration of compounds was 1 m. the sch2 group acts to link the pyrimidine and thiazole rings of our compounds. we tested a variety of alternatives to this linker, such as its deuterated analog (scd2), for the purpose of a kinetic isotope study. we reasoned that if the linker was implicated in hydrolytic metabolism, then, because of the kinetic isotope effect, a deuterated (scd2) analog would show an improvement in metabolic stability. the deuterium analogs (s1, s8, s9, s13) had affinity similar to their isotopologues, as expected (supporting information table s1 and data not shown). however, the deuterated compounds failed to show an improvement in the pet l - fac liver assay, indicating that a hydrolytic mechanism is probably not involved in the metabolism of the sch2 linker. we also tested the replacement of the sulfur atom of the sch2 group with a methylene group (ch2ch2). replacing the sulfur atom of the linker with a carbon atom resulted in a considerable decrease in dck affinity and metabolic stability (supporting information table s1 and data not shown). we next tested a linker in which the methylene was substituted to contain a methyl group (sch(ch3)). these racemic methyl - linker compounds showed very promising biological results and increased metabolic stability (see supporting information schemes 1 and 2 for the synthesis of compounds 9 and 10). therefore, we carefully examined the synthetic route in an attempt to reduce the synthetic steps and improve the total yield. we succeeded in developing a six - step synthetic route toward 11 in an overall yield of 43% (scheme 1). commercially available 3-hydroxy-4-methoxybenzonitrile a was subjected to an aqueous ammonium sulfide solution under basic conditions to provide thioamide b. cyclization to form the thiazole core of c was achieved via condensation of thioamide b with 4-bromopentane-2,3-dione in refluxing ethanol. introduction of a peg chain into the phenyl ring of compound d with 13-chloro-2,5,8,11-tetraoxatridecane under basic conditions was achieved in 89% yield. reduction of the resulting ketone - containing compound with diisobutylaluminum hydride (dibal - h) afforded racemic secondary alcohol e in high yield. the acyl chloride was reacted in crude form with 4,6-diamino-2-mercaptopyrimidine to generate product 11r / s. reagents and conditions : (a) (nh4)2s (20% in h2o), pyridine, et3n, 60 c, 85% ; (b) 4-bromopentane-2,3-dione, etoh, reflux, 95% ; (c) 13-chloro-2,5,8,11-tetraoxatridecane, cs2co3, dmf, 50 c, 89% ; (d) dibal - h, dcm, 78 c, 92% ; (e) socl2, dcm, 0 c to rt ; (f) 4,6-diamino-2-mercaptopyrimidine, k2co3, dmf, 75 c, 65% in last two steps. ch(ch3) linker was introduced to a compound that contained the propyl group at the thiazole ring 5-position (compound 9) and to a compound that, instead of the propyl group, contained a methyl (compound 10) (figure 5a). as mentioned above, the rationale for compound 10 was the predicted improvement in metabolic stability. interestingly, whereas compounds with a propylthiazole ring previously showed tighter binding to dck compared to the analogous methylthiazole compounds, we now measured better binding with the methyl - containing compound 10 to the propyl - containing compound 9 (figure 5b). hence, the proximity of the thiazole - ring substituent (propyl or methyl) to the methyl - linker substituent resulted in the larger propyl group being not as accommodating in the dck active site. despite the improved in vitro binding parameters for 10 over 9, the cell - based assay yielded similar ic50 values, yet consistent with 10 being superior (figure 5b). both compounds were synthesized as the racemic mixture (r / s) ; the addition of a methyl group (arrow) to the methylene linker group makes these compounds chiral. whereas 9 has a propyl group at the thiazole ring 5-position (rt), 10 has a methyl group. (b) in vitro (ic50 and ki) and cell (ic50) properties for 9 and 10. (c) the propyl group at the thiazole ring makes 9 bind as a single molecule to binding site position 1 of dck (see text for details). notably, despite forming the enzyme inhibitor with racemic 9, in the crystal structure we observe only the r - isomer (compound 9 in yellow, pdb code 4q1d, fo a theoretical model of the s - isomer (gray) demonstrates that only the r - isomer fits the electron density. (d) the methyl group at the thiazole ring permits two molecules of 10 to bind to dck : one to position 1 and one to position 2. in position 1 we observe only the r - isomer (10r - p1, cyan, pdb code 4q1e ; fo fc omit map contoured at 2 in green). a theoretical model of the s - isomer at position 1 (gray) clearly demonstrates that only the r - isomer fits the electron density (red arrow). (e) in position 2 we observe only the s - isomer (10s-p2, plum, pdb code 4q1e ; fo fc omit map contoured at 1.5 in green). a theoretical model of the r - isomer at position 2 (gray) clearly demonstrates that only the s - isomer fits the electron density (red arrow). both compounds 9 and 10 were prepared as racemic mixtures ; the introduced linker - methyl group makes that position a new chiral center (arrow, figure 5a). to elucidate which of the two enantiomers is the active dck inhibitor, we determined the crystal structure of dck in complex with compounds 9 and 10 (solved at 2.0 and1.85 resolution, respectively, table 1). as expected, compound 9 binds as a single molecule to dck, specifically at position 1, because of the presence of the propyl group in the thiazole ring. interestingly, despite the fact that a racemic mixture of 9 was used to form the complex to dck, the crystal structure provides unambiguous evidence for the r - isomer binding at position 1 (figure 5c and supporting information figure s5). likewise, inspection of the structure of the complex between racemic 10 and dck shows that the r - isomer occupies the most relevant position 1 binding site (figure 5d and supporting information figure s5). since compound 10 contains the methyl substituent in the thiazole ring, which allows for a molecule to also occupy position 2, we observe compound 10 at that position as well. however, whereas it is the r - isomer of 10 that binds to position 1, it is the s - isomer that binds to position 2 (figure 5e and supporting information figure s5). we previously concluded that position 1 is the critical binding site for this family of inhibitors. this would suggest that the measured in vitro inhibition values of racemic 10 are reflecting the preferential binding of the r - isomer. to test this, we synthesized compound 11, which is a slight modification of 10 (the nature of the phenyl group substituents) but notably had the racemic mixture separated to yield the pure isomers 11r and 11s (figure 6a). we determined the in vitro binding affinities of the enantiomerically pure compounds and observed that 11s has 400-fold weaker binding affinity relative to 11r (figure 6b). this result provides clear evidence that the r - form is responsible for the tight interaction with dck. this result also validates our structure - based interpretation that position 1 is the one most relevant inhibitor binding site for dck inhibition and that position 2 is occupied because of the high concentration of the inhibitor used in the crystallization setups. (a) schematic representation of compounds 11s, 11r, and 12r (r or s designate the chirality of the linker methylene carbon ; arrows point at the added methyl group). (b) in vitro (ic50 and ki) and cell (ic50) properties for 11s, 11r, and 12r. the r - isomer of both 11 and 12 is responsible for the observed inhibition of the enzyme. (c) dck was crystallized in the presence of enantiomerically pure 12r, and the enzyme inhibitor complex structure was solved (pdb code 4q1f). fc omit map (1.6) for the position 1 binding site clearly shows the presence of 12r (brown). despite the thiazole methyl group in 12r (which is compatible with molecules also binding to position 2) this is consistent with the results with compound 10 (figure 5) that showed that only the s - isomer binds to positon 2. having discovered that the r - isomers of compounds 9, 10, and 11 are responsible for the dck inhibition, we set out to develop an asymmetric synthesis (scheme 2). the chiral synthesis developed by our group for compound 12r, which is a close analog of 10, features a chiral corey shibata (cbs) reaction of ketone d. chiral alcohol e was synthesized according to this method with an enantiomeric excess of 96%, as determined via chiral hplc. employing mesic or tosic anhydride to give the sulfonates under different basic conditions such as et3n, pyridine, or dmap resulted in elimination to the alkene, presumably due to the stability of the secondary benzylic - like carbocation. the use of trifluoroacetic anhydride (tfaa) at 0 c converted alcohol e into the corresponding trifluoroacetate (tfa) f without a significant decrease in the % ee of the ester. finally, compound f was reacted with 4,6-diamino-2-mercaptopyrimidine to generate 12r in 61% yield over two steps with an enantiomeric excess of 40%. presumably, a portion of the reaction occurs via a direct sn2 pathway, while another part occurs via an sn1 pathway, and thereby racemized material was obtained. chiral resolution via recrystallization generated 12r with an enantiomeric excess of over 90%. likewise, (s)-()-2-methyl - cbs - oxazaborolidine was used in the cbs reduction to synthesize 12s. reagents and conditions : (a) (nh4)2s (20% in h2o), pyridine, et3n, 60 c, 85% ; (b) 4-bromopentane-2,3-dione, etoh, reflux, 96% ; (c) n-(2-bromoethyl)methanesulfonamide, cs2co3, dmf, 50 c, 82% ; (d) (r)-(+)-2-methyl - cbs - oxazaborolidine, bh3thf complex, thf, 78 c, 77%, (96% ee) ; (e) tfaa, dcm, 0 c, (f) 4,6-diamino-2-mercaptopyrimidine, dmf, 80 c, 61% in last two steps. compound 12r (figure 6a) was measured to have very similar in vitro binding affinities to 11r (figure 6b). significantly, just as the affinity of 11s was much reduced relative to 11r, the affinity to dck of 12s was much reduced relative to 12r. this reiterated the preference of dck for compounds that contain the r - isomer of the linker. we expected 12r to bind only at position 1 based on the previous structure with compound 10 (observing 10r bound at position 1) and the kinetic results using enantiomerically pure 11s, 11r, 12s, and 12r (observing higher affinities for the r - isomers) and since the crystals were formed with the enantiomerically pure 12r. additionally, lacking the s - isomer, we expected a vacant position 2 binding site. indeed, the crystal structure of the dck12r complex revealed a single inhibitor molecule at position 1 (figure 6c). this result suggests that the r - isomer has very low affinity to the binding site at position 2. notably, while the interaction between the r - isomer and dck is limited to the position 1 binding site, this does not diminish the binding affinity for the enzyme. what could be behind the dramatic selectivity of the dck position 1 binding site for the r - isomers of the inhibitors ? likewise, what prevents the r - isomer from binding at position 2, while this binding site is compatible with the binding of the s - isomer ? the simple explanation would involve steric considerations relating the inhibitor and enzyme, where the chiral methyl group of the linker clashes with enzyme residues in the case of one isomer but not the other. however, inspection of the crystal structures solved with compounds 10(r / s) and 12r does not support this interpretation ; we could model the s - isomer bound to position 1 (figure 5d) and the r - isomer bound at position 2 (figure 5e) with no apparent clashes. comparison of the binding mode between 10r and 10s reveals that the relative orientation of the pyrimidine ring to the thiazolephenyl part is strikingly different between the r and s isomers (figure 7a and figure 7b). that is, by a change of the angles of the linker that connects the pyrimidine ring to the thiazole ring, each isomer has adjusted its conformation to best fit its binding site (i.e., induced fit). this demonstrates that the enzyme dictates the relative orientations between the pyrimidine ring, linker, and the thiazolephenyl rings. it also shows that the relative orientation between thiazole and phenyl rings (being coplanar) is largely unchanged, not surprising because of the resonance between the rings. chiral selectivity is due to conformational selection by the enzyme s binding site. (a) observed orientation of 10r (cyan) at position 1 (10r - p1, pdb code 4q1e) and 10s (plum) at position 2 (10s - p2) upon dck binding. note the different relative orientations of the thiazole and pyrimidine rings between 10r and 10s. (c) the conformation of 10r (10r - p1) is dictated by the position 1 binding site. in this conformation the distance between the chiral linker methyl group and the thiazole ring methyl group is 4.2. (d) the theoretical model of 10s binding with the same conformation as 10r in position 1 (10s - p1) shows that the homologous distance is reduced to 2.5. (e) the conformation of 10s (10s - p2) is dictated by the position 2 binding site. in this conformation the distance between the chiral linker methyl group and the thiazole ring methyl group is 4.4. (f) the theoretical model of 10r binding with the same conformation as 10s in position 2 (10r - p2) shows that the homologous distance is reduced to 2.6. (g) for 10r - p1, the observed torsion angle between the thiazole ring and the linker is 59. scanning possible torsion angles shows that this value represents a low energy conformation of 10r. (h) for 10s - p1, the observed torsion angle is 189. this value corresponds to a high - energy conformation. (i) for 10s - p2, the observed torsion angle is 326. scanning possible torsion angles shows that this value is at a low energy conformation of 10s. (j) for 10r - p2, the observed torsion angle is 147. this value corresponds to a high - energy conformation. to further probe the observed chiral selectivity, we constructed a theoretical model of 10s binding at position 1 with the same orientation as 10r. whereas the observed distance between the chiral methyl of the linker and the thiazole ring methyl group for 10r in position 1 is 4.2 (figure 7c), for the modeled 10s bound to position 1, that distance would be an unfavorable 2.5 (figure 7d). likewise, whereas the observed distance between the chiral methyl and the thiazole methyl for 10s in position 2 is 4.4 (figure 7e), for the modeled r - isomer adopting the same conformation as 10s, that distance would be an unfavorable 2.6 (figure 7f). hence, the strict chiral selection to either position 1 or position 2 is due to the enzyme dictating a particular inhibitor orientation that is vastly different between the binding sites. in the case of position 1, that orientation is not compatible with the s - isomer, and for position 2, that orientation is not compatible with the r - isomer. using computer simulations, we obtained a qualitative estimate of the conformational penalty incurred by 10r and 10s upon binding with the protein. the conformational penalty is the energy difference between the preferred solution - phase geometry of a substrate and the geometry that it assumes upon binding : e = esolution ebound. each enantiomer was docked with the solvated protein at position 1 and allowed to equilibrate (see details in experimental section and supporting information figure s6). the equilibrated, docked inhibitor structures were removed from the protein, and their energies were assessed with the semiempirical pddg / pm3 method. unbound structures of 10r and 10s were optimized in implicit solvent to determine their low - energy solution - phase conformations. as with the bound structures, the resulting energies were used to obtain qualitative conformational penalties for each enantiomer. the conformational penalty for 10s was almost twice the conformational penalty for 10r (45 kcal / mol larger penalty for 10s), further demonstrating that 10r needs to undergo a much less unfavorable structural rearrangement in order to bind with the protein at position 1. another way of considering this issue is to examine the energy of the inhibitor as a function of rotation around the bond that connects the thiazole ring to the chiral linker atom (bond marked with in figure 7c f). for 10r bound to dck at position 1, the observed dihedral angle that specifies this rotation is 59 and fits a low energy conformation (figure 7 g). in contrast, the modeled s - isomer at this binding site would have a torsion angle of 189, which is clearly a high - energy conformation (figure 7h). the same pattern is observed for position 2, with the s - isomer binding to dck with a torsion angle of 326, which is a low energy conformation, while the modeled r - isomer at that position is a high - energy conformation (figure 7i and figure 7j). hence, the chiral selectivity does not come directly from the enzyme sterically favoring one isomer over the other. rather, the enzyme dictates a particular conformation, and the selectivity comes from one isomer being able to adopt that particular conformation, whereas the energy penalty for the other isomer precludes its binding. in addition to explaining the chiral selectivity for the compounds discussed here, this understanding can be used for the design of chiral molecules that bind to either binding site. specifically, the prediction would be that replacing the thiazole methyl group with a hydrogen atom would eliminate any steric clash to the chiral methyl group, and hence either isomer could bind to either inhibitor binding site. we first determined the metabolic stability of 12r in a standard microsomal liver clearance assay. the nadph - dependent t1/2 of 12r was 37-fold longer than that of our previous lead compound 2 (table 2). we then tested compound 12 in mice, using our previously described positron emission tomography (pet) assay. whereas our earlier lead compound 2 retained only 25% inhibition of dck activity 4 h after dosing by intraperitoneal injection, compound 12 (given as the racemic mixture) exhibited > 50% inhibition of dck activity at this time point (figure 8a). furthermore, 8 h after treatment with compound 12, dck inhibition was still above 30%. we then determined the pharmacokinetic properties of compound 12 to compare with our previous lead compounds 1 and 2. as shown in figure 8b, the pharmacokinetic properties of compound 12 were significantly improved relative to the previously published values for compounds 1 and 2. collectively, these findings demonstrate that introduction of the chiral linker plus replacement of the thiazole ring propyl substituent by a methyl group yields a dck inhibitor with improved metabolic stability. in vivo evaluation of compound 12. (a) quantification of pet probe, f - l - fac, uptake in the liver of c57bl/6 female mice treated with compounds 12 (25 mg / kg) via intraperitoneal injection. dose formulation : 50% peg / tris, ph 7.4. data are mean values sem for at least n = 5 mice / time point. c57bl/6 female mice were dosed via intraperitoneal injection with 50 mg / kg compound 12 formulated in 50% peg / tris, ph 7.4. data are mean values sem for n = 4 mice / time point. structural and inhibition studies of the compounds discussed here, performed using both the purified recombinant enzyme and a cell - based assay, revealed and rationalized the essential determinants for binding to dck and also guided the type and placement of substituents. these compounds contain a propyl group at the 5-position of the thiazole ring, since, as shown earlier, the propyl substituent provides improved affinity for dck compared to compounds with a methyl group at that position. unfortunately, this affinity - strengthening propyl group compromised the metabolic stability relative to compounds containing a methyl group at that position. this forced us to revert to the weaker - binding, but more metabolically stable, scaffold of a methyl group at the thiazole ring. with the goal of improving metabolic stability, we tested a chiral methylene methyl sulfur linker between the thiazole and pyrimidine moieties. this linker was found to confer two positive effects : (1) in terms of affinity for dck, the modified linker compensated for the lack of the thiazole propyl group, and (2) the compounds exhibited improved metabolic stability. the interaction of dck with compounds containing this linker is specific to the r - isomer. this was proven by the dck - inhibitor crystal structure and by comparing the binding affinities of the r versus s enantiomers. the new lead compound 12r is a promising dck inhibitor, which by perturbing the dntp pools and inducing dna replication stress overload could be used in combination with other drugs to specifically trigger synthetic lethality in cancer cells. general laboratory reagents were purchased from fisher (pittsburgh, pa, usa) and sigma - aldrich (st. louis, mo, usa). (800 north five points road, west chester, pa 19380, usa) performed the separation of r and s enantiomers. unless otherwise noted, reactions were carried out in oven - dried glassware under an atmosphere of nitrogen using commercially available anhydrous solvents. 4,6-diamino-2-mercaptopyrimidine was obtained from drying the hydrate over dynamic vacuum at 110 c for 20 h. all other reagents obtained from commercial suppliers were reagent grade and used without further purification unless specified. reactions and chromatography fractions were analyzed by thin - layer chromatography (tlc) using merck precoated silica gel 60 f254 glass plates (250 m). visualization was carried out with ultraviolet light, vanillin stain, permanganate stain, or p - anisaldehyde stain. flash column chromatography was performed using e. merck silica gel 60 (230400 mesh) with compressed air. h and c nmr spectra were recorded on a arx500 (500 mhz), avance 500 (500 mhz), or avance 300 (300 mhz) spectrometers. chemical shifts are reported in parts per million (ppm,) using the residual solvent peak as the reference. the coupling constants, j, are reported in hertz (hz), and the resonance patterns are reported with notations as the following : br (broad), s (singlet), d (doublet), t (triplet), q (quartet), and m (multiplet). electrospray mass spectrometry data were collected with a waters lct premier xe time - of - flight instrument controlled by masslynx 4.1 software. samples were dissolved in methanol and infused using direct loop injection from a waters acquity uplc into the multimode ionization source. analytical hplc analysis was performed on a knauer smartline hplc system with a phenomenex reverse - phase luna column (5 m, 4.6 mm 250 mm) with inline knauer uv (254 nm) detector. mobile phase : a, 0.1% tfa in h2o ; b, 0.1% tfa in mecn. percent enantiomeric excess (% ee) values were determined via chiral hplc with a chiralpak ia-3/ia polysaccharide - based immobilized type column (3 m, 4.6 mm 150 mm) with inline knauer uv (310 nm) detector. mobile phase : a, 0.1% tfa in hexanes ; b, 0.1% tfa in propanol. eluent gradient : 50% phase a and 50% phase b. chromatograms were collected by a ginastar (raytest usa, inc. ; wilmington, nc, usa) analog to digital converter and ginastar software (raytest usa, inc.). to a mixture of 3-ethoxy-4-hydroxybenzonitrile a (2.50 g, 15.3 mmol) in pyridine (35 ml) and triethylamine (2.5 ml) was added ammonium sulfide solution (20 wt % in h2o, 15.65 ml, 46.0 mmol). the organic layer was dried over anhydrous na2so4, concentrated in vacuo, and purified by flash column chromatography over silica gel (3:1 ethyl acetate / hexanes) to yield b (2.56 g, 13.0 mmol, 85%) as a yellow solid. h nmr (300 mhz, cdcl3) 7.68 (d, j = 2.1 hz, 1h), 7.48 (br s, 1h), 7.28 (dd, j = 8.5, 2.1 hz, 1h), 7.11 (br s, 1h), 6.89 (d, j = 8.5 hz, 1h), 6.03 (s, 1h), 4.21 (q, j = 6.9 hz, 2h), 1.47 (t, j = 6.9 hz, 3h) ; c nmr (125 mhz, acetone - d6) 200.5, 150.3, 145.8, 131.0, 121.0, 114.0, 112.6, 64.3, 14.1. a mixture of thioamide b (1.50 g, 7.6 mmol) and 4-bromopentane-2,3-dione (2.04 g, 11.4 mmol) in ethanol (40 ml) was stirred under refluxing conditions for 4 h. the resulting mixture was cooled and concentrated in vacuo to remove residual solvent. the crude residue was purified by flash column chromatography over silica gel (10:3 hexanes / ethyl acetate) to yield the desired thiazole intermediate c (2.00 g, 7.2 mmol, 95%) as a white solid. h nmr (300 mhz, cdcl3) 7.47 (d, j = 1.8 hz, 1h), 7.35 (dd, j = 8.2, 1.8 hz, 1h), 6.96 (d, j = 8.1 hz, 1h), 5.93 (s, 1h), 4.23 (q, j = 7.2 hz, 2h), 2.77 (s, 3h), 2.71 (s, 3h), 1.50 (t, j = 6.9 hz, 3h) ; c nmr (75 mhz, cdcl3) 196.0, 162.8, 148.9, 148.0, 146.3, 142.9, 125.9, 120.5, 114.8, 109.4, 64.9, 29.5, 14.9, 13.6. to a solution of thiazole intermediate c (1.66 g, 6.0 mmol) in dmf (35 ml) were added cs2co3 (3.13 g, 9.6 mmol) and 13-chloro-2,5,8,11-tetraoxatridecane (2.19 g, 12.0 mmol). after concentration to remove residual solvent, the resulting residue was washed with brine and extracted with ethyl acetate. the organic layer was washed with water three times, dried over anhydrous na2so4, and concentrated in vacuo, and the crude residue was purified by flash column chromatography over silica gel (1:1 ethyl acetate / hexanes) to yield desired ketone d (2.26 g, 5.3 mmol, 89%) as a white solid. h nmr (500 mhz, cdcl3) 7.48 (d, j = 2.0 hz, 1h), 7.38 (dd, j = 8.5, 2.0 hz, 1h), 6.94 (d, j = 8.5 hz, 1h), 4.244.20 (m, 2h), 4.17 (q, j = 7.0 hz, 2h), 3.933.89 (m, 2h), 3.793.75 (m, 2h), 3.703.63 (m, 4h), 3.573.53 (m, 2h), 3.37 (s, 3h), 2.77 (s, 3h), 2.71 (s, 3h), 1.47 (t, j = 7.0 hz, 3h) ; c nmr (125 mhz, cdcl3) 196.0, 162.5, 150.8, 149.4, 149.0, 143.1, 126.9, 119.8, 114.0, 111.4, 72.1, 71.1, 70.8, 70.7, 69.7, 69.0, 64.9, 59.2, 29.5, 15.0, 13.6. to a stirred solution of ketone d (1.06 g, 2.5 mmol) in ch2cl2 (35 ml) cooled to 78 c was added slowly diisobutylaluminum hydride (1.0 m in thf, 10 mmol, 10 ml). the mixture was allowed to warm to 23 c and stirred for 1 h. the mixture was cooled to 0 c and slowly quenched with a saturated aqueous solution of rochelle s salt. the cloudy solution was stirred for 1 h at 23 c until the solution became clear again. the resulting solution was extracted with ethyl acetate, washed with brine, dried over anhydrous na2so4, and concentrated in vacuo to give the desired alcohol e (978 mg, 2.3 mmol, 92%) as a pale yellow solid. h nmr (500 mhz, cdcl3) 7.44 (d, j = 2.0 hz, 1h), 7.33 (dd, j = 8.5, 2.0 hz, 1h), 6.89 (d, j = 8.5 hz, 1h), 4.91 (q, j = 6.5 hz, 1h), 4.224.17 (m, 2h), 4.13 (q, j = 7.0 hz, 2h), 3.913.86 (m, 2h), 3.763.72 (m, 2h), 3.693.61 (m, 4h), 3.553.51 (m, 2h), 3.35 (s, 3h), 2.37 (s, 3h), 1.52 (d, j = 6.0 hz, 3h), 1.44 (t, j = 7.0 hz, 3h) ; c nmr (125 mhz, cdcl3) 164.3, 155.1, 150.0, 149.0, 127.2, 125.8, 119.3, 113.8, 111.0, 71.8, 70.8, 70.6, 70.4, 69.5, 68.7, 64.6, 64.4, 58.9, 24.0, 14.7, 10.7. to a stirred solution of alcohol e (425 mg, 1.0 mmol) in ch2cl2 (8 ml) was added thionyl chloride (0.78 ml, 10.0 mmol) slowly at 0 c. the mixture was allowed to warm to 23 c and stirred for 1 h. after concentration in vacuo to remove residual solvent, the resulting crude residue was used directly for next step without any further purification because of the instability of chloride f. a mixture of crude chloride f from the previous step, 4,6-diamino-2-mercaptopyrimidine (625 mg, 4.0 mmol), and k2co3 (552 mg, 4.0 mmol) in dmf (7 ml) was stirred at 70 c for 1 h. the solution was cooled, concentrated in vacuo, and purified by flash column chromatography over silica gel (25:1 dichloromethane / methanol) to give the desired product ()-9 (357 mg, 0.65 mmol, 65% in two steps) as a white solid. h nmr (500 mhz, cdcl3) 7.49 (d, j = 2.0 hz, 1h), 7.35 (dd, j = 8.5, 2.0 hz, 1h), 6.90 (d, j = 8.5 hz, 1h), 5.24 (s, 1h), 5.02 (q, j = 7.0 hz, 1h), 4.58 (s, 4h), 4.224.18 (m, 2h), 4.15 (q, j = 7.0 hz, 2h), 3.913.87 (m, 2h), 3.783.75 (m, 2h), 3.693.63 (m, 4h), 3.563.53 (m, 2h), 3.37 (s, 3h), 2.50 (s, 3h), 1.81 (d, j = 7.0 hz, 3h), 1.46 (t, j = 7.0 hz, 3h) ; c nmr (125 mhz, cdcl3) 170.7, 163.8, 163.2 (2), 153.3, 149.9, 149.1, 127.9, 126.8, 119.4, 114.0, 111.3, 80.6, 71.9, 70.9, 70.7, 70.6, 69.7, 68.9, 64.7, 59.1, 37.7, 22.0, 14.8, 11.6 ; hrms - esi (m / z) [m + h ] calcd for c25h35n5o5s2h, 550.2158 ; found 550.2169. to a mixture of 3-hydroxy-4-methoxybenzonitrile a (3.00 g, 20.11 mmol) in pyridine (30 ml) and triethylamine (3 ml) was added ammonium sulfide solution (20 wt % in h2o, 20.7 ml, 60.3 mmol). the organic layer was dried over anhydrous na2so4, concentrated in vacuo, and purified by flash column chromatography over silica gel (3:1 ethyl acetate / hexanes) to yield b (3.13 g, 17.1 mmol, 85%) as a yellow solid. h nmr (500 mhz, acetone - d6) 8.77 (br s, 1h), 8.65 (br s, 1h), 7.85 (s, 1h), 7.59 (d, j = 2.5 hz, 1h), 7.56 (dd, j = 8.5, 2.3 hz, 1h), 6.94 (d, j = 8.5 hz, 1h), 3.88 (s, 3h) ; c nmr (125 mhz, acetone - d6) 200.7, 150.5, 145.7, 132.4, 119.5, 114.8, 110.2, 55.5. a mixture of thioamide b (2.75 g, 15.0 mmol) and 4-bromopentane-2,3-dione (4.03 g, 22.5 mmol) in ethanol (70 ml) was stirred under refluxing conditions for 4 h. the resulting mixture was cooled and concentrated in vacuo to remove residual solvent. the crude residue was purified by flash column chromatography over silica gel (10:3 hexanes / ethyl acetate) to yield the desired thiazole intermediate c (3.79 g, 14.4 mmol, 96%) as a white solid. h nmr (500 mhz, dmso - d6) 9.53 (br s, 1h), 7.34 (d, j = 2.0 hz, 1h), 7.26 (dd, j = 8.5, 2.0 hz, 1h), 6.98 (d, j = 8.5 hz, 1h), 3.80 (s, 3h), 2.66 (s, 3h), 2.57 (s, 3h) ; c nmr (125 mhz, dmso - d6) 195.2, 162.5, 150.1, 148.5, 147.1, 142.7, 125.6, 118.2, 112.9, 112.5, 55.9, 29.4, 13.2. to a solution of thiazole intermediate c (1.58 g, 6.0 mmol) in dmf (35 ml) were added cs2co3 (3.13 g, 9.6 mmol) and n-(2-bromoethyl)methanesulfonamide (2.18 g, 10.8 mmol). after concentration to remove residual solvent, the resulting residue was washed with brine and extracted with ethyl acetate. the organic layer was washed with water three times, dried over anhydrous na2so4, and concentrated in vacuo, and the crude residue was purified by flash column chromatography over silica gel (3:2 ethyl acetate / hexanes) to yield desired ketone d (1.89 g, 4.9 mmol, 82%) as a white solid. h nmr (500 mhz, cdcl3) 8.00 (s, 1h), 7.51 (d, j = 2.0 hz, 1h), 7.46 (dd, j = 8.5, 2.0 hz, 1h), 6.92 (d, j = 8.5 hz, 1h), 4.254.20 (m, 2h), 3.90 (s, 3h), 3.603.55 (m, 2h), 3.03 (s, 3h), 2.76 (s, 3h), 2.70 (s, 3h) ; c nmr (125 mhz, cdcl3) 195.8, 162.5, 151.5, 148.9, 147.8, 143.1, 126.4, 121.1, 112.4, 111.7, 69.1, 55.9, 42.7, 40.6, 29.4, 13.4. to a stirred solution of (r)-(+)-2-methyl - cbs - oxazaborolidine (6.7 ml of a 1.0 m solution in toluene, 6.7 mmol) in thf (26 ml) at 78 c under ar was added borane tetrahydrofuran complex (4.4 ml of a 1.0 m solution in thf, 4.4 mmol) followed by a solution of d (284 mg, 0.74 mmol) in thf (14 ml). after addition of the d solution with syringe pump for 6 h, the reaction mixture was stirred for another 20 min at 78 c. h2o (10 ml) and meoh (5 ml) were added, and the mixture was allowed to warm to room temperature. after concentration to remove residual solvent, the resulting residue was washed with brine and extracted with ethyl acetate. the organic layer was washed with water three times, dried over anhydrous na2so4, and concentrated in vacuo, and the crude residue was purified by flash column chromatography twice over silica gel with 3:2 ethyl acetate / hexanes and 40:1 dichloromethane / methanol as washing system separately to yield alcohol e (221 mg 0.57 mmol, 77%, 96% ee) as a white solid. h nmr (500 mhz, acetone - d6) 7.57 (d, j = 2.0 hz, 1h), 7.46 (dd, j = 8.5, 2.0 hz, 1h), 7.05 (d, j = 8.5 hz, 1h), 6.26 (br s, 1h), 5.024.95 (m, 1h), 4.21 (t, j = 5.5 hz, 2h), 3.88 (s, 3h), 3.57 (dt, j = 5.5, 5.5 hz, 2h), 3.04 (s, 3h), 2.48 (s, 3h), 1.50 (d, j = 6.0 hz, 3h) ; c nmr (125 mhz, acetone - d6) 162.9, 156.1, 151.3, 148.4, 127.1, 126.8, 119.7, 112.1, 111.4, 68.6, 64.1, 55.3, 42.6, 39.6, 23.0, 10.0. to a stirred solution of alcohol e (221 mg, 0.57 mmol) in ch2cl2 (13 ml) was added trifluoroacetic anhydride (0.66 ml, 2.9 mmol) slowly at 0 c. after being stirred at 0 c for 30 min, the mixture was allowed to warm to 23 c and stirred for another 30 min. after concentration in vacuo to remove residual solvent, the resulting crude residue was used directly for next step without any further purification because of the instability of the desired trifluoroacetate f. a mixture of crude chloride f from the previous step and 4,6-diamino-2-mercaptopyrimidine (112 mg, 0.86 mmol) in dmf (5 ml) was stirred at 80 c for 1 h. the solution was cooled, concentrated in vacuo, and purified by flash column chromatography over silica gel (25:1 dichloromethane / methanol) to give the couple of enantiomers 12r and 12s (178 mg, 0.35 mmol, 40% ee of 12r, 61% total yield in two steps) as a white solid. recrystallization of the enantiomers with meoh / acetone solvent system gave the 12r with > 93% ee. h nmr (500 mhz, acetone - d6) 7.55 (d, j = 2.0 hz, 1h), 7.48 (dd, j = 8.5, 2.0 hz, 1h), 7.06 (d, j = 8.5 hz, 1h), 6.26 (br s, 1h), 5.605.55 (m, 4h), 5.37 (s, 1h), 5.30 (q, j = 7.0 hz, 1h), 4.23 (t, j = 5.5 hz, 2 h), 3.89 (s, 3h), 3.58 (dt, j = 5.5, 5.5 hz, 2h), 3.05 (s, 3h), 2.52 (s, 3h), 1.74 (d, j = 7.0 hz, 3h) ; c nmr (125 mhz, dmso - d6) 168.0, 163.5 (2), 162.9, 153.6, 150.6, 147.8, 126.6, 126.2, 119.5, 112.3, 110.4, 79.0, 67.9, 55.7, 41.9, 36.1, 30.7, 22.2, 11.2 ; hrms - esi (m / z) [m + h ] calcd for c20h26n6o4s3h, 511.1256 ; found 511.1259 ; 12r []d + 340.0 (c 0.12, acetone) (93% ee)., we used the s74e - c4s - dck variant, which is the human dck protein where four solvent - exposed cysteines are mutated into serines (c4s). we showed that the c4s mutant generates better quality crystals without altering the three - dimensional conformation of the enzyme or its enzymatic activity. additionally, the enzyme contained the mutation of ser74 to glutamic acid (s74e) ; this mutation serves to mimic the phosphorylated state of this residue. when we refer to dck in this report, we mean the c4s - s74e - dck variant. dck was expressed in escherichia coli bl21 c41(de3) cells using a pet-14b vector ; the cells were grown in 2xyt medium and induced with 0.1 mm iptg for 4 h at 310 k. the cells were harvested, and the pellet was lysed by sonication. the lysate was cleared by centrifugation at 30 000 rev / min for 1 h at 277 k, and the supernatant was loaded onto a 5 ml histrap nickel - affinity column (ge healthcare). the column was washed with 300 ml of a buffer composed of 25 mm tris - hcl, ph 7.5, 500 mm nacl, 30 mm imidazole. the bound protein was eluted with the same buffer but containing 250 mm imidazole and was further purified by gel filtration using an s-200 column in a buffer consisting of 25 mm hepes, ph 7.5, 200 mm sodium citrate, 2 mm edta, 3 mm dtt. the protein fractions were pooled, concentrated, aliquoted, flash - frozen in liquid nitrogen, and stored at 193 k until use. the phosphorylation activity of dck was determined using a spectroscopic nadh - dependent enzyme - coupled assay. all measurements were taken in triplicate at 310 k in a buffer consisting of 100 mm tris, ph 7.5, 200 mm kcl, 5 mm mgcl2, 0.5 mm edta, 0.8 mm phosphoenolpyruvate, 0.4 mm nadh with 50 nm dck, and 1 mm atp. ic50 and ki were determined as described by us, and all data were fitted using the kaleidagraph software. these were performed in ccrf - cem acute lymphoblastic leukemia cells as previously described. pet studies to determine % inhibition of dck activity in vivo were performed as previously described. these assays were performed by cyprotex (watertown, ma) according to standard operating protocols. briefly, c57bl/6 female mice were treated with the dck inhibitors via intraperitoneal injection. the drugs were administered in 50% polyethylene glycol (peg 400)/50 mm tris - hcl, ph 7.5. five minutes after drug injection, whole blood (75 l) was obtained at various time points from the retro - orbital sinus using hematocrit capillary tubes. g for 5 min, and the supernatant (5 l) was transferred into a clean tube. calibration standards were prepared by spiking various amounts of 11 and 12 in 5 l of supernatant from the plasma of untreated mice to obtain final concentrations between 0.001 to 100 pmol/l. samples and the calibration standards were mixed with 500 l ice - cold acetonitrile / water (50/50, v / v) containing an internal standard (1). the residue was reconstituted in 100 l of acetonitrile / water (50/50, v / v). samples (5 l) were injected onto a reverse phase column (agilent zorbax rapid resolution high definition eclipse plus c18, 2.1 mm 50 mm, 1.8 m) equilibrated in water acetonitrile / formic acid, 95/5/0.1, and eluted (200 l / min) with an increasing concentration of solvent b (acetonitrile / formic acid 100/0.1, v / v : min/% acetonitrile ; 0/5, 2/5, 8/80, 9/80, 10/5, 12/5). the effluent from the column was directed to an electrospray ion source (agilent jet stream) connected to a triple quadrupole mass spectrometer (agilent 6460 qqq) operating in the positive ion mrm mode. the ion transitions for 1, 11, and 12 are 476.2334.5, 550.2408.2, and 511.1369.1 respectively. the peak areas for 11 and 12 were normalized to the peak area of the internal standard, and the plasma concentrations were computed using the standard curves generated by calibration standards spiked in plasma from untreated mice. approximated values of the area under the curve (auc), half - life (t1/2), maximum concentration in the plasma (cmax), and time to reach the maximum concentration (tmax) were calculated using boomer / multi - forte pk functions from microsoft excel. crystals of human dck in complex with inhibitors and udp were grown at 285 k using the hanging - drop vapor - diffusion method. all dck - inhibitor complexes were prepared as follows : 1 l of dck protein at 1017 mg / ml in complex with a 2.5-fold molar excess of inhibitor, and 2 mm udp and 5 mm mgcl2 were mixed with 1 l of reservoir buffer solution. the reservoir solution consisted of 0.91.5 m trisodium citrate dehydrate and 25 mm hepes, ph 7.5. prior to data collection, crystals were soaked in mineral oil for cryoprotection. diffraction data for dck in complex with compounds 48 were collected on the life sciences collaborative access team (ls - cat) beamline 21-id - g. data for all other complexes (compounds 912) were collected using the in - house x - ray source (rigaku ru-200 rotating anode) with a r - axis iv++ image plate detector. structures were determined by molecular replacement with molrep using the dck structure (pdb entry 4jln) as a search model. refinement was conducted using refmac, and model building was conducted using coot. all data sets were perfectly twinned, and iterative refinements were carried out using refmac with the twin option active. structural figures were prepared using the pymol molecular graphics system (version 1.6.0, schrdinger). the s - isomer in position 1 and the r - isomer in position 2 were generated by flipping the chirality of the linker carbon using maestro, version 9.1, schrdinger, llc, 2010. this program was also used to generate the torsion scans around the bond connecting the chiral linker carbon and the thiazole ring (torsion angle defined by cac cbc equilibration simulations were performed using the mcpro 2.0 software package with the opls - aa force field. the protein backbone and all bond lengths within the protein were held fixed. angles and torsions within 11 of the center of the bound molecule were allowed to vary. equilibration began with 5 10 configurations of solvent - only moves, followed by 10 10 configurations in which the protein and bound molecule were sampled, with additional solvent sampling at every tenth configuration. equilibrations were performed using metropolis monte carlo in the npt ensemble at 1 atm and 25 c. for the unbound structures, implicit solvent was simulated with the generalized born / surface area (gb / sa) method. | recently, we have shown that small molecule dck inhibitors in combination with pharmacological perturbations of de novo dntp biosynthetic pathways could eliminate acute lymphoblastic leukemia cells in animal models. however, our previous lead compound had a short half - life in vivo. therefore, we set out to develop dck inhibitors with favorable pharmacokinetic properties. we delineated the sites of the inhibitor for modification, guided by crystal structures of dck in complex with the lead compound and with derivatives. crystal structure of the complex between dck and the racemic mixture of our new lead compound indicated that the r - isomer is responsible for kinase inhibition. this was corroborated by kinetic analysis of the purified enantiomers, which showed that the r - isomer has > 60-fold higher affinity than the s - isomer for dck. this new lead compound has significantly improved metabolic stability, making it a prime candidate for dck - inhibitor based therapies against hematological malignancies and, potentially, other cancers. |
three cross - sectional surveys of cardiovascular disease risk factors and related health behavior were carried out in southeastern australia (14) to obtain rural data for comparison with the existing urban data. the first survey was conducted from august to october 2004 in limestone coast (lc, south australia), the second in february to march 2005 in corangamite shire (co, victoria), and the third in may to october 2006 in the wimmera region (wi, victoria). each survey used a stratified random sample of the population aged 2574 years drawn from the electoral roll. stratification was by sex and 10-year age - groups, with the exception of the combined 25- to 44-year age - group considered as one stratum. the original samples consisted of 1,120 individuals in lc, 1,000 in co, and 1,500 in wi. after excluding individuals who had died or had left the region, a total of 552 people in lc (participation rate 51%), 415 people in co (42%), and 596 people in wi (53%) participated in the study. the wi sample included an additional 127 subjects (participation rate 44%) from the age - group 7584 years. the survey methodology, as previously described (4), comprised self - administered questionnaires, physical measurements, and laboratory tests. a comparison of the socioeconomic background with population statistics available indicated that the participants closely resembled the true populations of the areas surveyed (4). the questionnaire, which included questions on health behavior, symptoms and diseases, medical history, socioeconomic background, and psychosocial factors, together with the invitation to attend the health check, was sent by mail to all selected participants. health checks were carried out in local health centers or other survey sites by specially trained nurses. in the health check, weight, height, waist and hip circumference, systolic and diastolic blood pressure, as well as fasting lipids and glucose were ascertained, and bmi was computed as described in more detail elsewhere (4). the venous blood samples were drawn after an overnight fast of at least 10 h and analyzed at the flinders medical centre clinical trials laboratory, which is internationally accredited for lipid measurement under the centres for disease control lipid standardization program (atlanta, ga) (4). first, the most recent ncep atp iii (1) criteria require three or more of the following : waist circumference 102 cm for men and 88 cm for women ; fasting glucose 5.6 mmol / l or medication for high blood glucose ; systolic blood pressure 130 mmhg, diastolic blood pressure 85 mmhg, or antihypertensive medication ; triglycerides 1.7 second, the international diabetes federation (idf) (2) criteria specify central obesity with a waist circumference 94 cm for men and 80 cm for women of europid origin, plus two or more of the following : fasting plasma glucose 5.6 mmol / l or previously diagnosed type 2 diabetes ; systolic blood pressure 130 mmhg, diastolic blood pressure 85 mmhg, antihypertensive medication ; plasma triglycerides 1.7 mmol / l ; and plasma hdl cholesterol < 1.03 depression and anxiety were measured by the hospital anxiety and depression scale (hads) (15). the instrument consists of seven items for anxiety (hads - a) and seven for depression (hads - d), reported by respondents over the last week. responses are scored on items from 0 to 3 : separate summed scores for anxiety and depression range from 0 to 21, where normal is 07, mild is 810, moderate is 1114, and severe is 1521. in the present study, we defined anxiety and depression as having a score in the mild to severe range (8). psychological distress was assessed by the kessler 10 measure (k10) (16), a 10-item measure of the anxiety and depression symptoms experienced in the most recent 4-week period. responses are recorded on a five - point scale, and the score is the sum of the responses. total scores are categorized into two levels of psychological distress : low (1015) and moderate the internal consistency coefficients for the hads - a, hads - d, and k10 in this study were = 0.82, 0.79, and 0.87, respectively. ethics approval for this study was obtained from the flinders university clinical research ethics committee. informed consent in writing was obtained from participants when they attended the health check component of the survey. internal consistency was determined by using cronbach 's. pearson correlation coefficients were used to assess the intercorrelations between depression, anxiety, and psychological distress. pearson test was used to test the associations of depression, anxiety, and psychological distress with the presence of metabolic syndrome. independent t tests were used to compare mean age and alcohol consumption for patients with metabolic syndrome and for healthy subjects. multivariate analysis of covariance was used to test differences between individuals with the metabolic syndrome and individuals without the metabolic syndrome for psychological distress, anxiety, and depression. ancova was used to examine the association between depression and the five components of metabolic syndrome simultaneously. analyses were adjusted for age, sex, smoking status, alcohol intake, physical activity, marital status, and education. the questionnaire, which included questions on health behavior, symptoms and diseases, medical history, socioeconomic background, and psychosocial factors, together with the invitation to attend the health check, was sent by mail to all selected participants. health checks were carried out in local health centers or other survey sites by specially trained nurses. in the health check, weight, height, waist and hip circumference, systolic and diastolic blood pressure, as well as fasting lipids and glucose were ascertained, and bmi was computed as described in more detail elsewhere (4). the venous blood samples were drawn after an overnight fast of at least 10 h and analyzed at the flinders medical centre clinical trials laboratory, which is internationally accredited for lipid measurement under the centres for disease control lipid standardization program (atlanta, ga) (4). the most recent ncep atp iii (1) criteria require three or more of the following : waist circumference 102 cm for men and 88 cm for women ; fasting glucose 5.6 mmol / l or medication for high blood glucose ; systolic blood pressure 130 mmhg, diastolic blood pressure 85 mmhg, or antihypertensive medication ; triglycerides 1.7 mmol / l ; and hdl cholesterol < 1.03 mmol / l for men and < 1.30 mmol / l for women. second, the international diabetes federation (idf) (2) criteria specify central obesity with a waist circumference 94 cm for men and 80 cm for women of europid origin, plus two or more of the following : fasting plasma glucose 5.6 mmol / l or previously diagnosed type 2 diabetes ; systolic blood pressure 130 mmhg, diastolic blood pressure 85 mmhg, antihypertensive medication ; plasma triglycerides 1.7 mmol / l ; and plasma hdl cholesterol < 1.03 mmol / l for men and < 1.29 depression and anxiety were measured by the hospital anxiety and depression scale (hads) (15). the instrument consists of seven items for anxiety (hads - a) and seven for depression (hads - d), reported by respondents over the last week. responses are scored on items from 0 to 3 : separate summed scores for anxiety and depression range from 0 to 21, where normal is 07, mild is 810, moderate is 1114, and severe is 1521. in the present study, we defined anxiety and depression as having a score in the mild to severe range (8). psychological distress was assessed by the kessler 10 measure (k10) (16), a 10-item measure of the anxiety and depression symptoms experienced in the most recent 4-week period. responses are recorded on a five - point scale, and the score is the sum of the responses. total scores are categorized into two levels of psychological distress : low (1015) and moderate high (1650). the internal consistency coefficients for the hads - a, hads - d, and k10 in this study were = 0.82, 0.79, and 0.87, respectively. ethics approval for this study was obtained from the flinders university clinical research ethics committee. informed consent in writing was obtained from participants when they attended the health check component of the survey. internal consistency was determined by using cronbach 's. pearson correlation coefficients were used to assess the intercorrelations between depression, anxiety, and psychological distress. pearson test was used to test the associations of depression, anxiety, and psychological distress with the presence of metabolic syndrome. independent t tests were used to compare mean age and alcohol consumption for patients with metabolic syndrome and for healthy subjects. multivariate analysis of covariance was used to test differences between individuals with the metabolic syndrome and individuals without the metabolic syndrome for psychological distress, anxiety, and depression. ancova was used to examine the association between depression and the five components of metabolic syndrome simultaneously. analyses were adjusted for age, sex, smoking status, alcohol intake, physical activity, marital status, and education. for 1,345 men and women aged 2584 years, information was available for the metabolic syndrome, hads - d, hads - a, k10, smoking status, alcohol intake, and physical activity (table 2). a total of 409 (30.4%) participants met the ncep atpiii criteria (1) for metabolic syndrome. a total of 90 participants (6.7%) had diabetes (based on self - reported diabetes or fasting glucose 7.0 mmol / l) : 71 (5.3%) with and 19 (1.4%) without the metabolic syndrome. the characteristics of the 409 participants with and 936 without the metabolic syndrome are presented in table 2, which also shows the characteristics of the 338 subjects without diabetes who had the metabolic syndrome. when comparing all 409 metabolic syndrome participants with those without the metabolic syndrome, no sex - based prevalence differences were found. participants with metabolic syndrome were older (mean 60.5 vs. 55.0 years, p < 0.001). the correlations between depression (hads - d) and anxiety (hads - a), depression and psychological distress (k-10), and anxiety and psychological distress were 0.59 (95% ci 0.560.63), 0.66 (0.630.69), and 0.72 (0.690.74), respectively, and were all significant (p < 0.001). participants with the metabolic syndrome were more likely to have moderate to severe depression (10 vs. 6.9%, p = 0.069) ; but the two groups were not significantly different in psychological distress (30.1 vs. 25.7%, p = 0.115) or anxiety (9.8 vs. 10.4%, p = 0.820). multivariate analysis showed that participants with metabolic syndrome by ncep atp iii criteria (1) had higher scores for depression compared with individuals without the metabolic syndrome (mean scores 3.41 vs. 2.95, p = 0.013 [table 3 ]) after adjusting for sex, smoking status, alcohol intake, and physical activity. when each of the components of the metabolic syndrome was considered for all participants, both the hdl cholesterol and waist circumference components were independently associated with depression (table 3). participants with lower hdl cholesterol had higher scores for depression compared with individuals with higher hdl cholesterol (mean scores 3.75 vs. 2.93, p = 0.003). participants with a larger waist circumference had higher scores for depression than individuals with smaller waist circumference (mean scores 3.38 vs. 2.86, p = 0.002). the 338 participants with the metabolic syndrome but without diabetes were similarly more likely to have moderate to severe depression (10.1 vs. 6.9%, p = 0.086 [table 2 ]). again, no significant differences between groups were found in psychological distress (30.5 vs. 25.7%, p = 0.108) or anxiety (9.2 vs. 10.4%, p = 0.604). association of low hdl cholesterol (mean depression scores 3.68 vs. 2.92, p = 0.004) and large waist circumference (mean depression scores 3.36 vs. 2.86, p = 0.003) with depression were also found (table 3). participants (n = 409) with the metabolic syndrome had higher scores for depression than individuals without the metabolic syndrome (mean scores 3.30 vs. 2.95, p = 0.035, data not shown) after controlling for covariates. in the group of 338 participants with the metabolic syndrome but without diabetes, we obtained the same association when using the idf criteria (mean scores 3.27 vs. 2.95, p = 0.070, data not shown). metabolic syndrome was associated with depression, anxiety, and psychological distress in the 409 participants with metabolic syndrome (p = 0.009) as well as the 338 participants without diabetes (p = 0.032) when combined in multivariate analyses and adjusted for the same covariates. in the present study, we have demonstrated an association between metabolic syndrome and depression. although the association is modest, it is important because of the increasing prevalence of metabolic syndrome and the effect that depression can have on the ability of patients to successfully make lifestyle changes and comply with medication required for hypertension and dyslipidemia. the association is demonstrated here in a general population to our knowledge for the first time, whereas earlier studies (table 1) used subgroups of populations (813,17). this distinction is important because many individuals with metabolic syndrome have diabetes, which itself is known to be associated with depression (5). metabolic syndrome has been defined in several ways that involve quantitative anthropometric, clinical, and laboratory measurements (1,2). for the primary assessment, we chose ncep atp iii (1) criteria, since these criteria were used in most of the previously reported studies (8,9,1113,17). in addition, we used the more recently described idf criteria (2), with a lower cutoff point for waist circumference, and showed that the association was consistent across both definitions. of the components of metabolic syndrome, increased waist circumference was associated with depression, as reported in another study using a restricted population sample (9). this association is present regardless of the diabetes status and remained even when adjusted for significant covariates. we also found a significant independent relationship between low hdl cholesterol level and depression (table 3). this finding appears to be consistent with other recent research (17), but the underlying mechanism remains unknown. there has been little consistency in the psychological tests used in previous studies to measure depression (table 1). assessment varies from individual interview instruments (e.g., the structured clinical interview for dsm disorders or the hopkins symptom checklist) to self - reported epidemiological measures (e.g., centre for epidemiological studies depression scale) (table 1). most measures include somatic components of depression, and the length of recall ranges from present symptoms to past months., we used the hads, which has been designed for and validated in medical patients. it measures the presence of cognitive and affective components of depression and, unlike other instruments, excludes many of the somatic symptoms of depression (fatigue, loss of appetite and weight, sleep disturbance, psychomotor changes) that may overlap with physical problems. in addition, the length of recall is limited to the past week. the hads is widely used in population health studies and screening in primary care, although it has only been used in one reported study (13) linking metabolic syndrome and depression. the pathophysiological basis for the association between metabolic syndrome and depression is likely to be complex and to involve the inflammatory state that has been described as a consequence of central obesity (18). bjrntorp (7) postulated that psychosocial factors, including depression, can activate the hpa axis, producing hypersecretion of corticotrophin - releasing hormone, adrenocorticotropic hormone, and cortisol. this dysregulation of the hpa axis promotes deposition of visceral adipose tissue (6), which secretes inflammatory cytokines such as interleukin (il)-1 and il-6 and tumor necrosis factor (tnf)- (19,20). both il-6 and tnf- have been implicated in insulin resistance, which is considered to be the key factor in the metabolic abnormalities (21) of the metabolic syndrome. the proinflammatory response associated with depression may also have a direct effect on dyslipidemia (22). an alternative construct of the link between metabolic syndrome and depression places development of central obesity and activation of inflammatory processes as the initiating step. depression is seen as being a consequence of this immune activation (23). in this model, development of depression is analogous to sickness behavior, which can be associated with viral infection or other causes of immune activation. dysregulation of the hpa axis can occur via cytokine - induced stimulation of the central noradrenergic stress system (24). the main limitation of our study is the cross - sectional design, which does not allow for the demonstration of the existence of hpa axis activation and an inflammatory state in participants with central obesity. establishment of this link requires both longitudinal investigation and further analysis of blood samples, which would allow direct examination of the link between depression, inflammatory state, and metabolic syndrome. another limitation of this study is the rural population ; the association still needs to be demonstrated in urban groups as well as those who have greater cultural and socioeconomic diversity. while the characteristics of participants in our study closely resembled those of the local populations surveyed (4), it is possible that depressed individuals may have been less likely to participate. if this is the case, the present findings could over- or understate the association. in summary, our data show an association between the metabolic syndrome and the cognitive and affective components of depression in a general population, where the prevalence of depression in individuals with the metabolic syndrome is 50% higher. contrasting other studies, conclusions we have made are unlikely to be attributed to idiosyncrasies of the sample. based on the findings in this study, awareness of depressive symptoms as part of metabolic syndrome could be important in clinical management as in other chronic diseases. acknowledgment of depressive symptoms by the practitioner and the patient should improve ability to undertake lifestyle changes with adjustment of physical activity and food intake, as well as adherence to medications that are likely to be compromised by depression. identification and management of depression should therefore precede or accompany other measures in the management of metabolic syndrome. it is also possible that treatment of metabolic syndrome with lifestyle changes will ameliorate depression through reduction of visceral adiposity and inflammation. intervention studies to address this hypothesis could provide further insight into the relationship between depression, central obesity, and inflammation. | objective there is a recognized association among depression, diabetes, and cardiovascular disease. the aim of this study was to examine in a sample representative of the general population whether depression, anxiety, and psychological distress are associated with metabolic syndrome and its components.research design and methods three cross - sectional surveys including clinical health measures were completed in rural regions of australia during 20042006. a stratified random sample (n = 1,690, response rate 48%) of men and women aged 2584 years was selected from the electoral roll. metabolic syndrome was defined by the third report of the national cholesterol education program expert panel on detection, evaluation, and treatment of high blood cholesterol in adults, adult treatment panel iii (ncep atp iii), and international diabetes federation (idf) criteria. anxiety and depression were assessed by the hospital anxiety and depression scale and psychological distress by the kessler 10 measure.resultsmetabolic syndrome was associated with depression but not psychological distress or anxiety. participants with the metabolic syndrome had higher scores for depression (n = 409, mean score 3.41, 95% ci 3.123.70) than individuals without the metabolic syndrome (n = 936, mean 2.95, 95% ci 2.763.13). this association was also present in 338 participants with the metabolic syndrome and without diabetes (mean score 3.37, 95% ci 3.063.68). large waist circumference and low hdl cholesterol showed significant and independent associations with depression.conclusionsour results show an association between metabolic syndrome and depression in a heterogeneous sample. the presence of depression in individuals with the metabolic syndrome has implications for clinical management. |
burkitt 's lymphoma (bl) is a very high grade b cell neoplasm of the lymphoreticular system and is classified under nonhodgkin 's lymphoma (nhl). it was first described and published by denis burkitt, termed the lesion a sarcoma involving the jaws by noticing the lesions on the faces of central - african children. according to its geographic distribution, incidence, magnitude, and risk factors there are three types of bl : endemic bl, sporadic bl (sbl), and hiv infection associated bl. endemic bl is seen in equatorial africa and papua new guinea with estimated incidence rate of 5 - 15 cases/100,000 and mortality rates over 5.7/100,000. epstein - bar virus (ebv) is strongly associated in the development of endemic bl and cofactors may include equatorial humid weather of having malarial endemic, chromosomal abnormalities, immune defects, and protein energy deficits. endemic bl affects mainly facial skeleton in young children with peak incidence of 5 - 7 years, but can secondarily involve abdominal organs and bone marrow. central nervous system (cns) involvement in endemic bl is reported in one - third of the cases and may lead to headaches, paraplegia, and cranial nerve defects. sporadic bl is seen outside the african region in the rest of the world with estimated incidence rate of 0 - 8.8 cases in north america, 0 - 4.9 cases in far and middle east asia, and 0 - 4.6 cases in europe/100,000. the indian incidence varied from 0 to 1 case/100,000 with highest at bangalore and lowest at poona registries. the incidence rates of nhl have risen all over the world including india in the last 30 years. time - space clustering studies suggested the combination of genetic susceptibility and specific undetermined environmental factors plays a role in the tumor formation. sbl affects mainly abdominal organs, bone marrow infiltration, peripheral lymph node deposits, and waldeyer 's ring in older children and young adults with peak incidence of 10 - 12 years. head and neck involvement is seen in 10 - 30% of reported case series commonly in the form of cervical lymphadenopathy. facial bones and other extranodal sites of head and neck are involved in fewer than 10% of cases. we present the first reporting indian case of maxillary sbl with neuro - orbital involvement. a 21-year - old pharmacy student was referred by his general dentist to the department of oral medicine and radiology for the evaluation of swelling in the right side cheek region of 2 months duration. the swelling was asymptomatic and showed a gradual increase in size to attain the present dimensions. extraoral examination showed a mildly diffused, firm and nontender swelling in the right side maxillary region [figure 1 ]. single right submandibular lymphnode was palpable, measuring 1 2 cm in size, soft, tender, and nonadherent. intraoral examination showed a firm, diffused, and nontender swelling on the right maxillary teeth bearing area measuring around 7 5 cm in size with anterioposterior extension from central incisor to second molar and mediolateral extension from midpalatine region to buccal sulcus [figure 2 ]. dental caries was seen in right maxillary first molar, but was not involving the pulp. no periodontal pockets and mobility of the teeth extraoral photograph showing mildly diffused swelling on the right side maxillary region intraoral photograph of showing diffused swelling on the right side maxillary teeth bearing area extending from central incisor to second molar intraoral periapical and maxillary occlusal radiographs showed intact lamina dura with mild diffused bony rarefactions [figure 3 ] and panoramic radiograph did nt show any boney abnormality [figure 4 ]. routine blood investigations were done and all the values were within normal limits apart from slightly raised erythrocyte sedimentation rate. histological specimen from incisional biopsy revealed a round cell malignant tumor [figure 5 ]. on immunohistochemistry (ihc) examination, many tumor cells showed positive reaction to cd79 and cd20 (l26), some tumor cells showed positive reaction to cd45 (leukocyte common antigen) and cd45ro (uchl1), atypical tumor cells showed negative reaction to cd 3 and cd45ro (uchl1), and cd 10 was inconclusive. considering the histopathology, ihc reactions favored nhl consistent with bl [figure 6 ]. maxillary occlusal radiograph showing mildly diffused rarefactions of the right side palatal bone orthopantomogram showing no bone changes in the right side maxillary region photomicrograph stained with hematoxylin and eosin showing atypical lymphoid infiltrate in the connective tissue, 10 photomicrograph showing positive reaction of many tumor cells to cd20 marker four weeks after, the incisional biopsy, patient presented to the department with further increase in the maxillary swelling and diplopia of the right eye. the patient was then referred to the department of medical oncology of the regional cancer center, chennai for further evaluation and definitive management. on ophthalmic examination, patient had right side sixth cranial nerve and medial rectus palsy. computed tomography (ct), axial section showed a tumor mass involving the right side maxilla, and sagittal section showed the mass extended to the inferior wall of the right orbit [figure 7 ]. ultrasonography (usg) of the abdomen and lymph nodes showed features of mild hydronephrosis and a lobulated hypoechoic mass measuring 7.8 7.3 3.5 cm with cystic spaces in the right iliac region, anterior to the right common iliac vessels suggested mass of nodal origin. smear from bone marrow aspiration showed slightly hypercellular with 24% lymphocytes and 3% blast cells. complete blood picture and biochemical analysis were done and all values were in normal limits apart from raised serum glutamic oxaloacetic transaminase (sgot) and serum glutamic - pyruvic transaminase (sgpt). computed tomography, axial section showing tumor mass involving the right side maxilla the present case was classified as group c (any cns involvement and/or bone marrow involvement) under french, american, british staging system for childhood b - large and bl. chemotherapy regimen was started according to lmb 89 protocol group c. patient received prephase cyclophosphamide, oncovin, prednisone (cop) followed by induction phase cop adriamycin, methotrexate (copadm) i and ii. patient showed significant regression of both maxillary and abdominal mass with improvement of sixth nerve palsy, but developed severe neutropenia (neutrophils 0%, eosinophils 0%, monocytes 10.7%, lymphocytes 14.5%, and granulocytes 74.8%) which was rescued with granulocyte colony stimulating factor [figure 8 ]. patient received consolidation phase cyve i and ii (cytarabine (ara - c), vp-16 (etoposide) followed by copadm maintenance. smear from cerebrospinal fluid showed few small groups of atypical lymphoid cells suggestive of cns involvement. high dose chemotherapy regimen and radiotherapy was considered. however, patient died a week later from multisystem organ failure. sporadic bl is the fastest growing malignancy of the lymphoreticular system to affect humans with 80% of its cells undergoing mitosis at any point of time and has a potential ability to double in size every day. the differential diagnosis of acute swelling in the maxilla as initial and solo presentation without local factors is extremely difficult. it is very important for the oral medicine specialist to be familiar with such clinically presented rarities in the maxilla such as langerhan 's disease, eosinophilic granuloma, osteosarcoma, rhabdomyosarcoma, multiple myeloma, leukemia 's, malignant lymphomas or other rarest conditions possible because of the fact that they are the first one to come across and early diagnosis and prompt referral is imperative to prevent dissemination and to obtain favorable prognosis. there are few cases of sbl involving extranodal sites of head and neck region are reported and the sites of involvement include maxilla, mandible, palate, cheeks, tongue, gingiva, lower lip, tonsils, nasopharynx, maxillary sinus, orbit, ethmoid, sphenoid, mastoid, occipital, frontal bone, and thyroid. clinical features of sbl may vary from no signs and symptoms to life - threatening airway obstruction according to the severity, anatomic location, and temporal presentation of the disease. the clinical features in the jaws and surrounding structures may include facial swelling, exophytic growth, gingival hyperplasia, teeth mobility, teeth displacement, premature tooth eruption, pain, and/or sensory disturbances. pain is the most common presenting symptom and tooth pain as solo presentation secondary to dental tissue infiltrate is reported. mental nerve neuropathy as one of the clinical symptoms consists to pressure effect of the tumor has been described in the literature. two reports of mental nerve neuropathy or numb chin syndrome as only presentation of bl have been documented. our case showed unusual presentation ; despite the aggressive maxillary tumor extending to orbit did not show any common presenting signs and symptoms such as tooth mobility, tooth displacement, pain, and sensory disturbances except facial swelling. intraoral radiography showing break in the lamina dura around the teeth is one of the earliest radiological changes followed by numerous ill - defined small foci of radiolucent areas, which coalesce to form large radiolucent areas. radiological changes in the jaws may precede clinical involvement in sbl and some proposed radiological investigations for patients with unexplained features suspicious of sbl. the present case did not show any marked radiological changes apart from mild diffused rarefactions on occlusal radiograph despite of extensive tumor suggests radiological features may not have preceded the clinical involvement in the present case. the most common abdominal site of involvement is bowel and adjacent mesentery followed by retroperitoneum, hepatomegaly, splenomegaly, ileocaecal region, uterus, ascites, and adrenal mass in descending order. the less common right iliac region is involved in our case and usg showed hypoechoic mass with cystic spaces. no cases of hypoechoic mass with cystic spaces at abdominal sites on usg were reported in the literature. several blood investigations are consistently altered with sbl include anemia, thrombocytopenia, sgot and sgpt, alkaline phosphatase, serum lactic dehydrogenase (ldh), serum uric acid, and blood urea nitrogen. elevated serum ldh level is indicator for severity of tumor spread and also considered consistent with recurrent tumors. the onset and progression of tumor was rapid with no notable elevated hematological indicators apart from increased sgot and sgpt. cns involvement is uncommon in sbl and presented as babinski response in our case babinski response is plantar extension reflex suggestive of disease of spinal cord and brain. sbl with orbital involvement mainly affects older adults and our patient is youngest of all reported case. the presenting orbital signs and symptoms include eye pain, eyelid swelling, chemosis, altered visual acuity, proptosis, ptosis, diplopia, restricted ocular motility, and afferent papillary defect. the orbital involvement in our case presented initially as diplopia of the right eye followed by the right side sixth cranial nerve and medial rectus palsy. the present case is the first patient of sbl associated with sixth nerve and medial rectus palsy and was not reported previously in the literature. the prognosis of most of sbl with orbital involvement is poor and only one patient out of eight, including the present case was alive at 5 years posttreatment. the combination of neuro - orbital involvement resulted in extremely poor prognosis in the present case. the aggressive sbl patients may not necessarily show the common presenting clinical signs and symptoms, radiological findings, and varied laboratory panel as were reported in the literature, but not seen with our patient. the incidence of sbl is increasing worldwide ; varied and aggressive presentations can be documented like neuro - orbital involvement in our case. an unexplained aggressive clinical presentation must have a high index of suspicion for sbl. a delayed treatment in the present case resulted in neuro - orbital dissemination contributed to poor prognosis essentials the importance of early diagnosis, and definitive care of sbl patients. | burkitt 's lymphoma (bl) is the fastest growing malignancy of the lymphoreticular system to affect humans and has a potential ability to double in size every day. a case of maxillary sporadic bl (sbl) associated with neuro - orbital involvement in an indian male is presented. sbl initially presented as maxillary swelling with no obvious dental and periodontal changes. histological specimen from incisional biopsy revealed a round cell malignant tumor and immunohistochemistry reactions favored nonhodgkin 's lymphoma consistent with bl. four weeks later, patient presented with orbital involvement as diplopia, sixth cranial nerve palsy, and medial rectus palsy. chemotherapy regimen according to lmb 89 protocol was started. during chemotherapy regimen patient showed bradycardia and babinski response, suggestive of central nervous system involvement. sbl associated with orbital involvement is extremely rare and only seven cases have been reported. our case showed unusual presentation ; despite the aggressive tumor did not show any common clinical, radiological, and hematological findings. we also discussed the role of oral medicine specialist, importance of early diagnosis, and prompt referral in management of maxillary sbl. |
traditionally, most rna molecules were regarded as carriers conveying information from the gene to the translation machinery. the most prominent exceptions to this are transfer rna (trna) and ribosomal rna (rrna), both of which are directly involved in the process of translation. however, since the late 1990s, it has been widely acknowledged that other types of non - protein - coding rna molecules are present in organisms ranging from bacteria to mammals, which affect a large variety of processes including plasmid replication, phage development, bacterial virulence, chromosome structure, dna transcription, rna processing and modification, development control and others (116). these observations suggest that the traditional view of the structure of the genetic regulatory systems in organisms is far from complete. therefore, further research on non - protein - coding rna will give us a new framework for considering and understanding the genomic programming of biological complexity. however, the unsystematic naming of non - protein - coding rnas may be an impediment to effective research. the term small rnas (srnas) has been predominantly used for such rnas in bacteria, whereas the term non - coding rnas (ncrnas) has been the most common term for eukaryotic rnas of this kind (17,18). to have a common term for all such rnas, we have opted to apply the term ncrna to all these functional rnas, irrespective of the realm of life in which they might appear. the understanding of the importance of ncrnas in basic cellular processes is ever increasing, and new members and classes of ncrnas are continuously being reported. thus, over the years, several databases have been established to collect, organize and classify ncrna sequences and information. some databases are intended to collect only certain category of ncrnas, such as srp rnas, tmrnas or rnase p rnas, whereas others, such as the small rna database, the non - coding rna database and the rfam database, have collected ncrnas of several categories (1924). however, even in the latter kind of databases certain ncrna members or classes are missing. another problem with all the current databases is that the classification systems for ncrnas used nowadays are not uniform and only a few attempts have been made to integrate the various classification systems. in these classification systems, some ncrna groups are named according to cellular localizations, such as snrnas, snornas or scrnas, some are named according to functions, like prnas (package rnas), grnas (guide rnas) or tmrnas (transfer - messenger rnas), and others again are simply labeled according to their sedimentation coefficients (6s rna, 5.3s rna, etc.). furthermore, because of this lack of integration, one type of ncrna often appears under several names or in more than one category (7,12,2530). noncode comprises almost all ncrnas now publicly available (except trnas and rrnas) that are either confirmed experimentally or predicted computationally. the first release of noncode (v1.0) contains 5339 non - redundant sequences from 861 organisms, including eukaryotes, eubacteria, archaebacteria, virus and viroids. furthermore, to integrate existing classification systems, a new classification system labeled the process function class (pfclass) has been introduced, based on the cellular process and function in which a given ncrna is involved. pfclass provides a unified classification system and a concise functional annotation of ncrnas. according to the cellular process involved, the pfclass classification system is the first attempt of a unified classification system for ncrnas. it is our hope that this integrated system will help in clearing up the classification problem. in conclusion, the aim of the noncode database is to be a unified gateway to search, retrieve and update information about ncrnas in order to facilitate research on ncrnas, gene networks and functional genomics. through a user - friendly web interface at http://noncode.bioinfo.org.cn, genbank entries were the major source of data, and the pubmed database was used as the starting point for the data collection (31). the publications that matched with these queries were then examined and the ncrna sequences were extracted from the obtained literature. by reading the filtered literature, a new set of ncrna keywords were gained and added into the keywords table. this new keywords table was used to filter the genbank bct, inv, mam, phg, pln, pri, rod, vrl and vrt divisions automatically, and the filtered result was then manually confirmed. all the data are integrated and organized in such a manner that users can efficiently query and browse information. one significant characteristic of noncode is its content of additional information on the ncrnas obtained from the related literature. (i) for each sequence filtered from genbank, we manually checked whether or not it represented an actual ncrna and assigned the confirmed sequence an accession number (ncid, i.e. ncrna i d). (ii) basic information name, alias, length, organisms, references, etc.of confirmed sequences was collected from genbank. (iii) additional information concerning function, cellular role, cellular location, etc. each ncrna has also been annotated with one of the five specific mechanisms (sequence base pairing, structural complementarity, spatial blocking, catalysis or epimodification), through which it exerts its function. (iv) according to our pfclass classification system, one or more of the 26 pfclasses were assigned to all ncrnas. moreover, a subset of 1114 ncrnas have been divided into nine additional categories according to whether they are specific to gender or tissue or associated with tumors and diseases, etc. (v) to visualize the location of an ncrna in the genome or in a specific dna fragment, along with regulatory elements in the flanking sequences, genbank annotations were used to create figures for all ncrnas. (vi) each ncrna sequence was checked for redundancies using perl scripts, and each cluster of redundant sequences was given a non - redundant accession number (uniqid, i.e. unique ncrna i d). (vii) the secondary structures of non - redundant ncrna sequences were predicted using the vienna rna package (32). ever since the beginning of ncrna research there has not been in place any integrated system for classification, and therefore, exists a considerable measure of confusion with respect to naming of ncrnas. therefore, when the noncode database was established it was carefully considered as how to establish classification criteria that might increase the usefulness of the database resource. the cellular process and function of an ncrna was chosen as the basic criterion for a unified classification system called pfclass in noncode. when labeled according to this system, each kind of ncrna is named after its cellular process and corresponding function. the actual category is given according to two or three levels of keywords connected by an underscore. the first keyword will be dna, rna or protein, representing a cellular process in which either of the three molecular types is a crucial component. the second keyword describes the actual process, and if the ncrna is involved in a complex process with several aspects, a third keyword may further indicate a more specific function of the ncrna. for example, the snrna u1 will be assigned to the pfclass rna_processing_splicing, and rnase p rnas to the pfclass rna_processing_cleavage (for details see table 1). the pfclass classification system represents the first attempt of a unified classification system for ncrnas. in the future, as our understanding of ncrnas deepen, and the content of noncode further expands, steps will be taken to further extend and perfect the pfclass system in order to increase its usefulness. to further harmonize the exchange of data between different systems, application of gene ontology (go) (33) annotation on our pfclass system will be considered. genbank entries were the major source of data, and the pubmed database was used as the starting point for the data collection (31). the publications that matched with these queries were then examined and the ncrna sequences were extracted from the obtained literature. by reading the filtered literature, a new set of ncrna keywords were gained and added into the keywords table. this new keywords table was used to filter the genbank bct, inv, mam, phg, pln, pri, rod, vrl and vrt divisions automatically, and the filtered result was then manually confirmed. all the data are integrated and organized in such a manner that users can efficiently query and browse information. one significant characteristic of noncode is its content of additional information on the ncrnas obtained from the related literature. briefly, seven steps were carried out after the genbank screening. (i) for each sequence filtered from genbank, we manually checked whether or not it represented an actual ncrna and assigned the confirmed sequence an accession number (ncid, i.e. ncrna i d). (ii) basic information name, alias, length, organisms, references, etc.of confirmed sequences was collected from genbank. (iii) additional information concerning function, cellular role, cellular location, etc. each ncrna has also been annotated with one of the five specific mechanisms (sequence base pairing, structural complementarity, spatial blocking, catalysis or epimodification), through which it exerts its function. (iv) according to our pfclass classification system, one or more of the 26 pfclasses were assigned to all ncrnas. moreover, a subset of 1114 ncrnas have been divided into nine additional categories according to whether they are specific to gender or tissue or associated with tumors and diseases, etc. (v) to visualize the location of an ncrna in the genome or in a specific dna fragment, along with regulatory elements in the flanking sequences, genbank annotations were used to create figures for all ncrnas. (vi) each ncrna sequence was checked for redundancies using perl scripts, and each cluster of redundant sequences was given a non - redundant accession number (uniqid, i.e. unique ncrna i d). (vii) the secondary structures of non - redundant ncrna sequences were predicted using the vienna rna package (32). ever since the beginning of ncrna research there has not been in place any integrated system for classification, and therefore, exists a considerable measure of confusion with respect to naming of ncrnas. therefore, when the noncode database was established it was carefully considered as how to establish classification criteria that might increase the usefulness of the database resource. the cellular process and function of an ncrna was chosen as the basic criterion for a unified classification system called pfclass in noncode. when labeled according to this system, each kind of ncrna is named after its cellular process and corresponding function. the actual category is given according to two or three levels of keywords connected by an underscore. the first keyword will be dna, rna or protein, representing a cellular process in which either of the three molecular types is a crucial component. the second keyword describes the actual process, and if the ncrna is involved in a complex process with several aspects, a third keyword may further indicate a more specific function of the ncrna. for example, the snrna u1 will be assigned to the pfclass rna_processing_splicing, and rnase p rnas to the pfclass rna_processing_cleavage (for details see table 1). the pfclass classification system represents the first attempt of a unified classification system for ncrnas. in the future, as our understanding of ncrnas deepen, and the content of noncode further expands, steps will be taken to further extend and perfect the pfclass system in order to increase its usefulness. to further harmonize the exchange of data between different systems, application of gene ontology (go) (33) annotation on our pfclass system will be considered. till date, more than 10 000 sequences filtered from genbank by our in - house program have been manually examined. the current release (v.1.0) of noncode contains a total of 6232 entries assigned to 26 pfclasses, and covers 109 traditional classes such as snrna, snorna, microrna and rnase p rna. basic information on each entry is provided, including accession number in genbank, traditional class, name, pfclass, organism, reference, uniqid (accession number without redundancy in noncode) and ncid (accession number with redundancy in noncode), all of which can be used as keywords for data search. noncode also provides additional information on function and cellular role, cellular location, chromosomal information, alternative names, secondary structure and whether or not the ncrna has undergone splicing. each ncrna has also been annotated with one of the five specific mechanisms (sequence base pairing, structural complementarity, spatial blocking, catalysis or epimodification), through which it exerts its function. figures showing genomic locations for all ncrnas and their regulatory elements have been included, and a subdivision into nine additional classes (outside the pfclass system) has also been applied to a number of ncrnas. noncode also offers an efficient search option, allowing recovery of sequence, related publications and other information. in the near future (i) for a number of ncrnas, information on function, location, etc. is still lacking, and this information will be completed as soon as it becomes available. (ii) as the information on ncrnas increases and the content of noncode further expands, the pfclass system will be further extended and perfected in order to increase its usefulness. go annotation on the pfclass system will also be considered seriously, with the aim of harmonized exchange of data between the different systems. (iii) additional services such as blast alignment, ncrnas prediction and possibilities for submission and registration of users ' sequences will be provided. in addition, two large - scale screens for novel ncrnas in caenorhabditis elegans and human tissues are being carried out in our laboratory (y. wang, z.y. he, unpublished data), and the results will be added in the next version of noncode. noncode is thus designed to adapt and to reflect the most current information on ncrnas available. it will continue to grow in both content and functionality, and will be updated every six months to include any new data from literature and genbank. we are indebted to all of our colleagues who focus much of their research efforts on the analyses of ncrnas. kscx2 - 2 - 27, national sciences foundation of china grant nos 39890070 and 60496320, the national high technology development program of china under grant no. 2002aa231031, national key basic research & development program 973 under grant nos 2002cb713805 and 2003cb715900, and beijing science and technology commission grant no. h010210010113. | noncode is an integrated knowledge database dedicated to non - coding rnas (ncrnas), that is to say, rnas that function without being translated into proteins. all ncrnas in noncode were filtered automatically from literature and genbank, and were later manually curated. the distinctive features of noncode are as follows : (i) the ncrnas in noncode include almost all the types of ncrnas, except transfer rnas and ribosomal rnas. (ii) all ncrna sequences and their related information (e.g. function, cellular role, cellular location, chromosomal information, etc.) in noncode have been confirmed manually by consulting relevant literature : more than 80% of the entries are based on experimental data. (iii) based on the cellular process and function, which a given ncrna is involved in, we introduced a novel classification system, labeled process function class, to integrate existing classification systems. (iv) in addition, some 1100 ncrnas have been grouped into nine other classes according to whether they are specific to gender or tissue or associated with tumors and diseases, etc. (v) noncode provides a user - friendly interface, a visualization platform and a convenient search option, allowing efficient recovery of sequence, regulatory elements in the flanking sequences, secondary structure, related publications and other information. the first release of noncode (v1.0) contains 5339 non - redundant sequences from 861 organisms, including eukaryotes, eubacteria, archaebacteria, virus and viroids. access is free for all users through a web interface at http://noncode.bioinfo.org.cn. |
idiopathic infertility is a condition that refers to those men who have abnormal semen parameters without an identifiable cause based on history, physical examination, and currently available laboratory and radiographic examination. the fact that is impossible to determine the precise etiology of this form of infertility does not allow for making a rational treatment but only an empirical one ; spermatogenesis is supported acting on various pathways. a wide number of agents have been proposed as specific treatment for men with infertility but there is no strong evidence of any therapy and nowadays there is still no consensus between various societies that are researching in this specific field. oxidative stress (os) is defined as the unbalance between production of free radicals, molecules characterized by high reactivity due to one or more unpaired electrons in the external orbital, and antioxidant defenses in the biological systems. nowadays, it is widely accepted as an important pathogenetic mechanism in different diseases. among free radicals the most important and studied are reactive oxygen species (ros), of which the most in vivo production occurs most of all during oxidative processes of energetic substrates in the mitochondrial respiratory chain [2, 3 ]. however, other important kinds of free radicals exist, besides ros, among which nitrogen reactive species are the most studied. an augmented ros production can be the consequence of an augmented electronic flow in the respiratory chain, when it is activated from an augmented energetic demand or an augmented contribution of substrates, as what occurs in obesity. in leukocytes and many other cytotypes, as endothelial and mesangial cells, fibroblasts, thyrocytes, oocytes, leydig cells, adipocytes, epstein - barr infected cells and neoplastic cells, ros generation has been assessed to have a positive physiologic functional role, different from respiratory burst. however an uncontrolled production of free radicals was linked to many pathologic events, as rheumatoid arthritis and myocardial infarction, and in general ros damage occurs in inflamed tissues, characterized from cellular lysis and intracellular content release. moreover in diabetes mellitus, oxidation, accompanying glycation in vivo, supports the formation of more permanent, irreversible chemical modifications on different kinds of molecules ; metalcatalyzed autoxidation of glucose in the presence or absence of protein is paralleled by the generation of reactive oxygen species. the formation of glycoxidation products in vivo depends on the relative glucose concentrations, but also on the local oxidative environment. on the other hand in diabetic patients antioxidant capacity it is possible to characterize different cellular defensive mechanisms against the free radical damage which act in the endoplasmic network, mitochondria, plasmatic membrane, peroxisomes, and cytosol as well as extracellular ambient. the first mechanism is the prevention of production or the rapid inactivation of free radicals, due to the activity of several enzymes, like catalase, peroxidase glutathione complex, and superoxide dismutase (sod), or of transition - metals binding proteins, like transferrin, ferritin and ceruloplasmin. the second mechanism determines an interruption of propagation of the lipid peroxidation chain by a reaction with the intermediate radicals and the consequent their neutralization. scavengers, which can be water soluble, as albumin, bilirubin, ascorbic acid, urates and thiols, or liposoluble, as vitamin e and coenzyme q10, the only liposoluble antioxidant synthesized in the living organisms. the mobility of scavengers, particularly the liposoluble ones, and above all at membrane level, allows for intercepting radicals and transformin them in more stable molecules and therefore stopping the damage chain. the third defensive mechanism uses processes which remove molecules damaged by oxidative attack, allowing the reconstitution of normal structures (e.g., specific phospholipases remove the peroxidized fatty acids, making possible the reacylation of damaged molecule by an acyl - coa and the respective enzyme). recently a lot of methods have been developed in order to measure the total antioxidant status in biological fluids. total antioxidant capacity (tac) is a measurement of the nonenzymatic antioxidants, that are primarily extracellular, as ascorbate, urates, albumin, tocopherol, and glutathion. they are chain - breaking molecules able to block the propagation chain of lipid peroxidation and to prevent the amplification of radical generation and the subsequent biochemical damage. differently from enzymes, they are consumed at the moment in which they act and this fact could explain the reduction in their levels in biological fluids producing ros (e.g., the high levels of ros and a low tac in the seminal fluid of infertile males suggests oxidative stress is associated with a variety of etiologies of male infertility). tac is considered an index of the antioxidant status of a biological sample better than the measurement of one or more specific antioxidants, that could not carefully reflect the combined effect of the various antioxidants and their collaboration during the oxidative stress. we have applied the method of rice - evans and miller, with some modifications, to different endocrine diseases and the results are summarized in the following paragraphs. it consists in tac evaluation by using the system metmyoglobin - h2o2, as source of radicals, in presence of the chromogen 2,2-azinobis-(3-ethylbenzothiazoline-6-sulphonate), the radical form of which is spectroscopically detectable after a latency time (lag) due to antioxidants presence and therefore proportional to their content. the involvement of os in male infertility is well established, but studies on the relationships between sperm quality, seminal antioxidant, and os continue to be published [12, 13 ]. an increase in seminal ros is associated with sperm dna fragmentation ; the better parameter to identify such condition should be ros - tac score. recently it has been shown that not only seminal but also blood antioxidants can be considered biochemical markers to support sperm quality evaluation. different prooxidative conditions can negatively influence sperm viability and motility, sharing os as common end, including exposure to radiation, extern temperature, drugs and toxins, heavy metals, smoking, biological hazards, and electromagnetic [16, 17 ]. finally, new mechanisms have been recently discovered, such a ros - induced uncoupling between electron transport and atp synthesis, by evaluation of sperm mitochondrial respiratory activity with a polarographic assay in hypotonically treated sperm cells ; on the other hand, mitochondrial independent mechanisms, such as alteration of atp utilization or contractile apparatus of the flagellum, have been hypothesized, since menadione (mitochondrial generator of superoxide) and hydrogen peroxide caused an immediate disruption of motility, with delayed or no decrease in atp content, respectively, in a model of boar sperm. considering all the reported data, antioxidant therapies have a strong physiopathological rationale for the employment in idiopathic male infertility. there is evidence that 17--e2 induces ros production in mitochondria as signal - transducing messengers. in this way it activates the binding of three oxidant - sensitive transcription factors : ap-1, creb, and nuclear respiratory factor 1. however, the production of ros seems to exert negative effect and explain a sex - related differential longevity. estrogens bind to e - receptors and, via activation of map kinase and nf-b pathways, result in an upregulation of antioxidant enzymes. moreover, the oxidative damage of mitochondrial dna is fourfold higher in males than females. findings in transgenic animal overexpressing mn - sod or catalase strongly support this view. e2 increases glutathione levels in different cellular cns models, both neuronal and glial cells. in other models (rat liver microsomes, with lipid peroxidation determined by measuring tbars after exposure to various prooxidants) catechol estrogens appeared the most potent antioxidants, even if estrogens and catechol estrogens interact with the peroxidative process at different levels. protective effects of estrogens on ter - butylhydroperoxide - induced hepatocyte damage were studied in vitro, suggesting that this protection may be related not only to their antioxidant activity against free radicals but also to the maintenance of the normal redox status of cells, which partially restores intracellular gsh levels. neutrophil granulocytes play an important role in atherogenesis also through their free radical generation. despite the conflicting results in the literature [27, 28 ], a suppressive effect on superoxide anion production of human neutrophil granulocytes by 17--estradiol, progesterone and testosterone was demonstrated. despite these effects, estrogen - like compounds mediate dna damage by ros generation in human lymphocytes and sperm. in ovariectomized wistar rats, administration of estradiol reduced mda, together with a slight decline in catalase and sod, with no modification of vitamins a and e. the different effects of estrogens on the glandular and aglandular portions of endometrium were studied in ovariectomized ewes : treatment with e2 decreased sod1, catalase and gpx in both the endometrial tissues, and gsr only in the glandular part. progesterone did not influenced sod2, catalase, gpx and gsr but induced a sod1 decrease in the aglandular tissue. as far as vascular system is concerned, estrogen depletion after ovariectomy in rats induced, via oxidative stress, the activation of heme - oxygenase 1 (ho-1), an inducible stress protein. no / inos system contributes to the induction of ho-1, which may subsequently suppress inos activity. an interesting review on the cellular and molecular mechanisms underlying vasculoprotective action of estrogens explains how e2 can act through a nongenomic stimulation of membrane / intracellular mediators and/or the classical genomic pathway. in this way recently, the effect of oxidative stress in female reproduction has been reviewed, and it appears to be involved in the pathophysiology of infertility and assisted fertility. oxidant status of the cell modulates angiogenesis, which is critical for follicular growth, corpus luteum formation, endometrial differentiation and embryonic growth. erythrocyte gsh - px is influenced during the menstrual cycle : higher gsh - px activity was found from the later follicular to early luteal phase. a significant correlation was observed between e2 and gsh - px cycle - related changes, while no significant cycle phase - dependent variation was found in pyruvate kinase activity. estrogen treatment reduces peroxidation of neuronal synaptic membrane in postmenopausal women, thus preventing neurovascular and neurodegenerative disorders. a large number of neurological and psychiatric diseases like parkinson, sla, dementia and schizophrenia show an enhanced ros production and beneficial effects due to antioxidant properties of estrogen have been shown. even if these data indicate that e2 is a potent preventative agent against neurodegenerative diseases, by activating antioxidant defense systems, scavenging ros, limiting mitochondrial protein damage, improving electron transport chain activity and reducing mitochondrial dna damage, the high oxidative cellular environment in neurodegeneration makes e2 a poor agent for the treatment of a overt disease. oxidative stress stimulates the production of the hydroperoxide - dependent hydroxylation of estradiol to the catechol estrogen metabolites, which can undergo ros producing redox cycling, setting up a self - generating toxic cascade offsetting any antioxidant / antiapoptotic effects generated by the parent estradiol. additional factors, related to the disease, can further complicate such a condition : for instance a dysregulation of the catecholamine system could alter comt - catalyzed methylation, preventing removal of redox cycling catechol estrogens from the system enhancing prooxidant effects of e2. initially attributed to ldl lowering and hdl increasing, this protective effect is centered on decreased ldl oxidation. a direct effect on arterial tissue and a modulation of vascular reactivity through no and prostaglandin synthesis, based on both receptorial and immediate nongenomic mechanisms, are also involved. recently another mechanism was hypothesized implicating the esterification of estrogens in hdl and the transfer to ldl. despite these mechanisms, two recent placebo - controlled studies on women with chd failed to show beneficial effects of hormone replacement therapy on coronary events, even if mutations in thrombogenic genes may represent an important confounding factor. postmenopausal women develop visceral obesity and insulin resistance and are at increased risk for type 2 diabetes. in the management of menopausal disturbances both natural steroid and selective estrogen receptor modulators (serms) could be useful for antioxidant activity, but both positive and negative effects have to be considered when evaluating the role of estrogen in cardiovascular disease [44, 45 ]. these drugs differ from pure receptor agonists and antagonists because their action is different in various tissues, granting the possibility to selectively inhibit or stimulate estrogen - like action in various tissues. both clomiphene citrate and tamoxifen (tmx) have been suggested as empiric treatments for male infertility, with different reports in the last decades. tmx has reached a leading role as a suitable gonadotropin stimulator rising directly and indirectly the testicular function with a clear advantage on clomiphene that, according to its estrogenic effect, would seem to lower the sex - hormone binding protein and testosterone levels depressing spermatogenesis [4650 ]. the effects of different serms on hypothalamic - pituitary - testicular axis have been investigated in men with idiopathic oligozoospermia : tmx (20 mg / daily), toremifene (60 mg / daily) and raloxifene (60 mg / daily), administration resulted in a significant increase in gonadotropin levels, which was more marked for tmx and toremifene compared with raloxifene. several medical trials assessed the efficacy of tmx in infertility treatment, but the results were not strong enough to justify general acceptance. similarly, a multicentric who study showed only an 8% increase in pregnancy rate in couples receiving clomiphene versus placebo. another meta - analysis of antiestrogen therapy (clomiphene and tamoxifen), including randomized and placebo - controlled studies, concluded that such a treatment had no significant influence on pregnancy rate. another meta - analysis, which excluded some previously considered studies (in which vitamin c was used as placebo) and included other randomized controlled trials, showed an overall beneficial effect on pregnancy outcome (pooled or : 2.42, 95% ci 1.48, 3.94 p = 0.00004). a modest but significant increase in sperm concentrations and a statistically significant improvement in sperm motility were shown in the same study. tmx seems to have a strong effect on sperm count and concentration in eugonadal patients but it does not improve other semen values such as volume, ph, motility, morphology, and viability and this could be related to its effect on seminiferous tubules where it should ameliorate first steps of spermatogenesis. a recent article suggests a better effect of tmx in patients with low fsh levels before starting the therapy suggesting the need for a well - functioning hypothalamic - pituitary - gonadal axis. the tmx therapy has been supplemented with low dose of testosterone undecanoate (tu), a weak androgen that in this context does not interact with the axis but improves other sperm parameters, except for sperm count, and the chances of conception. the efficacy of tmx association with coenzyme q10 has been investigated in a group of 183 patients affected by idiopathic oligoasthenozoospermia, randomly assigned to different treatment schedules : tmx + coq10, tmx, or coq10 alone. in the first two groups, as expected, fsh, lh, and testosterone levels significantly increased ; sperm motility and % of morphologically normal forms were significantly higher in tmx + coq10 and coq10 groups and slightly increased, but without statistical significance, in the tmx group. however, due to the role of ros in male infertility and the role of estrogens in modulating antioxidant systems, as reviewed in the previous paragraph, they could also have a pathophysiological basis to explain their positive effects. no studies are reported on the effects of tmx on antioxidant capacity in seminal plasma. the role of antioxidants in male infertility treatment is well recognized [59, 60 ] ; other therapies can influence tac suggesting the possibility that hormonal therapy, at least in part, has beneficial effects through the balance between ros and antioxidants. we previously reported that a treatment with coq10 is able to increase seminal plasma tac, also in low dose schedule (100 mg / daily), slightly influencing seminal parameters of varicocele patients. on the other hand, a fsh treatment (225 ui / week for three months) induced a trend toward increase in endogenous seminal plasma coq10 levels in oligoasthenozoospermic subjects. recently we have investigated the effects of tmx administration (20 mg daily for a three - month period) in a group of 5 patients, aged 2732 years, affected by idiopathic infertility. criteria of exclusion were varicocele, primary or secondary hypogonadism, and low urinary tract inflammation. they were oligospermic, with a mean basal sperm concentrations of 14.7 10.3 10/ml, 32.8 22.3% of progressive motile cells, and 30.0 7.8% normal morphology. lag values were significantly increased by hormonal treatment (120.0 14.1 versus 102.5 10.6 sec). these very preliminary observations need to be validated in a large sample and in double - blind, placebo - controlled study. however they again suggest the possible role of estrogens as modulators of antioxidants system in human semen and furnish a further rationale, on biochemical basis, for antiestrogens in male infertility. | among treatments proposed for idiopathic male infertility, antiestrogens, like tamoxifen, play a possible role. on the other hand, oxidative stress is a mechanism well recognized for deleterious effects on spermatozoa function. after reviewing the literature on the effects of estrogens in modulation of antioxidant systems, in both sexes, and in different in vivo and in vitro models, we suggest, also on the basis of personal data, that a tamoxifen treatment could be active via an increase in seminal antioxidants. |
world health organization (who) has described mood disorders as the major health issues of the 21st century. bipolar mood disorder (bmd) is a chronic disease, with acute attacks, and has a lifetime prevalence of almost 2% to 4%. such disorder, which is associated with various disabilities, can be detected in the people of every social class and race. more attention has been paid to the diagnosis of bmd in children and adolescents and the rate of diagnosis in such age group has been increased. moreover, 13% to 28% of the patients with bmd have disease onset before the age of 13 years and 50% to 66% before the age of 18 years. have conducted several studies focusing on adolescents suffering from such disease and their family since 2001. research has shown that having a family member with a mood disorder affects the whole family and leads to loss of their abilities and adaptability. such deficiencies, not only cause stress in the family members during the acute attacks, but they also affect the course of the disease. in the literature review conducted by steele. (2010), the rate of depression and anxiety in the family members who take care of bmd sufferers has been reported 40% to 55%. the family members of such patients experience many difficulties and pressures during the periods of treatment, rehabilitation and recovery. furthermore, their quality of life (qol) is threatened and, in some cases, they feel depressed and anxious as well. hence, managing the disease demands a direct interaction between the patients family members and mental health professionals. families of adolescents with bmd, who take care of the patients, often feel isolated in their struggle to cope with the disease and seek help for their children. therefore, focusing on the patients family is considered as a logical starting point since the adolescents live with their primary family who are responsible for taking care of them. accordingly, family - focused education is a psycho - educational method and a skills training approach for the families with patients suffering from bmd. family psycho - educational intervention could significantly decrease psychological problems of family caregivers if it is combined with common mental health care. family - focused psycho - education is an effective treatment strategy as its positive effects on children and adolescents with bmd have been proven in the review studies. family - focused psycho - education is a new approach developed for the families of children with such mood disorder to help them cope with the disease. research has revealed that not only this type of education has been effective for adults with bmd, but it also could decrease the severity of the disease, improve relationships and increase problem solving skills and adaptability in the families. another study demonstrated that such an intervention could enhance the knowledge and information about the disease in bmd sufferers and their families. during their one - year follow - ups, the researchers observed a decrease in the psychological problems caused by care - giving and high emotional expression. currently, qol is one of the issues of interest to international communities and researchers. who has also paid special attention to the development of health care assessment, beyond its traditional criteria such as mortality and morbidity, to evaluate the influential power of physical and psychological diseases on the ability of performing daily activities. the researchers believe that physical health is influenced by psychological growth and mental health promotion is based on the prevention and treatment of emotional stresses. functioning in other domains of qol therefore, performing interventions on such patients assists inimproving qol, accelerating recovery, reducing length of hospital stay and eventually health care costs. otherwise, reduced mental health can negatively affect qol and it leads to job loss, family disruption, impaired interpersonal communication, and inability to perform personal, family and social responsibilities. hence, assessing qol in medical environment can be valuable due to some reasons. the techniques and measurement tools used to assess qol could provide information which has been neglected or not achieved by traditional analysis of treatment outcomes. assessing qol as one part of treatment outcomes helps the clinicians and therapists to realize the slight differences that exist in individuals responses to treatment. several studies have also confirmed the effectiveness of community - based interventions, home follow - ups, the role of supportive services, rehabilitation and clinical interventions in the improvement of qol in the patients. so far, a few studies have been done on the adolescents with bmd and their families especially in iran while no study in iran has focused on the families of the patients with such disorder. therefore, considering the importance and early onset of bmd and the fact that adolescence is a sensitive period, we aimed to evaluate the effect of family - centered education on mental health and qol of families with adolescents suffering from bmd referred to medical centers affiliated to shiraz university of medical sciences. this intervention was performed on 40 families (38 mothers and 2 fathers) of the adolescents with bmd which referred to the psychiatric clinics affiliated to shiraz university of medical sciences during 2012 - 13. significant results after the comparison between and within groups shows an adequate sample size (post power analysis > 75%). they were randomly assigned to intervention and control groups using block randomization method (block size : 4) (figure 1). design and protocol of the study inclusion criteria were having an adolescent aged 12 - 18 with confirmed diagnosis of bmd by a child and adolescent psychiatrist and lack of any other mental disorders such as mental retardation, epilepsy, schizophrenia, and drug abuse. however, exclusion criteria were unwillingness to participate in the study and absence more than two sessions in the educational program. data were collected using general health questionnaire (ghq), quality of life assessment questionnaire (short form-36) and demographic questionnaire. demographic data included age, sex and educational level of the adolescent and his / her parents, a history of physical or psychological diseases in the family as well as the number of siblings and birth order. the questionnaires were completed by all participants three times : before, immediately after and one month after the intervention. it is a 28-item self - report questionnaire which contains 4 subscales measuring somatic symptoms : anxiety, sleep disorder, social dysfunction, and depression. each sub - scale contains 7 items and each item is scored on a 4-point likert scale. the score for each subscale ranges from 0 to 21. the total score, which is obtained by summing up the scores of all subscales, ranges from 0 to 84 with lower scores indicating higher general health status. the reliability and validity of the questionnaire have been assessed in various studies and also estimated and confirmed in iran. by conducting a pilot study, yaghoubi estimated the sensitivity and specificity of the questionnaire as 86.5% and 82%, respectively and the cut - off score as 23 based on a likert - scale scoring. the reliability coefficient of the questionnaire was estimated 88% using test - retest and cronbach s alpha. we used the persian version of chq-28, which was translated by yaghoubi and palahang (1995), under the supervision of dr. short form of qol questionnaire (sf-36) is a 36-item self - report instrument designed to measure health - related qol across eight dimensions of physical functioning, role limitations caused by physical and emotional problems, bodily pain, general health, mental health, social functioning, emotional problems, energy / fatigue, and health changes. the scores on each dimension range from 0 to 100 with higher scores reflecting higher quality in that dimension. the questionnaire has been extensively used and its validity and reliability were estimated in various studies. furthermore, internal consistency of the questionnaire was estimated by calculating cronbach s alpha coefficient which was reported between 0.72 and 0.94. in iran, this study was approved by ethics committee of shiraz university of medical sciences (no ct-92 - 6718). all the families in the intervention group participated in a family - centered educational program consisting of six 90-minute sessions per week for 6 weeks. 20 participants in the intervention group were divided into groups of 4 and all of them attended the workshops and received the same content. the educational workshops were directed by a psychiatric nurse and a child and adolescent psychiatry specialist. to provide the intervention protocol, we used fristad s educational package (2003) and family educational package on bmd in children and adolescents prepared by mahmoudi - gharaei. family educational package included information about the nature and symptoms of mood disorders, therapeutic methods, complications and their course, appropriate interactions with patients, the effect of disease on the family, family s reactions to the disease, compatibility with the disease, common problems in the family with a patient suffering from mood disorder and problem - solving techniques. during the sessions, visual aids equipment was used in order to attract and preserve the attention of the participants. the contents were explained to them using powerpoint slides, graphs and images related to bmd sufferers. the educational workshops included lectures, presentations and interactive discussions as well as questions and answers. the first 15 minutes of each session was devoted to presenting a summary of the previous session and continued by presenting new contents, the mothers questions and their talks about the situations they encountered in this regard. at the end of each session, a training booklet containing the presented contents in that session, and after the end of the 6th session, a booklet containing the key points presented during the whole sessions were given to the participants who attended the educational workshops. chi - square and one - way anova were used to compare demographic variables based on the quantitative or qualitative nature of the variables. multi - sample repeated measures anova was used to assessthe effect of time and group on the scores of qol and mental health questionnaires. single factor multivariate anova and bonferroni post hoc tests were used for within - group comparisons and the independent t - test for between - group comparisons. this intervention was performed on 40 families (38 mothers and 2 fathers) of the adolescents with bmd which referred to the psychiatric clinics affiliated to shiraz university of medical sciences during 2012 - 13. significant results after the comparison between and within groups shows an adequate sample size (post power analysis > 75%). they were randomly assigned to intervention and control groups using block randomization method (block size : 4) (figure 1). design and protocol of the study inclusion criteria were having an adolescent aged 12 - 18 with confirmed diagnosis of bmd by a child and adolescent psychiatrist and lack of any other mental disorders such as mental retardation, epilepsy, schizophrenia, and drug abuse. however, exclusion criteria were unwillingness to participate in the study and absence more than two sessions in the educational program. data were collected using general health questionnaire (ghq), quality of life assessment questionnaire (short form-36) and demographic questionnaire. demographic data included age, sex and educational level of the adolescent and his / her parents, a history of physical or psychological diseases in the family as well as the number of siblings and birth order. the questionnaires were completed by all participants three times : before, immediately after and one month after the intervention. it is a 28-item self - report questionnaire which contains 4 subscales measuring somatic symptoms : anxiety, sleep disorder, social dysfunction, and depression. each sub - scale contains 7 items and each item is scored on a 4-point likert scale. the score for each subscale ranges from 0 to 21. the total score, which is obtained by summing up the scores of all subscales, ranges from 0 to 84 with lower scores indicating higher general health status. the reliability and validity of the questionnaire have been assessed in various studies and also estimated and confirmed in iran. by conducting a pilot study, yaghoubi estimated the sensitivity and specificity of the questionnaire as 86.5% and 82%, respectively and the cut - off score as 23 based on a likert - scale scoring. the reliability coefficient of the questionnaire was estimated 88% using test - retest and cronbach s alpha. we used the persian version of chq-28, which was translated by yaghoubi and palahang (1995), under the supervision of dr. short form of qol questionnaire (sf-36) is a 36-item self - report instrument designed to measure health - related qol across eight dimensions of physical functioning, role limitations caused by physical and emotional problems, bodily pain, general health, mental health, social functioning, emotional problems, energy / fatigue, and health changes. the scores on each dimension range from 0 to 100 with higher scores reflecting higher quality in that dimension. the questionnaire has been extensively used and its validity and reliability were estimated in various studies. furthermore, internal consistency of the questionnaire was estimated by calculating cronbach s alpha coefficient which was reported between 0.72 and 0.94. in iran, this study was approved by ethics committee of shiraz university of medical sciences (no ct-92 - 6718). all the families in the intervention group participated in a family - centered educational program consisting of six 90-minute sessions per week for 6 weeks. 20 participants in the intervention group were divided into groups of 4 and all of them attended the workshops and received the same content. the educational workshops were directed by a psychiatric nurse and a child and adolescent psychiatry specialist. to provide the intervention protocol, we used fristad s educational package (2003) and family educational package on bmd in children and adolescents prepared by mahmoudi - gharaei. family educational package included information about the nature and symptoms of mood disorders, therapeutic methods, complications and their course, appropriate interactions with patients, the effect of disease on the family, family s reactions to the disease, compatibility with the disease, common problems in the family with a patient suffering from mood disorder and problem - solving techniques. during the sessions, visual aids equipment was used in order to attract and preserve the attention of the participants. the contents were explained to them using powerpoint slides, graphs and images related to bmd sufferers. the educational workshops included lectures, presentations and interactive discussions as well as questions and answers. the first 15 minutes of each session was devoted to presenting a summary of the previous session and continued by presenting new contents, the mothers questions and their talks about the situations they encountered in this regard. at the end of each session, a training booklet containing the presented contents in that session, and after the end of the 6th session, a booklet containing the key points presented during the whole sessions were given to the participants who attended the educational workshops. chi - square and one - way anova were used to compare demographic variables based on the quantitative or qualitative nature of the variables. multi - sample repeated measures anova was used to assessthe effect of time and group on the scores of qol and mental health questionnaires. single factor multivariate anova and bonferroni post hoc tests were used for within - group comparisons and the independent t - test for between - group comparisons. the age range of the participants was 12 - 18 years and their meansd ages were 162.7and16.951.46 in the intervention and control group, respectively. there were no statistically significant differences between the two groups in terms of demographic variables. the majority of the participants in both groups had primary education and moderate monthly income level. the results of independent t - test and chi - square test showed that both groups were matched on demographic variables. the first part of table 1 displays the mean qol score before, immediately after and one month after the intervention. the results of multi - sample repeated measure anova for qol score showed that the interaction between the variables of group and time was significant (p<0.001), reflecting the fact that changes in qol scores over time were not similar in both groups (figure 2). therefore, subgroup analyses were used to compare the groups at each time point (between - group analysis) and comparing the responses between time points for each group (within - group analysis). comparison of the mean change scores of qol and mental health between the intervention and control groups before, immediately after and one month after the intervention the mean changes of qol increased in the intervention group and decreased in the control groups over the time (time 1 : before intervention, time 2 : immediately after intervention, time 3 : one month after intervention) the results of between - group analysis (table 1) indicated that the mean qol score in the control group was significantly greater than that of the intervention group before the intervention (p<0.001). however, it was not significantly different between the groups immediately after (p=0.756) and one month after the intervention (p=0.177) in this group. within - group analysis showed that the mean qol score increased in the intervention group over the measurement time points (figure 2). however, it decreased at two follow - up time points when compared to the baseline. changes in the mean scores of qol and mental health before, immediately after and one month after the intervention the values 0, 1 and 2 in the time column indicate baseline (before), immediately after intervention and one month after the intervention, respectively a significant grouptime interaction effect indicated that the pattern of changes in the mean mental health were different in the groups (p<0.001). the mean changes of mental health increased in the intervention group and decreased in the control groups over time the second part of table 1 shows between - group comparisons in each time point measured. the mean scores in the intervention group (48.9011.37) were significantly greater than those of the control group (29.6514.56) (p<0.001). contrary to the baseline, the mean mental health score was significantly lower for the intervention group both immediately and one month after the intervention when compared to the control group (both p<0.001). the results of pairwaise comparisons for within - group analysis are presented in table 2. although it decreased over time in the intervention group the first part of table 1 displays the mean qol score before, immediately after and one month after the intervention. the results of multi - sample repeated measure anova for qol score showed that the interaction between the variables of group and time was significant (p<0.001), reflecting the fact that changes in qol scores over time were not similar in both groups (figure 2). therefore, subgroup analyses were used to compare the groups at each time point (between - group analysis) and comparing the responses between time points for each group (within - group analysis). comparison of the mean change scores of qol and mental health between the intervention and control groups before, immediately after and one month after the intervention the mean changes of qol increased in the intervention group and decreased in the control groups over the time (time 1 : before intervention, time 2 : immediately after intervention, time 3 : one month after intervention) the results of between - group analysis (table 1) indicated that the mean qol score in the control group was significantly greater than that of the intervention group before the intervention (p<0.001). however, it was not significantly different between the groups immediately after (p=0.756) and one month after the intervention (p=0.177) in this group. within - group analysis showed that the mean qol score increased in the intervention group over the measurement time points (figure 2). however, it decreased at two follow - up time points when compared to the baseline. changes in the mean scores of qol and mental health before, immediately after and one month after the intervention the values 0, 1 and 2 in the time column indicate baseline (before), immediately after intervention and one month after the intervention, respectively a significant grouptime interaction effect indicated that the pattern of changes in the mean mental health were different in the groups (p<0.001). the mean changes of mental health increased in the intervention group and decreased in the control groups over time the second part of table 1 shows between - group comparisons in each time point measured. the mean scores in the intervention group (48.9011.37) were significantly greater than those of the control group (29.6514.56) (p<0.001). contrary to the baseline, the mean mental health score was significantly lower for the intervention group both immediately and one month after the intervention when compared to the control group (both p<0.001). the results of pairwaise comparisons for within - group analysis are presented in table 2. although it decreased over time in the intervention group caregivers of bmd sufferers may experience difficulties different from those with other psychological diseases due to cyclic nature of the disease. also, the patient s mood tends to fluctuate between two opposite poles of mania and depression. although the patients may experience recovery phases, the caregivers and family members are constantly concerned about suicidal thoughts and disease recurrence in the patients and changes in the nature of their disease (59%). all studies have reported family dysfunction, despair and helplessness in the families living with such patients. in a review article, more than 24 articles about caregivers of the patients with bmd were analyzed, in which 21 articles reported psychiatric distress in the caregivers. the family members must be qualified enough and have sufficient competencies to handle such task. to achieve this goal, the families need to receive the required trainings on strategies of interacting with the patient, methods of medication use and dealing with the symptoms of the disease and the resulting behaviors. likewise, training the families helps to reduce disease recurrence rate and provide a relaxed and convenient environment for the patients and their family members. our finding also showed that family - centered psycho - education is essential and effective in this regard. the result of another study revealed that family psycho - education could significantly decrease the sense of pressure and family burden immediately after and one year after the intervention. they also found that depression scores significantly decreased in the patients families and relatives who received such intervention. furthermore, we observed a significant difference in the intervention group in terms of the family s psycho - education before, immediately after and one month after the intervention. qol is one of the most important areas in the life of the families with psychiatric patients, especially those with bmd, being vulnerable to negative effects. actually, qol is characterized as an individual s specific perception of life satisfaction, physical health, social and family health, hope, etiquette and mental health. in the case of depressive disorders and bmd like other diseases, improving the qol of the patients and their families another study applied an intervention which included combinations of education about the illness, family support, crisis intervention and problem solving skills training. since life satisfaction and psychological welfare are measures of qol, these types of interventions could improve qol in the participants. family - centered education was used in relation with the disease, family support, crisis intervention, interaction with the patient, reaction to the symptoms of the disease and problem solving skills training. accordingly, qol improved in our participants in the intervention group immediately after and one month after the intervention. in the control group, however, no significant difference was observed. another study showed that family interventions could improve psychological well - being and comfort, symptoms of the disease, interaction with the patient and family members and eventually qol since the mentioned factors are all subscales of qol. researchers believe that physical health can be influenced by psychological growth and mental health promotion is based on prevention and treatment of emotional stress. functioning in other domains of qol is threatened as the level of mental health decreases. research findings suggest that children suffering from behavioral disorders and psychiatric diseases can negatively affect their parents. a study on mental health status in family caregivers of the patients with psychiatric disorders revealed that 35% of the respondents reported some sorts of mental health problems. accordingly, in the present study, family - centered education was used to promote mental health in the families of the adolescents with bmd. we observed a statistically significant difference in the intervention group in this regard immediately after and one month after the intervention. the family caregivers, who take care of a family member with psychiatric disorders, have several needs and nurses can help them meet their needs by proper planning and applying nursing procedures after identifying and prioritizing the needs. all members of the caregiving team are responsible for supporting the families with the patients suffering from psychiatric diseases and satisfying their needs ; however, nurses are in a special position and have a key role in this regard since they are the main support of family members in hospitals. the number of the recruited participants was few due to less referral of adolescents with bmd and late diagnosis of their disease. further research with larger sample size could be effective in confirming the results of the present study and making changes in the mean scores of qol and mental health. a follow - up period of more than one month which is repeated over time is recommended to confirm our results. the study findings confirmed the effectiveness of family - centered psycho - education program on mental health and quality of life of the families of adolescents with bipolar mood disorder. further research is recommended to be done on families in which the father or mother have bmd and have adolescents with bmd. | background : bipolar mood disorder (bmd) is a type of mood disorder which is associated with various disabilities. the family members of the patients with bmd experience many difficulties and pressures during the periods of treatment, rehabilitation and recovery and their quality of life (qol) is threatened. in the present study, we aimed to evaluate the effect of family - centered education on mental health and qol of families with adolescents suffering from bmd.methods:in this randomized controlled clinical trial performed on 40 families which were mostly mothers of the adolescents with bmd referred to the psychiatric clinics affiliated to shiraz university of medical sciences during 2012 - 13. they were randomly assigned to intervention and control groups.results:the results of single factor multivariate anova / single - factor multivariate analysis of variance and bonferroni post hoc tests showed that the interaction between the variables of group and time was significant (p<0.001). the mean of qol and mental health scores increased in the intervention group, but it decreased in the control group at three measurement time points.conclusion:the study findings confirmed the effectiveness of family - centered psychoeducation program on mental health and quality of life of the families of adolescents with bipolar mood disorder.trial registration number : irct201304202812n15 |
since its discovery and introduction, the electroencephalogram (eeg) was viewed with a great enthusiasm as the only methodology allowing a direct, online view of the brain at work. the enormous complexity of the eeg signal should not surprise us since, the eeg is a direct correlate of brain function, and the brain is a complex system. so far it offers appreciable promise as a means to characterize significant deviations from the natural aging found in alzheimer and other dementias. since the 1970s, first with the introduction of structural imaging technologies such as computer - assisted tomography (cat) and magnetic resonance imaging (mri), and then with the development of regional metabolic - perfusion methods such as positron emission tomography (pet), single photon emission - computed tomography (spect), and the ability to map oxygen consumption and regional blood flow in specific neural locations with functional magnetic resonance imaging (fmri), eeg has been supplanted in basic and clinical studies. these new techniques produce noninvasive views of in vivo brain anatomy with considerable resolution that contributed to their clinical and, therefore, economic utility. however, these functional brain imaging methods, despite their high spatial resolution for anatomical details, are relatively limited in their temporal resolution when measuring functional brain activation (seconds to minutes). thus, these more recent neuroimaging techniques can not discriminate the activation of different relays within a distributed network either in series or in parallel. over the years, several improvements have been introduced to eeg measures in part, because neuroelectric signals can track information processing with millisecond precision. therefore, even if the eeg is affected by the problem of low spatial resolution when compared to other techniques (e.g., fmri and pet), its high temporal resolution makes it possible to highlight the mechanism of temporal synchronization of the cortical pyramidal neurons. compared to fmri and pet, the advantage of using eeg is the possibility to evaluate the physiological mechanisms of cortical neural synchronization at the basis of the emerging brain feature : brain oscillations. it should be noted that a high temporal resolution is crucial for the study of an emerging property of brain activity, namely, the spontaneous and event - related oscillatory activity at different frequencies ranging at 24 hz (delta), 48 hz (theta), 813 hz (alpha), 1330 hz (beta), and > 30 hz (gamma). each of these frequencies conveys peculiar physiological information on brain functional state during sleep and wake periods. among the main purposes of modern neuroscientific research are the identification of patterns of neuronal activity underlying cognitive function and the finding of global functional indexes quick to be automatically computed towards clinical applications. it is, therefore, important to implement techniques that may measure natural brain aging and discriminate it from neurodegeneration [4, 5 ]. recently, greater attention has been focused on the application of quantitative eeg (qeeg) and/or event - related potentials (erps) as suitable clinical markers of early stage of disease or its progression. this is likely a result of recent improvements in the ease of the technology used and in the access to sufficient computing power and algorithms necessary for rapid processing of very complex raw datasets. examples of recent technological advances include a reduction in the size (and portability) of eeg amplifiers and the development of high - density array nets that do not require skin abrasion to places with low impedance. it has been reported that a positive erp peaking 600 ms after the zerotime of stimuli to be encoded (p600) was reduced in patients with alzheimer 's disease (ad) and mild cognitive impairment (mci), particularly in those mci patients who subsequently converted to ad [7, 8 ]. furthermore, a positive erp peaking 300 ms after the zerotime of oddball stimuli (p300) was found reduced in patients with dementia. thus, there exist theoretical and empirical reasons for the application of erps as a measure of individual variation of cognitive function along pathological aging. it has been shown that it is sensitive to alzheimer 's disease processes during its early stages. however, recording of erps requires a peculiar setup between the stimulation device and eeg machine, about 4060 minutes of time for the examination in the patient, and technicians able to carry out engaging experimental conditions. in this regard, recording of resting state eeg rhythms represents a procedure much easier and rapid that does not require stimulation devices. the present paper outlines the impact of eeg techniques for the measurement of physiological and pathological brain aging and provides a comprehensive analysis of brain aging by the analysis of resting state eeg rhythms in elderly subjects with various degrees of cognitive decline. its major goal is to highlight the emerging neurophysiological findings important to determine whether these techniques provide sufficient innovative and potentially useful information for the assessment of normal aging and dementia, both at the group- and at the single - subject levels. furthermore, it is to underline the practical utility of the eeg technique as global functional indexes quickly evaluable for automatic computation towards clinical applications. advanced eeg analysis techniques can illustrate changes in specific rhythms oscillating at various frequencies over time, provide quantitative measurements of individual rhythms, and reduce the effects of volume currents from far - field generators [10, 11 ]. hence, eeg signals generated from extracerebral sources (e.g., electrocardiogram, electromyogram, electroretinogram, eye movement etc.) can be isolated from those produced by the brain, providing a direct measure of the recorded neuroelectric signals. eeg coherence or synchronicity of rhythmic signals from separate electrodes, in different frequency bands, generated in different cortical areas, can also be measured. the high - resolution eeg technique has markedly enhanced the spatial resolution of the conventional eeg from about 69 cm to 2 - 3 cm by the use of spatial enhancement methods such as laplacian transformation with a regularized 3d spline function. this method reduces the low spatial eeg frequencies contributed by volume conduction and eliminates electrode reference influence [1215 ]. compared to other linear or nonlinear modelling analysis techniques of cortical sources of eeg - meg, surface laplacian estimation provides a rough representation of the neural currents without an explicit model of the generators (i.e., shape, number and location) by using a model of the head as a volume conductor [12, 13 ]. however, surface laplacian methods can not disentangle the activity of two spatially adjacent cortical zones such as primary somatosensory and motor areas that are contiguous across the central sulcus or deep cortical sources in secondary somatosensory and insular cortices. surface laplacian estimation is also unreliable when computed at the borders (i.e., temporo - parietal electrodes). its maxima often overlie cortical sources of eeg potentials, since the influence of tangential relative to radial oriented generators is greater [12, 13, 16 ]. spectral coherence analysis indexes the temporal synchronization of two eeg time series among electrodes in the frequency domain and permits characterization of linear functional corticocortical connectivity. eeg spectral coherence is a normalized measure of the coupling between two electroencephalographic signals at any given frequency [17, 18 ]. it is commonly interpreted as an index of functional coupling [19, 20 ], mutual information exchange, functional coordination, and integrity of cortical neural pathways. its basic theoretical assumption is that when the activity of two cortical areas is functionally coordinated, the eeg rhythms of these cortical areas show linear correlation and high spectral coherence. in general, decreased coherence reflects reduced linear functional connections and information transfer (i.e., functional uncoupling) among cortical areas or modulation of common areas by a third region. in contrast, coherence increase is interpreted as augmented linear functional connections and information transfer (i.e., functional coupling), which reflects the interaction of different cortical structures for a given task. it has been repeatedly demonstrated that perceptive, cognitive, and motor processes are associated with enhanced eeg spectral coherence [2326 ], as a function of the extension and type of the neural networks engaged [27, 28 ]. finally, the direction of the information flow within the eeg rhythms between pairs of electrodes can be estimated by a directed transfer function (dtf) [2934 ]. there are different methods to solve the noninvasive localization of the neuronal generators responsible for measured eeg phenomena (i.e., the source reconstruction of the electromagnetic brain scalp signals). low - resolution electromagnetic tomography algorithm (loreta) software, which can be freely downloaded by internet (http://www.unizh.ch/keyinst/newloreta/loreta01.htm), has been successfully used in recent eeg studies on pathological brain aging [3540 ]. loreta computes 3d linear solutions (loreta solutions) for the eeg inverse problem within a 3-shell spherical head model including scalp, skull, and brain compartments [4143 ]. loreta solutions consisted of voxel z - current density values able to predict eeg spectral power density at scalp electrodes. as it is a reference - free method of eeg analysis, one can obtain the same loreta source distribution for eeg data referenced to any reference electrode including common average. furthermore, it can be also used from data collected by low spatial sampling (e.g., 19 electrodes) when cortical sources are estimated from resting eeg rhythms [4447 ]. a normalization of the data was obtained by normalizing the loreta current density at each voxel with the power density averaged across all frequencies (0.545 hz) and across all voxels of the brain volume. after the normalization, the solutions lost the original physical dimension and were represented by an arbitrary unit scale. this procedure reduced intersubjects variability and was used in previous eeg studies [3640 ]. resting state cortical eeg rhythms typically change across physiological aging, with gradual modifications in profile and magnitude of the spectra power ; in detail, it was observed a marked amplitude decrease of alpha (813 hz) and a global slowing of the background eeg, which increases in power and spatial distribution in the slower delta (24 hz) and theta (48 hz) rhythms [4851 ]. a recent study in a large sample of healthy subjects (n = 215, 1885 years) confirmed an age - dependent power decrement of posterior low - frequency alpha (alpha 1 ; 810.5 hz) and delta rhythms. aging effects on parieto - occipital alpha rhythms presumably reflect the activity of dominant oscillatory neural network in the resting awaken brain. this activity is modulated by thalamocortical and corticocortical interactions facilitating / inhibiting the transmission of sensorimotor information and the retrieval of semantic information from cortical storage [27, 53, 54 ]. in the condition of awaken rest, alpha 1 frequency noteworthy, there is consensus that alpha rhythms represent the dominant resting oscillations of the adult, awaken human brain [5458 ] and have been linked to intelligent quotient, memory, and cognition. whereas high - frequency alpha rythms reflect the oscillation of specific neural systems for the elaboration of sensorimotor or semantic information [50, 55, 56 ]. over the course of natural aging, the power decrease of the occipital alpha rhythms might be associated with changes in the cholinergic basal forebrain system function, which sustain the excitatory activity in the cholinergic brainstem pathway. therefore, that assessment of nonlinear eeg interactions is important, as this method can provide information on the strength, direction, and topography of the interdependencies. spatial organization of nonlinear interactions between different brain regions has been investigated to compare anterior - posterior intrahemispheric and left - right interhemispheric interactions across physiological aging. differences were found in the rates of interdependencies between the left prefrontal and the right parietal regions between young and elderly, suggesting that the aging brain engages the right parietal region to assist the pre - frontal cortex. dementia is one of the most frequent chronic diseases of the elderly, and it is characterized by loss of intellectual and behavioral abilities that interfere with daily functioning. dementia incidence tends to increase with age affecting over 30% of people after age 85 [61, 62 ]. consequently, social costs for managing dementia are expected to rise becoming an important social problem. important neuropathological features indicating alzheimer 's dementia (ad) include brain cortical and subcortical atrophy leading to ventricular enlargements primarily due to neuronal loss in the temporal and parietal structures. among the primary markers of alzheimer 's disease, microscopic signs including neurofibrillary tangles (intracellular aggregates of tau protein filaments) and amyloid plaques (extracellular aggregates of amyloid beta - peptides) that are dispersed throughout the cerebral cortex and basal ganglia, particularly concentrated in the hippocampus, entorhinal cortex, and postcentral parietal neocortex. tangles are mainly found in hippocampal and parahippocampal limbic structures, whereas amyloid plaques are largely diffuse throughout the cortex. a neurophysiological hallmark of brain aging is a progressive impairment of use - dependent synaptic plasticity and of synaptic connectivity between neurons and its association with the degree of dementia. however, in preclinical conditions, plastic compensatory remodelling appears to continue that maintains neural function so that the neuronal and synaptic death can occur in the absence of dementia symptoms for an unknown period of time that might take for years or decades. when compared to the resting state eeg rhythms of healthy normal elderly (nold) subjects, ad patients showed an amplitude increase of widespread delta and theta sources and an amplitude decrease of posterior alpha (813 hz) and/or beta (1330 hz) sources [35, 47, 6669 ]. the observation of these abnormalities of the eeg rhythms could allow a discrimination among different dementia diagnoses for instance, a marked decline of posterior slow - frequency alpha power shows peculiar features in mild ad subjects when compared to cerebrovascular dementia, frontotemporal dementia and normal elderly subjects with similar cognitive impairment. these eeg abnormalities have been associated with altered regional cerebral blood flow / metabolism and with impaired global cognitive function as evaluated by mmse [68, 7072 ]. of note, early stages of ad (even preclinical) are typically associated with slowing down of resting occipital alpha rhythms, namely, a decrease of the individual alpha frequency (iaf) peak in power density. the iaf peak, defined as the frequency associated with the strongest eeg power at the extended alpha range, should be always taken into account in eeg studies in ad subjects, since power changes in theta and alpha bands might be dependent phenomena. furthermore, the conventional partition of eeg power into many conventional frequency bands allows the comparison of the results with those of most of the field studies but may prevent the separation of independent eeg rhythms or sources. despite the evidence of abnormal cortical rhythms in mci and ad, eeg analysis alone is unable to allow a diagnosis of disease. regard, several studies have shown a strict relationship between genetic risk factors such as apolipoprotein e 4 genotype (apo - e 4) and late - onset ad. apo - e 4 has been found to affect eeg rhythms in ad patients, it is associated with abnormalities of resting state eeg rhythms in ad [7476 ] with relatively specific eeg measures. compared to ad patients with 2 and 3, ad patients with 4 showed higher theta and lower beta spectral power. furthermore, the ad apoe 4 carriers patients were characterized by higher theta power and lower beta power at baseline, whereas they were characterized by higher delta power and lower alpha power at 3 years at followup. moreover, ad patients with apoe 4 has been related to selective decrease in functional corticocortical connectivity, which was suggested by the reduction of right and left temporoparietal, right temporofrontal, and left occipitoparietal alpha eeg coherence. eeg power per se does not capture one of the main features of ad, namely, the impairment of functional neural connectivity. it has been reported that ad patients present a reduced linear coupling of resting state eeg rhythms among cortical regions, as revealed by spectral eeg coherence [22, 74, 7780 ], suggesting a linear temporal synchronicity of coupled eeg rhythms from simultaneously engaged neural sources. such findings imply that functional coupling of cortical rhythms at certain frequency bands might be interesting features of ad and that abnormality of cortical eeg coherence may be a fine - grained marker of ad, which is supposed to reflect a disease of cerebral networks sub - serving global cognition. it could be speculated that this impaired pattern of eeg functional coupling is modulated by cholinergic systems and that a decrease of cortical eeg coherence is characterized by defective basal forebrain cholinergic inputs to cortex and hippocampus. most eeg studies of ad have reported a prominent decrease of alpha band coherence [22, 65, 74, 7780, 8285 ] this result also has been found to be associated with apoe genetic risk, which is hypothesized to be mediated by cholinergic deficit. however, delta and theta band coherence changes in ad are not homogeneous, as some studies demonstrate contradictory results with either a decrease or an increase of slow - band eeg coherence [22, 79, 82, 86 ]. these conflicting results might be due to the use of coherence markers from single electrode pairs rather than for the total coherence as obtained averaging the eeg spectral coherence across all combinations of electrode pairs. the latter may better take into account frequency band - by - frequency band the global impairment of brain networks and cognition along the ad process, which is supposed to be a disease affecting the functional integration within cerebral neural networks sub - serving cognition. in a recent study, the results show that the delta total coherence is higher in the ad than in the mci and in the mci than in the nold group. furthermore, the alpha1 total coherence is lower in the ad group than in the mci and nold groups. this evidence confirms that the functional coupling of resting eeg rhythms is progressively abnormal in amnesic mci and ad subjects. to improve the functional coupling evaluation, eeg and meg data have been analyzed with procedures inspired by the theory of nonlinear dynamics, which provides a measure of signal dynamic coordination. the ad patients have significantly lower dimensional complexity of eeg than age - approximated non - demented controls. thus it may be associated with deficient information processing in the brain injured by ad. brain rhythms loose the usual modulation in complexity as observed by eyes - open versus eyes - close comparisons, as a reflection of neuronal death, deficiency in neurotransmission, and/or loss of connectivity in local neuronal networks [89, 90 ]. nonlinear analysis has also been used to model brain flexibility in information processing, defined as the capability to affect state of information processing from identical initial conditions. ad patients show a decrease in information processing flexibility, such that eeg complexity decrease in ad might be attributable to decreased nonlinear dynamics that are associated with cognitive decline. among the techniques for nonlinear brain dynamics, synchronization likelihood combines sensitivity to linear and nonlinear functional coupling of eeg / meg rhythms. this measure has been shown to be significantly decreased at alpha and low beta bands when comparing ad to mci and/or nold subjects [23, 9193 ]. in addition to the corticocortical uncoupling progression, a decrease of synaptic coupling is likely to contribute to reducing selective eeg coherence for faster rhythms, as observed in healthy humans by transient use of a cholinergic synaptic blocker like scopolamine. animal models suggest that acetylcholine loss produces a decrease of high - frequency eeg couplings and an increase of slow - frequency couplings. loss or a significant drop in eeg synchronization in faster rhythms has also been correlated with decreased mmse scores in mci and ad patients. linear and nonlinear eeg analyses improve classification accuracy of ad compared to unaffected controls, and these methods correlate with disease severity [23, 88, 91 ]. a significant increase of delta and theta power in conjunction with decrease of alpha and beta power over a period of 30 months from diagnosis have been found. the length of the followup is of paramount importance and indicates the reason for a lack of findings over a 12-month period. the major question in this context is which is the physiological mechanism at the basis of abnormal resting brain rhythms in mci and ad ? abnormality of resting eeg rhythms may originate from impairment in the cholinergic neural projections from basal forebrain, which is a pivotal aspect of ad. furthermore, the cholinergic basal forebrain has been found to be responsive to the treatment with cholinesterase inhibitors more for ad than other dementias. long - term (1 year) treatments of acetylcholinesterase inhibitors (achei) demonstrate less temporal and occipital alpha reduction for responders compared to nonresponders and a combined effect on delta and low alpha [37, 101 ]. hence, increasing cholinergic tone was related to restoring temporal and occipital alpha rhythms in responders. brain cholinergic systems also appear to improve primarily cerebral blood flow with a functional impact on attentional and memory functions. assessing preclinical dementia is of keen interest as a clinical research issue, since mci often precedes frank dementing illness. as the selective cognitive impairments characteristic of mci are primarily memory - related and not severe enough to exceed standard clinical criteria for ad, their prodromal qualities do not greatly impair daily functioning and can be identified by refined clinical and neuropsychological evaluation. consistent mci symptoms 35 years following their identification either remain stable or decrease in 30%50% of the cases, whereas the remaining cases progress toward a frank ad condition or, less frequently, to other dementias. the mci condition has often been considered a precursor of ad despite the fact that not all the mci patients develop the alzheimer disease. epidemiological and clinical followup studies confirm that mci reflects a transition state towards mild ad and prompts the idea that early identification of mci patients can facilitate rehabilitative or pharmacological interventions to slow down the disease progression [103105 ]. figure 1 illustrates mci effects for low - frequency alpha (810.5 hz) activity from parietal, occipital, and limbic areas that demonstrate an intermediate magnitude in mci compared to mild ad and normal elderly. increase of slow eeg power coupled with a decrease in alpha activity is linked to cognitive performance decline in mci compared to nold. more important, the spectral magnitude of these sources is correlated negatively with mmse scores across subjects of the three groups, suggesting that eeg evidence of alpha power decrease in mci compared to normal subjects is related to behavioral cognition [66, 84, 106109 ]. the relative spectral magnitude decrease of posterior low - frequency alpha sources in mci may be related to an initial selective impairment of the cholinergic basal forebrain, which could induce a sustained increase of the excitatory activity in the cholinergic brainstem pathway [59, 94, 95 ]. tms studies indicate that the cortex of ad patients is hyperexcitable and that such hyperexcitability even may offer clues for the differential diagnosis from other dementias in which the cholinergic deficit is not predominant. as a consequence, the increased excitability of thalamocortical connections would desynchronize the resting alpha rhythms and enhance the cortical excitability. hence, changes of low - frequency alpha power in mci and mild ad suggest a progressive impairment of the thalamocortical and corticocortical systems that govern visual attention. this hypothesis is consistent with clinical findings of increasing deficits of visuospatial abilities in mci and mild ad. similarly, limbic sources imply a progressive impairment of thalamocortical and corticocortical systems regulating attention tone for memory functions. decreases in corticothalamic modulation and increase of slow eeg rhythms correlated to progressive cortical hypoperfusion have been found in ad [72, 111 ]. abnormal delta and alpha sources in the posterior brain regions could, therefore, index the progressive decline of cognitive visuospatial functions across mci and mild ad thereby supporting a transition between these conditions [103105 ]. an intriguing aspect includes the peculiar magnitude increase of the parieto - occipital high - frequency alpha sources (alpha 2, 10.513 hz) in mci compared to mild ad and normal elderly. furthermore, prospective studies have demonstrated that increased delta / theta activity, decreased alpha and beta, and slowed mean frequency may be predictors of progression from mci to dementia [66, 84 ]. these findings imply that neuroelectric indices could be developed for the preclinical assessment of dementia, as their acquisition are inexpensive, easily implemented, entirely non - invasive, and very well suited for large - scale screening and followup of at - risk populations. the hypothesis that presence of apoe 4 affects sources of resting eeg rhythms in mci and ad was assessed in 89 mci with 34.8% 4 incidence and 103 ad with 50.4% 4 incidence. alpha 1 and 2 sources in occipital, temporal, and limbic areas were of lower amplitude in subjects carrying the apoe 4 allele. for ad homozygous for apoe 4 allele, abnormal temporo - parietal and occipitoparietal eeg or meg rhythms were found [74, 88 ]. however, in addition to apoe 4 allele, another important genetic risk factor for late - onset ad is haplotype b of cst3 (the gene coding for cystatin c a neurotrophic protein), which was investigated to establish eventual links with cortical rhythmicity. eeg measures were obtained from 84 mci with 42% b haplotype and 65 ad with 40% b haplotype. slow alpha (from parietal, occipital, and temporal areas) and fast alpha (from occipital areas) power were statistically lower in cst3 b carriers. a trend was observed for occipital delta power sources as stronger in cst3 b carriers than in noncarriers for both mci and ad patients. association between the presence and amount of hippocampus atrophy in ad and mci subjects and changes in sources of posterior slow rhythms have been observed by eeg and whole - head meg [114116 ]. less known is the relationships between impairment of white matter and slow rhythms across the continuum from mci to ad. this issue has been addressed with eeg assessments in mci (n = 34) and ad (n = 65) cases. delta activity was related to the amount of cortical atrophy revealed by mri voxel - to - voxel volumetry of lobar brain volume (white and gray matter) such that as delta power increased, brain volume decreased. thus, changes in brain structure and function could be found for mci and ad patients. as life expectancy and elderly populations in western countries are increasing, the incidence of mci that may predict ad or vascular dementia is rising. cognitive impairment associated with mci or ad is associated with decreased power and coherence in the alpha / beta band, at least at the group level. this observation suggests the occurrence of a functional disconnection among cortical areas, since both power and coherence in the delta and theta bands increase with cortical deafferentiation from subcortical structures. however, the extent to which features of neuroelectric activity can be used to predict the conversion from mci to ad in single subjects is as yet unclear. in a seminal eeg study, a multiple logistic regression of theta power (3.57.5 hz), mean frequency, and interhemispheric coherence has been able to to predict decline from mci to ad at long term for with an overall predictive accuracy of about 90%. furthermore, spectral eeg coherence or other eeg features have shown to contribute to the discrimination of nold from mild ad with 89%45% of success, from mci to ad with 92%78% of success, and the conversion of mci subjects to ad with 87%60% of success [66, 79, 84, 119124 ]. investigated whether combined analysis of eeg power and coherence provide early and reliable discrimination of mci subjects who will convert to ad after a relatively brief followup. cortical connectivity using spectral coherence measures and loreta was evaluated to characterize eeg sources at baseline in 69 mci cases that were reassessed clinically after about 14 months. at followup, 45 subjects were classified as stable mci (mci stable), whereas the remaining 24 had converted to ad (mci converted). results showed that at baseline, frontoparietal midline coherence as well as delta (temporal), theta (parietal, occipital, and temporal), and low - frequency alpha (central, parietal, occipital, temporal, and limbic) sources were stronger in mci converted than mci stable subjects. cox regression modeling showed low midline coherence, and weak temporal source was associated with 10% annual rate ad conversion, while this rate increased up to 40% and 60% when strong temporal delta source and high midline gamma coherence were observed, respectively. this outcome indicates that quantitative eeg is able to predict with a good approximation mci progression to ad in the short run. the present paper highlights the use of modern eeg techniques that report assessment of physiological and pathological brain aging. application of these techniques allows the quantification of the power and functional coupling of resting state eeg rhythms at scalp electrodes and mathematical cortical sources. the results reviewed in the present paper suggest that these quantitative indexes of resting state eeg rhythms might reflect neurodegenerative processes along preclinical and clinical stages of ad. moreover, risk factors including genetic causes correlate with neurophysiological findings to reinforce their causative role in diagnosis and prognosis of pathologic brain aging. unfortunately, this remarkable literature suffers from the partial lack of integration of various eeg techniques such as analysis of power density and functional coupling (i.e., spectral coherence, and directed transfer function) within a unique frame of goal - directed test for evaluation of physiological brain aging and discrimination from abnormal scenarios heralding neurodegeneration. in the near future, systematic evaluation of ad and other dementing disorders relative to normal aging using refined and integrated eeg techniques will help to coalesce these methodologies and improve diagnostic utility. if this approach can provide clinically useful information at the individual level, such methods should prompt design of an instrument widely available for large - scale population - based screening studies. future studies should find which are qeeg markers for early diagnosis, prognosis, and monitoring of alzheimer disease and explore the clinical utility of this methodological approach. the global structural and functional indexes are quick to be automatically computed towards clinical applications. | physiological brain aging is characterized by synapses loss and neurodegeneration that slowly lead to an age - related decline of cognition. neural / synaptic redundancy and plastic remodelling of brain networking, also due to mental and physical training, promotes maintenance of brain activity in healthy elderly subjects for everyday life and good social behaviour and intellectual capabilities. however, age is the major risk factor for most common neurodegenerative disorders that impact on cognition, like alzheimer 's disease (ad). brain electromagnetic activity is a feature of neuronal network function in various brain regions. modern neurophysiological techniques, such as electroencephalography (eeg) and event - related potentials (erps), are useful tools in the investigation of brain cognitive function in normal and pathological aging with an excellent time resolution. these techniques can index normal and abnormal brain aging analysis of corticocortical connectivity and neuronal synchronization of rhythmic oscillations at various frequencies. the present review suggests that discrimination between physiological and pathological brain aging clearly emerges at the group level, with suggested applications also at the level of single individual. the possibility of combining the use of eeg together with biological / neuropsychological markers and structural / functional imaging is promising for a low - cost, non - invasive, and widely available assessment of groups of individuals at - risk. |
a literature search of the medline database (from january 1966 through july 2007) was conducted using the medical subject headings diabetes mellitus, type 2 ; drug therapy, combination ; drug combinations ; sulfonylurea compounds ; acetohexamide ; chlorpropamide ; tolbutamide ; tolazamide ; glyburide ; glipizide ; biguanides ; and metformin and keyword glimepiride. studies were also identified through a search of references cited in the original published studies and relevant review articles. the contents of 299 abstracts or full - text manuscripts identified during the literature search were reviewed independently by two investigators in duplicate to determine whether they met the criteria for inclusion. when there were discrepancies between investigators for inclusion or exclusion, a third investigator conducted additional evaluation of the study and the discrepancies were resolved in conference. the following inclusion criteria were used for study selection : 1) observational study that investigated the relationship between combination therapy with metformin (biguanides) plus sulfonylureas and risk of cvd and/or mortality, 2) adjusted relative risk (rr) or equivalent (i.e., hazard ratio, odds ratio) and corresponding variance or equivalent reported, and 3) diagnosis of type 2 diabetes established using the standard criteria for the time of the study. a standardized abstraction form was used to record the following information : study title, first author 's name, year of publication, study country, study years, name of cohort, study design (prospective or retrospective cohort study or case - control study), duration of follow - up, characteristics of the study population (sample size, distribution of age, race, and sex, mean diabetes duration, mean a1c), type of reference group, and confounding factors controlled for. the rr of cardiovascular mortality / morbidity and/or all - cause or cause - specific mortality associated with combination therapy and their corresponding cis or ses were abstracted. the number of events for all - cause mortality and cardiovascular mortality / morbidity were abstracted. rrs were used as the measure of association between combination therapy of metformin and sulfonylurea and cvd and all - cause mortality. the rrs of each study were weighted by the inverse of their variance. to stabilize the variances and to normalize the distributions, the rrs and corresponding ses from each of the individual studies when necessary, ses were derived from the cis provided in each original study. the primary data for time to event analyses were not available for the combined cohort. therefore, for the overall analysis, rr estimates and 95% cis for all - cause mortality and cvd associated with combination therapy were pooled irrespective of the reference group used. subgroup analyses were conducted by reference group (diet, sulfonylurea monotherapy, or metformin monotherapy). both fixed - effects and dersimonian and laird random - effects models were used to calculate the pooled rr of cvd and all - cause mortality associated with combination therapy (9). although both models yielded similar findings, results from the random - effects model are presented herein owing to significant heterogeneity among the studies. we used cardiovascular mortality and all - cause mortality, as well as a composite end point of cvd hospitalizations (the first cardiovascular event either fatal or nonfatal event), or mortality as our study outcomes. for this study, we first weighted both of the rrs by the inverse of their variance and then pooled the rrs by using a fixed - effects model to obtain an overall estimate for the study. begg 's rank correlation test was used to examine the association between effect estimates and their variances, and egger 's linear regression test, which regresses z statistics on the reciprocal of the se for each study, was used to detect publication bias (11,12). additionally, each study was omitted one at a time to evaluate the influence of that study on the pooled estimate. all analyses were performed using stata version 8.2 (stata, college station, tx). rrs were used as the measure of association between combination therapy of metformin and sulfonylurea and cvd and all - cause mortality. the rrs of each study were weighted by the inverse of their variance. to stabilize the variances and to normalize the distributions, the rrs and corresponding ses from each of the individual studies when necessary, ses were derived from the cis provided in each original study. the primary data for time to event analyses were not available for the combined cohort. therefore, for the overall analysis, rr estimates and 95% cis for all - cause mortality and cvd associated with combination therapy were pooled irrespective of the reference group used. subgroup analyses were conducted by reference group (diet, sulfonylurea monotherapy, or metformin monotherapy). both fixed - effects and dersimonian and laird random - effects models were used to calculate the pooled rr of cvd and all - cause mortality associated with combination therapy (9). although both models yielded similar findings, results from the random - effects model are presented herein owing to significant heterogeneity among the studies. we used cardiovascular mortality and all - cause mortality, as well as a composite end point of cvd hospitalizations (the first cardiovascular event either fatal or nonfatal event), or mortality as our study outcomes. for this study, we first weighted both of the rrs by the inverse of their variance and then pooled the rrs by using a fixed - effects model to obtain an overall estimate for the study. begg 's rank correlation test was used to examine the association between effect estimates and their variances, and egger 's linear regression test, which regresses z statistics on the reciprocal of the se for each study, was used to detect publication bias (11,12). additionally, each study was omitted one at a time to evaluate the influence of that study on the pooled estimate. all analyses were performed using stata version 8.2 (stata, college station, tx). online appendix figure a1 (available at http://dx.doi.org/10.2337/dc08-0167) depicts the flow of studies in the meta - analysis. among 25 studies that met the inclusion criteria, eleven studies did not report cvd or mortality as an outcome, three studies were duplicated, and two involved multiple drug combinations. two studies examined the association between combination therapy of metformin and sulfonylurea in different groups of individuals according to which drug was given first, and these groups were treated as separate studies in the meta - analysis. the characteristics of the study participants and the design of the nine observational studies included in the meta - analysis are presented in table 1 (58,10,1316). six of the studies were retrospective cohort studies, two were prospective cohort studies, and one was a nested case - control study., two in canada, one in israel, and five in european countries. the number of participants in these studies ranged from 910 in the study by olsson. the mean follow - up time ranged from 2.1 to 7.7 years. among the nine studies, seven reported all - cause mortality, four reported cardiovascular mortality, and three reported cardiovascular hospitalizations. of the 101,733 participants included in these studies, 25,091 participants received a combination therapy of metformin and sulfonylurea. figure 1 depicts the results from the random - effects models pooling the adjusted rrs for all - cause mortality, cvd mortality, and cvd hospitalizations or mortality, respectively, associated with combination therapy of metformin and sulfonylurea. in addition, it shows the number of events associated with combination therapy in comparison with the control group for all - cause mortality, cvd mortality, and cvd hospitalizations or mortality. pooled rr estimates were not statistically significant for all - cause mortality or cvd mortality, while the use of combination therapy was significantly associated with an increased risk of cardiovascular hospitalizations or mortality. in sensitivity analyses, significant heterogeneity was present for studies reporting all - cause mortality (p 0.10 for all). in the study by evans. (5), participants of the reference group were used more than once in computing the pooled estimate. analyses were repeated omitting various combinations of this study, and no substantive changes in results were noted. furthermore, we conducted a sensitivity analysis in which those studies that did not adjust for duration of diabetes or previous cvd were excluded (6,8,13,14,17). rr estimates of all - cause mortality, cvd mortality, and cvd hospitalizations or mortality associated with combination therapy of metformin and sulfonylurea for subgroups defined according to the comparator treatment are presented in online appendix table a1. the estimated rrs were > 1.0 in all subgroups except for the association between all - cause mortality and combination therapy compared with sulfonylurea. compared with diet therapy, combination therapy significantly increased the rr of all - cause mortality, and combination therapy compared with metformin monotherapy significantly increased the rr of cvd hospitalizations or mortality. rr estimates of all - cause mortality, cvd mortality, and cvd hospitalizations or mortality associated with combination therapy of metformin and sulfonylurea for subgroups defined according to the comparator treatment are presented in online appendix table a1. the estimated rrs were > 1.0 in all subgroups except for the association between all - cause mortality and combination therapy compared with sulfonylurea. compared with diet therapy, combination therapy significantly increased the rr of all - cause mortality, and combination therapy compared with metformin monotherapy significantly increased the rr of cvd hospitalizations or mortality. in the current meta - analysis, combination therapy of metformin and sulfonylurea significantly increased the rr of cardiovascular hospitalization or mortality (fatal and nonfatal events) irrespective of the reference group (diet therapy, metformin monotherapy, or sulfonylurea monotherapy) used. however, there were no statistically significant effects of combination therapy of sulfonylurea and metformin on cvd mortality or all - cause mortality. these results may help clarify the conflicting findings of several large observational studies that examined the effect of combination therapy with metformin and sulfonylureas on the risk of cvd events among patients with type 2 diabetes, while the association of this combination with all - cause and cardiovascular mortality remains obscure. due to the progressive nature of type 2 diabetes, many patients are put on combinations of oral antihyperglycemic agents in order to meet glycemic goals. for instance, in the recommended algorithm, the combination of sulfonylurea and metformin is the second step in the management of patients with type 2 diabetes (18). it is likely that patients on combination therapy are likely to have either a more rapidly progressive form of the disease or a longer duration of diabetes, perhaps both. the reduction of blood glucose in high - risk obese patients with type 2 diabetes on metformin therapy alone in the ukpds was associated with a decrease in adverse cardiovascular events (2). however, when a combination of metformin and sulfonylurea was prescribed, there was an increased risk, which is in contrast with some of the observational studies. it may not only be important to reduce blood glucose, but also to consider the choice of agent used to make such a reduction. a recent meta - analysis has created much controversy about some of the newer medications used to reduce blood glucose by suggesting that rosiglitazone may be associated with an increased risk of myocardial infarction and possibly death (19). it is noteworthy that much of this increased risk with rosiglitazone was seen in combination therapies (20). however, the interim analysis of the rosiglitazone evaluated for cardiac outcomes and regulation of glycaemia in diabetes (record) trial has shown inconclusive results (21). our meta - analysis is important in the context of that study, as the combination of metformin and sulfonylurea is the comparator group to the rosiglitazone combinations. several observational studies have examined the association between combination therapy and risk of cvd and all - cause mortality. (5) carried out an analysis of a database of 400,000 people in scotland and identified 5,730 patients who were prescribed oral hypoglycemia agents between 1994 and 2001. patients treated with sulfonylureas alone or in combination with metformin appeared to have an increased rr of adverse cardiovascular outcomes compared with those treated with metformin alone. it was particularly disturbing to note that the combination of sulfonylurea with metformin seemed to abrogate the potential benefit of metformin on cvd outcome, as seen in the ukpds (2). (14) was carried out among 2,275 patients with type 2 diabetes and coronary artery disease, as part of the bezafibrate infarction prevention study. the patients were followed for over 7 years, and the authors demonstrated that cardiovascular events and mortality were the same whether glyburide, a sulfonylurea, or metformin was used for treatment. however, there was a significant time - related increased mortality when the combination therapy was used. olsson. (10) analyzed mortality in a small cohort of patients taking sulfonylureas alone or in combination with metformin and demonstrated a higher cardiovascular mortality in patients taking the combination than those taking sulfonylurea alone. in our meta - analysis, exclusion of the study by johnson. (15) led to a significant increased risk of cvd mortality associated with combination therapy of metformin and sulfonylurea. (15) reported a reduced risk of cvd mortality associated with combination therapy of metformin and sulfonylurea when compared with sulfonylurea monotherapy, but the study had many limitations. patients prescribed the combination therapy were 2.3 years younger than those prescribed metformin monotherapy and 5.8 years younger than those prescribed sulfonylurea monotherapy, a discrepancy that is difficult to explain. patients with more severe disease or intercurrent illnesses including hospitalization for cardiovascular events may have required insulin use and were therefore excluded from the study. in our analysis, we found a relatively greater association with fatal and nonfatal cvd events than in fatal events alone, suggesting that the incidence of cvd events may be increased with combination therapy, but there may have been a lower case - fatality rate. this contrasts with the recent data from the action to control cardiovascular risk in diabetes (accord) study (22) in which intensive treatment with multiple combinations of diabetes therapies was associated with decreased nonfatal cvd events but increased fatal events. it is impossible to determine the reason for this discrepancy, although it is possible that patients in the observational studies included in our analysis did not have a level of glycemia as low as that attempted in the accord trial. first, it is possible that patients needing such a combination have a more aggressive form of the disease and therefore more rapid deterioration in glycemic control over time. second, sulfonylureas are associated with weight gain, whereas metformin is associated with weight loss, as well as some improvement in a variety of cardiovascular risk factors. any weight gain induced by the combination may negate some of these beneficial effects and increase risks. other possible explanations include the known propensity of sulfonylureas to cause hypoglycemia. when used in combination with a drug like metformin, which may decrease hepatic glucose production, recovery from hypoglycemia hypoglycemia may increase the risk of cardiovascular abnormalities, including ischemia and a propensity to cause arrhythmias (23,24). there is also considerable controversy about the impact of sulfonylureas on ischemic preconditioning (25), but nothing is known about the effects of combination therapy. although a meta - analysis is not the best way to test the efficacy and safety of such a combination of treatments, it is highly unlikely that a large - scale clinical trial to test this hypothesis will be carried out. thus, we must rely on data from observational studies to arrive at conclusions and make appropriate recommendations. it is also unclear to what extent certain biases and methodological limitations, such as residual confounding, might exist in the studies included in this meta - analysis, since the majority of these studies were retrospective database analyses. in addition, the reference group varied among the studies. for instance, some studies used diet as the reference group, while others used sulfonylureas or metformin monotherapy as the reference group. finally, we observed substantial quantitative heterogeneity across the studies, but the small number of studies limited our ability to explore possible sources of this variability. additionally, findings from the subgroup analyses should be interpreted cautiously, as the number of studies examined was small. overall, our results provide a mix of reassurance and concern to prescribers of diabetes medications who use combination therapies to achieve good glycemic control. since sulfonylurea and metformin are likely the most widely used combination, it is possible that such use leads to early improvement in glycemic control, which, in itself, may lead to better microvascular outcomes. although is associated with lower mortality risk, in the ukpds, was associated with increased microvascular complications (2). therefore, one must balance the risks and benefits of medications used while making treatment decisions. we emphasize that this meta - analysis has limitations and serves to examine published data to generate hypotheses. we hope that our analysis will prompt the planning of future clinical trials to determine not only the value of good glycemic control, but also the safest and most cost effective way to achieve glycemic goals. clearly, we need further studies to assess the association of combination therapy of metformin and sulfonylurea with all - cause and/or cardiovascular mortality as well as to understand the potential mechanism of its deleterious effects. | objective observational studies assessing the association of combination therapy of metformin and sulfonylurea on all - cause and/or cardiovascular mortality in type 2 diabetes have shown conflicting results. we therefore evaluated the effects of combination therapy of sulfonylureas and metformin on the risk of all - cause mortality and cardiovascular disease (cvd) among people with type 2 diabetes.research design and methods a medline search (january 1966july 2007) was conducted to identify observational studies that examined the association between combination therapy of sulfonylureas and metformin on risk of cvd or all - cause mortality. from 299 relevant reports, 9 were included in the meta - analysis. in these studies, combination therapy of metformin and sulfonylurea was assessed, the risk of cvd and/or mortality was reported, and adjusted relative risk (rr) or equivalent (hazard ratio and odds ratio) and corresponding variance or equivalent was reported.resultsthe pooled rrs (95% cis) of outcomes for individuals with type 2 diabetes prescribed combination therapy of sulfonylureas and metformin were 1.19 (0.881.62) for all - cause mortality, 1.29 (0.732.27) for cvd mortality, and 1.43 (1.101.85) for a composite end point of cvd hospitalizations or mortality (fatal or nonfatal events).conclusions the combination therapy of metformin and sulfonylurea significantly increased the rr of the composite end point of cardiovascular hospitalization or mortality (fatal and nonfatal events) irrespective of the reference group (diet therapy, metformin monotherapy, or sulfonylurea monotherapy) ; however, there were no significant effects of this combination therapy on either cvd mortality or all - cause mortality alone. |
the following was an old conjecture of graham. conjecture 1.1let cp be the cyclic group of order p prime and let s be a sequence over cp of length p. if all (nontrivial) zero - sum subsequences of s are of the same length, then the number of distinct terms in s is at most 2. let cp be the cyclic group of order p prime and let s be a sequence over cp of length p. if all (nontrivial) zero - sum subsequences of s are of the same length, then the number of distinct terms in s is at most 2. in 1976, erds and szemerdi gave a proof of the conjecture for sufficiently large primes p. however, the proof was complicated enough that the details for small primes were never worked out. both in the paper of erds and szemerdi and in a later survey by erds and graham, the complexity of the proof was lamented. recently, a new proof, valid even for non - primes, was given by gao., using savchev and chen s recently proved structure theorem for zero - sum free sequences of long length in the cyclic group cn. however, as savchev and chen s result is fairly involved, they did not believe it to be the simple proof sought by erds, graham and szemerdi. in this paper, we give a short proof to the original conjecture of graham that uses only the cauchy davenport theorem and pigeonhole principle,. since the proof of the cauchy davenport theorem (known since 1813) is elementary and requires only a paragraph, our proof may perhaps qualify as simple. replacing the use of the cauchy davenport theorem with the devos goddyn mohar theorem (alternatively, the partition theorem from, could be used instead of devos goddyn mohar), we obtain an alternate proof, albeit not as simple, of the non - prime case. with only a little added effort, our method naturally yields an exhaustive list detailing the precise structure of s and shows that the result holds in an arbitrary finite abelian group, though the only additional group for which the hypotheses are non - void is c2c2 m. we state the main theorem in section 3, after introducing modern notation for sumsets, sequences and subsequence sums. we follow the notation of,, and concerning sumsets, sequences and subsequence sums. for the convenience of the reader less familiar with this notation let g be an abelian group, and let a, bg be nonempty subsets. we let g+a={g+aaa } and let ra, b(g) denote the number of representations of g = a+b as a sum with aa and bb. the stabilizer of a is h(a):={ggg+a = a}. the order of an element gg is denoted by ord(g), and we use h : gg / h to denote the natural homomorphism modulo h. we use cn to denote the cyclic group of order n. we let f(g) denote the free abelian monoid with basis g written multiplicatively. the elements of f(g) are then just multi - sets over g, but following long standing tradition, we refer to the sf(g) as sequences. we write sequences sf(g) in the form s = s1sr=gggvg(s),wherevg(s)0 and sig. we call |s|r=ggvg(s) the length of s, and vg(s)n0 the multiplicity of g in s. the support of s is supp(s){ggvg(s)>0}. a sequence s1 is called a subsequence of s if s1|s in f(g) (equivalently, vg(s1)vg(s) for all gg), and in such case, ss11 or s11s denotes the subsequence of s obtained by removing all terms from s1. we let h(s)max{vg(s)gg } denote the maximum multiplicity of a term of s. given any map :gg, we extend to a map of sequences, :f(g)f(g), by letting (s)(s1)(sr). if s = s1srf(g), with sig, then the sum of s is (s)i=1rsi=ggvg(s)g. we adapt the convention that the sum of the trivial / empty sequence is zero. we follow the usual notation for the set of subsequence sums : n(s)={(t)t|s and |t|=n}n(s)=i=1ni(s)andn(s)=i = n|s|i(s)and(s)=|s|(s). for a finite abelian group g, we define the davenport constant d(g) to be the minimal integer such that any sf(g) with |s|d(g) has 0(s). a basic argument shows that d(g)|g| (see [9, propositions 5.1.4 ]). we need the following result (see [9, theorem 5.2.10 ; lemma 5.2.9 ] and also [14, lemma 2.1 ]). proposition 2.1(ii) is a simple consequence of the pigeonhole principle, and we will only use proposition 2.1(i) in the trivial case |b|=k=2. proposition 2.1let g be an abelian group with a, bg finite and nonempty:(i)if |a+b||a|+|b|k, then ra, b(x)k for all xa+b ; (ii)if g is finite and |a|+|b||g|+k, then ra, b(x)k for all xg. let g be an abelian group with a, bg finite and nonempty:(i)if |a+b||a|+|b|k, then ra, b(x)k for all xa+b ; (ii)if g is finite and |a|+|b||g|+k, then ra, b(x)k for all xg. if |a+b||a|+|b|k, then ra, b(x)k for all xa+b ; if g is finite and |a|+|b||g|+k, then ra, b(x)k for all xg. theorem 2.2let g be an abelian group, let sf(g) be a sequence, and let nz+ with n|s|. let g be an abelian group, let sf(g) be a sequence, and let nz+ with n|s|. if h = h(n(s)), then(1)|n(s)|(gg / hmin{n, vg(h(s))}n+1)|h|. a particular case of the (general) devos goddyn mohar theorem is the much simpler cauchy davenport theorem,,,. let p be prime and let aicp, for i=1,,n, be nonempty. then |i=1nai|min{i=1n|ai|n+1,p}. let g be an abelian group, and let a, bg be nonempty subsets. we let g+a={g+aaa } and let ra, b(g) denote the number of representations of g = a+b as a sum with aa and bb. the stabilizer of a is h(a):={ggg+a = a}. the order of an element gg is denoted by ord(g), and we use h : gg / h to denote the natural homomorphism modulo h. we use cn to denote the cyclic group of order n. we let f(g) denote the free abelian monoid with basis g written multiplicatively. the elements of f(g) are then just multi - sets over g, but following long standing tradition we write sequences sf(g) in the form s = s1sr=gggvg(s),wherevg(s)0 and sig. we call |s|r=ggvg(s) the length of s, and vg(s)n0 the multiplicity of g in s. the support of s is supp(s){ggvg(s)>0}. a sequence s1 is called a subsequence of s if s1|s in f(g) (equivalently, vg(s1)vg(s) for all gg), and in such case, ss11 or s11s denotes the subsequence of s obtained by removing all terms from s1. we let h(s)max{vg(s)gg } denote the maximum multiplicity of a term of s. given any map :gg, we extend to a map of sequences, :f(g)f(g), by letting (s)(s1)(sr). if s = s1srf(g), with sig, then the sum of s is (s)i=1rsi=ggvg(s)g. we adapt the convention that the sum of the trivial / empty sequence is zero. we follow the usual notation for the set of subsequence sums : n(s)={(t)t|s and |t|=n}n(s)=i=1ni(s)andn(s)=i = n|s|i(s)and(s)=|s|(s). for a finite abelian group g, we define the davenport constant d(g) to be the minimal integer such that any sf(g) with |s|d(g) has 0(s). a basic argument shows that d(g)|g| (see [9, propositions 5.1.4 ]). we need the following result (see [9, theorem 5.2.10 ; lemma 5.2.9 ] and also [14, lemma 2.1 ]). proposition 2.1(ii) is a simple consequence of the pigeonhole principle, and we will only use proposition 2.1(i) in the trivial case |b|=k=2. proposition 2.1let g be an abelian group with a, bg finite and nonempty:(i)if |a+b||a|+|b|k, then ra, b(x)k for all xa+b ; (ii)if g is finite and |a|+|b||g|+k, then ra, b(x)k for all xg. let g be an abelian group with a, bg finite and nonempty:(i)if |a+b||a|+|b|k, then ra, b(x)k for all xa+b ; (ii)if g is finite and |a|+|b||g|+k, then ra, b(x)k for all xg. if |a+b||a|+|b|k, then ra, b(x)k for all xa+b ; if g is finite and |a|+|b||g|+k, then ra, b(x)k for all xg. theorem 2.2let g be an abelian group, let sf(g) be a sequence, and let nz+ with n|s|. let g be an abelian group, let sf(g) be a sequence, and let nz+ with n|s|. if h = h(n(s)), then(1)|n(s)|(gg / hmin{n, vg(h(s))}n+1)|h|. a particular case of the (general) devos goddyn mohar theorem is the much simpler cauchy davenport theorem,,,. let p be prime and let aicp, for i=1,,n, be nonempty. then |i=1nai|min{i=1n|ai|n+1,p}. lemma 3.1let g be an abelian group, let gg, and let rf(g) be nontrivial with(2)(r){g,2g, let g be an abelian group, let gg, and let rf(g) be nontrivial with(2)(r){g,2g, proofthe result is clear when |r|2, so we may assume that |r|3. in view of (2) and |r|ord(g)1, we have 0(r). note, since |r|ord(g)1, that (3) shows h0 as well. from 0(r) and (2) (note if r|rh1 with (r)=(r), then (rr1)=0 ; hence (r)(rh1)), we have (rh1)({g,2g,,|r|g}{(r)})({g,2g, h{g,0 }, 0{g,2g,,|r|g } and (r)=|r|g imply that |(rh1)|n21.in this case, we apply theorem 2.2 to r1(0r2s). however, assuming h(s)r1 and repeating the above arguments using (5) instead of (6) and using (10) instead of (9), we arrive at the same contradiction. thus (7) is established in both cases.factor s = glt, where tf(g), and let r|t be a maximal length subsequence (possibly trivial) such that (r)=|r|g. in view of (7), (8) and (5), it follows that(11)vg(s)=l = h(s)max{r, nr}n2|t| and 0(t) ; in particular, 0(r).step 2. then it follows in view of (11) that r = ord(g) and that g is the only element from hg in supp(s) (else we can find a zero - sum of length distinct from r). iteratively applying the definition of d(g / h)|g / h| to h(u1sgord(g)), beginning with u trivial, we find a zero - sum mod h subsequence u|sgord(g) with |u|n|h||g / h|+1. adding on an appropriate number of terms from gord(g) (note (gord(g))=h) yields a zero - sum subsequence u|s with |u|n|h||g / h|+2. if |h||h|=r, a contradiction. on the other hand, if |h|=n2, then we obtain the same contradiction unless |u|=n21, (u)=g and su1gn/2=g0h. thus, if there is some g0supp(t)h with g0g0, then swapping g0 for g0 in u yields a new u|sgord(g) with (u)h and |u|=n21 but (u)g, whence we obtain the contradiction as before. therefore, we instead see that all terms outside h in supp(s) are equal to g0. however, since all terms inside h in supp(s) are equal to g, this shows that |supp(s)|2. so we henceforth assume that ord(g)=n, in which case gcn is cyclic.since |r||t|rln2=ord(g)2 (the last inequality holds else the proof is complete, while the other inequalities follow from (11)), and since (r)=|r|g, it follows that (12)0{g,2g,,rg}(gl),(13)0{(r+1)g,(r+2)g,,(l+|r|)g}r+1(glr). hence l+|r|ord(g)1=n1 and |r|0, then every element has at least two representations.suppose 0(a+(ti))(a+(tj)) for some i n21. in this case, we apply theorem 2.2 to r1(0r2s). however, assuming h(s)r1 and repeating the above arguments using (5) instead of (6) and using (10) instead of (9), we arrive at the same contradiction. therefore we conclude that h(s)r > n2>nr, as claimed. factor s = glt, where tf(g), and let r|t be a maximal length subsequence (possibly trivial) such that (r)=|r|g. in view of (7), (8) and (5), it follows that (11)vg(s)=l = h(s)max{r, nr}n2|t| and 0(t) ; in particular, 0(r). then it follows in view of (11) that r = ord(g) and that g is the only element from hg in supp(s) (else we can find a zero - sum of length distinct from r). iteratively applying the definition of d(g / h)|g / h| to h(u1sgord(g)), beginning with u trivial, we find a zero - sum mod h subsequence u|sgord(g) with |u|n|h||g / h|+1. adding on an appropriate number of terms from gord(g) (note (gord(g))=h) yields a zero - sum subsequence u|s with |u|n|h||g / h|+2. if |h||h|=r, a contradiction. on the other hand, if |h|=n2, then we obtain the same contradiction unless |u|=n21, (u)=g and su1gn/2=g0h. thus, if there is some g0supp(t)h with g0g0, then swapping g0 for g0 in u yields a new u|sgord(g) with (u)h and |u|=n21 but (u)g, whence we obtain the contradiction as before. therefore, we instead see that all terms outside h in supp(s) are equal to g0. however, since all terms inside h in supp(s) are equal to g, this shows that |supp(s)|2. since |r||t|rln2=ord(g)2 (the last inequality holds else the proof is complete, while the other inequalities follow from (11)), and since (r)=|r|g, it follows that (12)0{g,2g,,rg}(gl),(13)0{(r+1)g,(r+2)g,,(l+|r|)g}r+1(glr). hence l+|r|ord(g)1=n1 and |r|0, then every element has at least two representations. suppose 0(a+(ti))(a+(tj)) for some i n2. in this case, assuming h(s)r1, we can (as before) find r1 nonempty sets a1,,ar1g such that i=1r1gaig=0r2sf(g). applying the cauchy davenport theorem to a1,,ar1, we find that r1(0r2s)=g, contradicting (5). therefore we conclude, in view of (21), that h(s)r > n2>nr, as claimed. first remark that ord(g)=n holds trivially for |g|=p prime, so step 2 is unnecessary. next, noting that the case n=2 is trivial, we can assume n3, and thus that n is odd. this eliminates the lengthy extra portion of step 3 needed to establish (14) when r = n2 with n even. also, the following argument, using the cauchy davenport theorem instead of the devos goddyn mohar theorem, can be used to establish (7). to show (7), we proceed in the same two cases. note that if there are two distinct g, gsupp(s) with multiplicity at least nr, then this contradicts (20) in view of n odd, whence we may assume otherwise. thus, assuming h(s)nr, it is easily seen that we can find nr1 nonempty sets a1,,anr1g such that i=1nr1gaig=0nr1sx1f(g), for some xsupp(s) (see [1, proposition 2.1 ]). applying the cauchy davenport theorem to a1,,anr1, we find that nr1(0nr1sx1)=g, whence nr1(0nr1s)=g, contradicting (6). therefore we may instead assume that (20) fails, i.e, (21)r > n2. in this case, assuming h(s)r1, we can (as before) find r1 nonempty sets a1,,ar1g such that i=1r1gaig=0r2sf(g). applying the cauchy davenport theorem to a1,,ar1, we find that r1(0r2s)=g, contradicting (5). therefore we conclude, in view of (21), that h(s)r > n2>nr, as claimed. | an old conjecture of graham stated that if n is a prime and s is a sequence of n terms from the cyclic group cn such that all (nontrivial) zero - sum subsequences have the same length, then s must contain at most two distinct terms. in 1976, erds and szemerdi gave a proof of the conjecture for sufficiently large primes n. however, the proof was complicated enough that the details for small primes were never worked out. both in the paper of erds and szemerdi and in a later survey by erds and graham, the complexity of the proof was lamented. recently, a new proof, valid even for non - primes n, was given by gao, hamidoune and wang, using savchev and chen s recently proved structure theorem for zero - sum free sequences of long length in cn. however, as this is a fairly involved result, they did not believe it to be the simple proof sought by erds, graham and szemerdi. in this paper, we give a short proof of the original conjecture that uses only the cauchy davenport theorem and pigeonhole principle, thus perhaps qualifying as a simple proof. replacing the use of the cauchy davenport theorem with the devos goddyn mohar theorem, we obtain an alternate proof, albeit not as simple, of the non - prime case. additionally, our method yields an exhaustive list detailing the precise structure of s and works for an arbitrary finite abelian group, though the only non - cyclic group for which the hypotheses are non - void is c2c2 m. |
a 7-year - old girl had been healthy until 7 days before she was admitted to niigata prefectural hospital, niigata, japan, on june 21, 2010, for cough and prolonged fever. on day 1 of her illness, the girl had visited her primary care physician and was prescribed azithromycin, a macrolide antimicrobial drug, for a lower respiratory tract infection. on day 7 of her illness, the girl s condition worsened acutely, with increased cough and fever, and she again visited her primary care physician. a chest radiograph showed pulmonary infiltrates in the left upper lung, and the patient was referred to our hospital on day 8 of her illness. the girl s history and family history were unremarkable. on hospital admission, the patient was alert and oriented. her temperature was 38.9c, heart rate was 101 beats / min, and oxygen saturation was 97%. the girl did not describe symptoms of myalgia, and physical examination did not show signs of erythema, hepatosplenomegaly, neurologic abnormalities, muscle weakness, or muscle atrophy. results of the initial laboratory test were as follows : leukocyte count, 6.2 10 cells / l (reference 3.08.6 10 cells / l) ; hemoglobin, 1.95 mmol / l (reference 1.672.31 mmol / l) ; platelet count, 23.3 10/l (reference 15.036.1 10/l) ; c - reactive protein, 27 mg / l (reference < 3.0 mg / l) ; aspartate aminotransferase, 161 iu / l (reference 1331 iu / l) ; alanine aminotransferase, 83 iu / l (reference 627 iu / l) ; lactate dehydrogenase, 691 iu / l (reference119229 iu / l) ; blood urea nitrogen, 3.2 mmol urea / l (reference 2.97.2 mmol urea / l) ; creatinine, 31.8 mol / l (reference 44.270.6 mol / l) ; sodium, 135 mmol / l (reference 138146 mmol / l) ; potassium, 4.1 mmol / l (reference 3.64.9 mmol / l) ; and chloride 96 mmol / l (reference 99109 mmol / l). a venous blood gas determination on room air showed a ph of 7.464 (reference 7.357.45kpa) and carbon dioxide partial pressure of 4.9 kpa (reference 4.76.0 kpa). levels of serum glucose, albumin, calcium, amylase, and bilirubin were normal (references 70109 mg / dl, 4.15.0 g / dl, 8.710.0 mg / dl, 39108 u / ml, and 0.30.9 mg / dl, respectively). creatine phosphokinase was elevated to 12,159 ng / ml (reference 45163 ng / ml). urinalysis showed blood 3 +, but analysis of urine sediment by microscopy showed no erythrocytes. the urine myoglobin level was 39,900 g / l (reference < 10 g / l). the serum concentration of cytokine interleukin (il)-18 on admission was 612 pg / ml (reference < 260 pg / ml), and the concentration of tumor necrosis factor (tnf-) was 3.48 pg / ml (reference < 1.79 pg / ml). the girl s fever did not respond to treatment with azithromycin, and she was given a tentative diagnosis of antimicrobial drug because the patient was < 8 years of age, she was started on treatment with tosufloxacin, a fluoroquinolone, granules for children (12 mg / kg / d) and steroid therapy (methylprednisolone, 1 mg / kg / d). the patient s fever resolved the next day, and her urine output was maintained with intravenous hydration. no signs or symptoms of muscle involvement developed during the patient s hospital stay. on day 16 after the patient was admitted to the hospital, results of laboratory testing showed improved values for creatine phosphokinase (1,855 ng / ml), aspartate aminotransferase (101 iu / l), alanine aminotransferase (162 pulmonary infiltrates seen on a chest radiograph had decreased substantially by day 16, and the patient was discharged from the hospital. on day 8 after discharge, her abnormal test results returned to normal, and her illness showed no signs of relapse. culture results for a respiratory sample obtained during hospitalization revealed normal bacterial flora, and the results for rapid diagnostic tests for influenza virus, adenovirus, and respiratory syncytial virus were negative at admission. the m. pneumoniae antibody titer by the particle agglutination test was 1,280 at admission, and 2 days later, the antibody titer had increased to 10,240. at hospital admission, with permission from the girl and her parents, a pharyngeal swab specimen was obtained to test for m. pneumoniae. the sample was sent to the laboratory of the kanagawa prefectural institute of public health, chigasaki, japan, where pcr and restriction fragment length polymorphism analysis were performed as described (1). a macrolide - resistant m. pneumoniae strain with an ag transition at position 2063 of the 23s rrna gene (designated a2063 g) was detected. laboratory test results from admission to 8 days after discharge are summarized in the table. this case of rhabdomyolysis in a 7-year - old girl is an unusual extrapulmonary manifestation of antimicrobial drug resistant m. pneumoniae infection. rhabdomyolysis is characterized by rupture and necrosis of muscle fibers, resulting in the release of cell breakdown products into the bloodstream and extracellular space. direct muscle injury is the most common cause of rhabdomyolysis, but a number of other causes are possible : hereditary enzyme disorders, drugs, toxins, endocrinopathies, malignant hyperthermia, neuroleptic malignant syndrome, heatstroke, hypothermia, electrolyte alterations, diabetic ketoacidosis and nonketotic hyperosmolar coma, severe hypothyroidism or hyperthyroidism, and bacterial or viral infections (2). bacterial and viral infections account for 5% of rhabdomyolysis cases in adults (3). because pathomechanisms other than infection can cause rhabdomyolysis (4,5), we can not say with certainty that m. pneumoniae infection caused this syndrome in the patient reported here. one possible mechanism of rhabdomyolysis is induction of inflammatory cytokines, such as tnf- and il-1. these cytokines can cause acute proteolysis in a variety of organs, including skeletal muscles (6,7), and m. pneumoniae can induce these cytokines (8). our patient had high levels of tnf- and il-18 during the acute phase of m. pneumoniae infection, and it is highly possible that these m. pneumoniae no other apparent cause, such as trauma, endocrine disorder, or infection, other than m. pneumoniae, was found for the development of rhabdomyolysis in this patient. a confounding factor in this case was that the extrapulmonary manifestation of m. pneumoniae infection was caused by an antimicrobial drug resistant strain of m. pneumoniae. approximately 15% of m. pneumoniae strains isolated from patients in japan are resistant to antimicrobial drugs (1,9,10), which may explain why extrapulmonary manifestations of antimicrobial drug however, the proportion of antimicrobial drug resistance in japan is increasing, so extrapulmonary manifestations of antimicrobial drug resistant m. pneumoniae infection might also increase. in conclusion, this case of rhabdomyolysis was associated with and, in the absence of any other apparent cause, appears to be attributable to infection with antimicrobial drug the development of a system that can be used in routine clinical practice to rapidly identify antimicrobial drug resistant m. pneumoniae would be highly beneficial in this era of increasing antimicrobial drug resistance. | we describe a case of rhabdomyolysis in a patient infected with antimicrobial drug resistant mycoplasma pneumoniae the patient s acute - phase serum levels of interleukin-18 and tumor necrosis factor were high, which suggests a pathogenic role for m. pneumoniae. in an era of increasing antimicrobial drug resistance, a system for rapidly identifying resistant m. pneumoniae would be beneficial. |
the single - coding exon of kcnj11 and the 39 exons of abcc8 were amplified and sequenced as described (7). reactions were analyzed on an abi 3730 capillary sequencer (applied biosystems, warrington, u.k.). sequences were compared with the reference sequences nm_000525 and nm_000352.2, which incorporate the alternatively spliced residue in exon 17 (l78208, l78224), using mutation surveyor 3.20 software (softgenetics, state college, pa). mutation testing was performed on parental dna extracted from peripheral leukocytes, and microsatellite analysis was used to confirm family relationships. human kir6.2 (genbank nm000525 ; e23 and i337) and rat sur1 (genbank l40624) were used. site - directed mutagenesis, synthesis of capped mrna, and preparation of xenopus laevis oocytes were performed as reported (33). oocytes were co - injected with 4 ng of wild - type or mutant sur1 mrna and 0.8 ng kir6.2 mrna, incubated in barth s solution and studied 14 days after injection. to simulate the heterozygous state, sur1 was coexpressed with a 1:1 mixture of wild - type and mutant kir6.2. for each batch of oocytes, all homomeric or heterozygous mutations were injected to enable direct comparison of their effects and to control for batch - to - batch variation in expression levels (see supplementary data). whole - cell currents were recorded using a two - electrode voltage clamp in response to voltage steps of 20 mv from a holding potential of 10 mv, in a solution containing (in mmol / l) 90 kcl, 1 mgcl2, 1.8 cacl2, and 5 hepes (ph 7.4 with koh) (33). metabolic inhibition was induced by 3 mmol / l sodium azide, the katp channel opener diazoxide (340 mol / l) was used to fully activate the channels, and tolbutamide (0.5 macroscopic currents were recorded from giant inside - out patches at 60 mv ; inward currents are shown as upward deflections of the current trace. the pipette (external) solution contained (in mmol / l) 140 kcl, 1.2 mgcl2, 2.6 cacl2, and 10 hepes (ph 7.4 with koh). the intracellular solution contained (in mmol / l) 107 kcl, 11 egta, 2 mgcl2, 1 cacl2, and 10 hepes (ph 7.2 with koh), as well as nucleotides as indicated. the mg - free intracellular solution contained (in mmol / l) 107 kcl, 1 k2so4, 10 egta, and 10 hepes (ph 7.2 with koh), as well as nucleotides as indicated. atp concentration - inhibition curves were constructed by alternating control and test solutions. to control for possible rundown, or activation by mgatp, the conductance in the test solution was expressed as the mean of that in control solution before and after nucleotide application. atp concentration - response curves were fit with : g / gc = 1/{1+[(atp)/ic50 ] } (eq. 1), where (atp) is the atp concentration, ic50 is the atp concentration at which inhibition is half maximal, and h is the slope factor. in some experiments (figs. 6 and 7), we used a conditioning prepulse of variable [mgatp ], followed by a test pulse of mgatp. current during the test pulse was expressed as a fraction of that in nucleotide - free solution. the single - coding exon of kcnj11 and the 39 exons of abcc8 were amplified and sequenced as described (7). reactions were analyzed on an abi 3730 capillary sequencer (applied biosystems, warrington, u.k.). sequences were compared with the reference sequences nm_000525 and nm_000352.2, which incorporate the alternatively spliced residue in exon 17 (l78208, l78224), using mutation surveyor 3.20 software (softgenetics, state college, pa). mutation testing was performed on parental dna extracted from peripheral leukocytes, and microsatellite analysis was used to confirm family relationships. human kir6.2 (genbank nm000525 ; e23 and i337) and rat sur1 (genbank l40624) were used. site - directed mutagenesis, synthesis of capped mrna, and preparation of xenopus laevis oocytes were performed as reported (33). oocytes were co - injected with 4 ng of wild - type or mutant sur1 mrna and 0.8 ng kir6.2 mrna, incubated in barth s solution and studied 14 days after injection. to simulate the heterozygous state, sur1 was coexpressed with a 1:1 mixture of wild - type and mutant kir6.2. for each batch of oocytes, all homomeric or heterozygous mutations were injected to enable direct comparison of their effects and to control for batch - to - batch variation in expression levels (see supplementary data). whole - cell currents were recorded using a two - electrode voltage clamp in response to voltage steps of 20 mv from a holding potential of 10 mv, in a solution containing (in mmol / l) 90 kcl, 1 mgcl2, 1.8 cacl2, and 5 hepes (ph 7.4 with koh) (33). metabolic inhibition was induced by 3 mmol / l sodium azide, the katp channel opener diazoxide (340 mol / l) was used to fully activate the channels, and tolbutamide (0.5 mmol / l) was used to block katp channels. macroscopic currents were recorded from giant inside - out patches at 60 mv ; inward currents are shown as upward deflections of the current trace. the pipette (external) solution contained (in mmol / l) 140 kcl, 1.2 mgcl2, 2.6 cacl2, and 10 hepes (ph 7.4 with koh). the intracellular solution contained (in mmol / l) 107 kcl, 11 egta, 2 mgcl2, 1 cacl2, and 10 hepes (ph 7.2 with koh), as well as nucleotides as indicated. the mg - free intracellular solution contained (in mmol / l) 107 kcl, 1 k2so4, 10 egta, and 10 hepes (ph 7.2 with koh), as well as nucleotides as indicated. to control for possible rundown, or activation by mgatp, the conductance in the test solution was expressed as the mean of that in control solution before and after nucleotide application. atp concentration - response curves were fit with : g / gc = 1/{1+[(atp)/ic50 ] } (eq. 1), where (atp) is the atp concentration, ic50 is the atp concentration at which inhibition is half maximal, and h is the slope factor. in some experiments (figs. 6 and 7), we used a conditioning prepulse of variable [mgatp ], followed by a test pulse of mgatp. current during the test pulse was expressed as a fraction of that in nucleotide - free solution. we identified novel heterozygous abcc8 mutations, e1507d (c.4521g > t) and e1507 g (c.4520a > g), in two male probands. this numbering refers to the l78208 human abcc8 isoform that encodes 1,582 amino acids and contains an additional residue in nbd1 ; consequently, e1507 in our sequence is equivalent to e1506 in that of huopio. proband 1 with the e1507d mutation weighed 2.9 kg at birth (42 weeks gestation) and was diagnosed as insulin - dependent at 10 weeks. at 30 weeks, the diabetes remitted and insulin treatment was withdrawn. proband 2 weighed 3.5 kg at birth (40 weeks gestation) and was diagnosed with diabetes at the age of 8 weeks. after an e1507 g mutation was identified, the patient was successfully transferred from insulin to glibenclamide at the age of 6 months. he was treated with glibenclamide (2.5 mg, once daily) for 4 months, at which time the diabetes remitted. a family history of diabetes had not been reported for either proband, and testing of parental samples demonstrated that the e1507d and e1507 g mutations had each arisen de novo in the proband. we analyzed the effects of mutations at e1506 of rat sur1 (equivalent to e1507 in the l78208 human abcc8 isoform) on the metabolic regulation of the katp channel by measuring whole - cell currents. wild - type (kir6.2-sur1) channels expressed in xenopus oocytes are normally closed, due to the high intracellular atp concentration ([atp]i), but can be opened by lowering [atp]i using a metabolic inhibitor, such as sodium azide (fig. the subsequent addition of the katp channel - opener diazoxide (0.34 mmol / l) increased the current only slightly, suggesting wild - type katp channels are almost fully activated by 3 mmol / l sodium azide. tolbutamide (0.5 mmol / l) almost completely blocked the current, confirming it flows through katp channels. c : representative whole - cell current amplitudes evoked by repeated voltage steps from 10 to 30 mv for wild - type (a, wt), and homomeric (b, home1506d) or heterozygous (c, hete1506d) kir6.2/sur1-e1506d channels. the bars indicate application of 3 mmol / l azide, 0.34 mmol / l diazoxide (dz), and 0.5 mmol / l tolbutamide (tb). scale bars are 0.5 a (y - axis) and 400 s (x - axis). d and e : mean steady - state whole - cell katp current amplitudes for wild - type homomeric (d) and heterozygous (e) mutant channels. currents were evoked by a voltage step from 10 to 30 mv before (control,) and after () application of 3 mmol / l azide, in the presence of 3 mmol / l azide plus 0.34 mmol / l dz (), and 3 azide + 0.5 mmol / l tb () for wt and sur1 mutant channels, as indicated. p 0.05, p 0.01, p 0.001 against wt (student t test). mutations that reduce the channel atp sensitivity normally increase the whole - cell current in the absence of metabolic inhibition, reflecting the fact that they are less blocked by resting [atp]i. a small but significant increase in resting current was observed for both homomeric kir6.2/sur1-e1506d (home1506d) and kir6.2/sur1-e1506 g (home1506 g) channels (fig. these currents were only slightly enhanced by metabolic inhibition but were further increased by diazoxide, suggesting azide - induced changes in nucleotide concentrations may be insufficient to fully activate the mutant channels. as previously reported (27), kir6.2/sur1-e1506k (home1506k) currents were minimal in both the absence and presence of azide but were slightly increased by diazoxide. the resting currents of heterozygous (het) e1506d and hete1506 g channels were significantly greater than wild - type channels, which may explain why these mutations cause neonatal diabetes. azide strongly activated all heterozygous channels, and in the additional presence of diazoxide there was a further increase. the maximal hete1506d current was not significantly different from the wild - type current, and that of het1506 g channels was only slightly smaller. these results suggest that hete1506d and hete1506 g channels retain metabolic sensitivity and that their functional expression is similar to wild - type channels. the maximal amplitude of hete1506k channels was reduced, as previously reported (27). tolbutamide (500 mol / l) blocked hete1506d currents by 95 1% (n = 8), hete1506 g currents by 95 1% (n = 6), and e1506k currents by 91 2% (n = 6) compared with 96 1% (n = 7) for wild - type channels. in the cell - attached configuration, the home1506d and home1506 g currents were both larger than wild - type or home1506k currents, consistent with the whole - cell resting currents (supplementary fig however, no significant difference was found between wild - type and mutant hete1506 channels. there was no difference in the amplitude of wild - type and home1506k katp currents recorded after patch excision (table 1, supplementary fig. 2b), indicating the smaller amplitude of home1506k whole - cell currents is not attributable to reduced channel expression. however, there was no difference in hete1506 g currents and a 300 mol / l mgatp markedly reduced the ability of 100 mol / l mgatp to block e1506d and e1506 g channels but had only a small effect on wild - type channels and no effect on the home1506k channels. atp sensitivity of wild - type and mutant channels after preconditioning with 10 mmol / l mgatp. a : representative home1506d currents recorded in response to different mgatp concentrations (as indicated), preceded by a preconditioning pulse in 10 mmol / l mgatp solution (indicated by the unlabeled solid line []) and followed by control (nucleotide - free) solution. the dashed lines (- - -) indicate the zero current. b d : mean relationship between mgatp concentration and katp current (i), expressed relative to that in the absence of nucleotide (ic), after 30-s preincubation in 10 mmol / l mgatp for wt (, n = 9), heterozygous (), and homomeric mutant () channels. channels were composed of kir6.2 and wild - type or mutant sur1 subunits, as indicated. c : home1506 g (n = 7), hete1506 g (n = 7). d : home1506k (n = 7), hete1506k (n = 5). 1, with the following parameters (in mol / l for ic50) : wild - type, ic50 = 28, h = 1.2 ; home1506d, ic50 = 78, h = 1.2 ; hete1506d, ic50 = 45, h = 1.2 ; home1506 g, ic50 = 105, h = 1.3 ; hete1506 g, ic50 = 45, h = 1.3 ; home1506k, ic50 = 18, h = 1.6 ; hete1506k, ic50 = 26, h = 1.4. a : representative home1506 g currents recorded in response to test pulses of 100 mol / l mgatp (unlabeled solid line []), preceded by a preconditioning pulse to a variable mgatp concentration (10 mol / l to 10 mmol / l, as indicated) and followed by control (nucleotide - free) solution. b : mgatp concentration during the conditioning prepulse plotted against the current during a 100 mol / l mgatp test pulse. current is expressed as a fraction of that in control (nucleotide - free) solution after the test pulse for wild - type (, n = 14), home1506d (, n = 15), home1506 g (, n = 10), and home1506k (, n = 8) channels. the lines are drawn through the points by eye. pre - exposure to 10 mmol / l mgadp, which fully blocked the wild - type and mutant channels, did not affect the ability of 100 mol / l mgatp to block any of the four channel types (data not shown). we identified novel heterozygous abcc8 mutations, e1507d (c.4521g > t) and e1507 g (c.4520a > g), in two male probands. this numbering refers to the l78208 human abcc8 isoform that encodes 1,582 amino acids and contains an additional residue in nbd1 ; consequently, e1507 in our sequence is equivalent to e1506 in that of huopio. proband 1 with the e1507d mutation weighed 2.9 kg at birth (42 weeks gestation) and was diagnosed as insulin - dependent at 10 weeks. at 30 weeks, the diabetes remitted and insulin treatment was withdrawn. proband 2 weighed 3.5 kg at birth (40 weeks gestation) and was diagnosed with diabetes at the age of 8 weeks. after an e1507 g mutation was identified, the patient was successfully transferred from insulin to glibenclamide at the age of 6 months. he was treated with glibenclamide (2.5 mg, once daily) for 4 months, at which time the diabetes remitted. a family history of diabetes had not been reported for either proband, and testing of parental samples demonstrated that the e1507d and e1507 g mutations had each arisen de novo in the proband. we analyzed the effects of mutations at e1506 of rat sur1 (equivalent to e1507 in the l78208 human abcc8 isoform) on the metabolic regulation of the katp channel by measuring whole - cell currents. wild - type (kir6.2-sur1) channels expressed in xenopus oocytes are normally closed, due to the high intracellular atp concentration ([atp]i), but can be opened by lowering [atp]i using a metabolic inhibitor, such as sodium azide (fig. the subsequent addition of the katp channel - opener diazoxide (0.34 mmol / l) increased the current only slightly, suggesting wild - type katp channels are almost fully activated by 3 mmol / l sodium azide. tolbutamide (0.5 mmol / l) almost completely blocked the current, confirming it flows through katp channels. c : representative whole - cell current amplitudes evoked by repeated voltage steps from 10 to 30 mv for wild - type (a, wt), and homomeric (b, home1506d) or heterozygous (c, hete1506d) kir6.2/sur1-e1506d channels. the bars indicate application of 3 mmol / l azide, 0.34 mmol / l diazoxide (dz), and 0.5 mmol / l tolbutamide (tb). scale bars are 0.5 a (y - axis) and 400 s (x - axis). d and e : mean steady - state whole - cell katp current amplitudes for wild - type homomeric (d) and heterozygous (e) mutant channels. currents were evoked by a voltage step from 10 to 30 mv before (control,) and after () application of 3 mmol / l azide, in the presence of 3 mmol / l azide plus 0.34 mmol / l dz (), and 3 azide + 0.5 mmol / l tb () for wt and sur1 mutant channels, as indicated. p 0.05, p 0.01, p 0.001 against wt (student t test). mutations that reduce the channel atp sensitivity normally increase the whole - cell current in the absence of metabolic inhibition, reflecting the fact that they are less blocked by resting [atp]i. a small but significant increase in resting current was observed for both homomeric kir6.2/sur1-e1506d (home1506d) and kir6.2/sur1-e1506 g (home1506 g) channels (fig. these currents were only slightly enhanced by metabolic inhibition but were further increased by diazoxide, suggesting azide - induced changes in nucleotide concentrations may be insufficient to fully activate the mutant channels. as previously reported (27), kir6.2/sur1-e1506k (home1506k) currents were minimal in both the absence and presence of azide but were slightly increased by diazoxide. the resting currents of heterozygous (het) e1506d and hete1506 g channels were significantly greater than wild - type channels, which may explain why these mutations cause neonatal diabetes. azide strongly activated all heterozygous channels, and in the additional presence of diazoxide there was a further increase. the maximal hete1506d current was not significantly different from the wild - type current, and that of het1506 g channels was only slightly smaller. these results suggest that hete1506d and hete1506 g channels retain metabolic sensitivity and that their functional expression is similar to wild - type channels. the maximal amplitude of hete1506k channels was reduced, as previously reported (27). tolbutamide (500 mol / l) blocked hete1506d currents by 95 1% (n = 8), hete1506 g currents by 95 1% (n = 6), and e1506k currents by 91 2% (n = 6) compared with 96 1% (n = 7) for wild - type channels. in the cell - attached configuration, the home1506d and home1506 g currents were both larger than wild - type or home1506k currents, consistent with the whole - cell resting currents (supplementary fig. however, no significant difference was found between wild - type and mutant hete1506 channels. there was no difference in the amplitude of wild - type and home1506k katp currents recorded after patch excision (table 1, supplementary fig. 2b), indicating the smaller amplitude of home1506k whole - cell currents is not attributable to reduced channel expression. in contrast, home1506d and home1506 g currents were both substantially smaller. however, there was no difference in hete1506 g currents and a 300 mol / l mgatp markedly reduced the ability of 100 mol / l mgatp to block e1506d and e1506 g channels but had only a small effect on wild - type channels and no effect on the home1506k channels. atp sensitivity of wild - type and mutant channels after preconditioning with 10 mmol / l mgatp. a : representative home1506d currents recorded in response to different mgatp concentrations (as indicated), preceded by a preconditioning pulse in 10 mmol / l mgatp solution (indicated by the unlabeled solid line []) and followed by control (nucleotide - free) solution. the dashed lines (- - -) indicate the zero current. b d : mean relationship between mgatp concentration and katp current (i), expressed relative to that in the absence of nucleotide (ic), after 30-s preincubation in 10 mmol / l mgatp for wt (, n = 9), heterozygous (), and homomeric mutant () channels. channels were composed of kir6.2 and wild - type or mutant sur1 subunits, as indicated. c : home1506 g (n = 7), hete1506 g (n = 7). d : home1506k (n = 7), hete1506k (n = 5). 1, with the following parameters (in mol / l for ic50) : wild - type, ic50 = 28, h = 1.2 ; home1506d, ic50 = 78, h = 1.2 ; hete1506d, ic50 = 45, h = 1.2 ; home1506 g, ic50 = 105, h = 1.3 ; hete1506 g, ic50 = 45, h = 1.3 ; home1506k, ic50 = 18, h = 1.6 ; hete1506k, ic50 = 26, h = 1.4. a : representative home1506 g currents recorded in response to test pulses of 100 mol / l mgatp (unlabeled solid line []), preceded by a preconditioning pulse to a variable mgatp concentration (10 mol / l to 10 mmol / l, as indicated) and followed by control (nucleotide - free) solution. b : mgatp concentration during the conditioning prepulse plotted against the current during a 100 mol / l mgatp test pulse. current is expressed as a fraction of that in control (nucleotide - free) solution after the test pulse for wild - type (, n = 14), home1506d (, n = 15), home1506 g (, n = 10), and home1506k (, n = 8) channels. pre - exposure to 10 mmol / l mgadp, which fully blocked the wild - type and mutant channels, did not affect the ability of 100 mol / l mgatp to block any of the four channel types (data not shown). our results provide a functional explanation for why mutation of e1506 in sur1 to lysine (k) results in hyperinsulinism, whereas mutation of the same residue to aspartate (d) or glycine (g) causes neonatal diabetes. this occurs because the mutations have opposite effects on the whole - cell katp currents ; hete1506d and hete1506 g increase resting whole - cell currents, whereas the hete1506k channels show no resting whole - cell currents and are activated less in response to metabolic inhibition. an increase in the resting -cell katp current would be expected to produce -cell hyperpolarization and reduce ca influx and insulin secretion evoked by glucose, thereby predisposing to diabetes. in contrast, a reduction in katp current will cause depolarization, maintained ca influx, and persistent insulin secretion, giving rise to hyperinsulinism. as reported for most other neonatal diabetes mutations in sur1 (18,21), the increase in the resting whole - cell currents was relatively small. such small changes are consistent with the relatively mild, transient form of diabetes and the absence of neurological symptoms (6,18). resting currents in the pancreatic -cell are likely larger than those measured in the oocyte, perhaps because of lower atp levels. it is not evident why the diabetes remits, but as is the case for other sur1 mutations causing transient neonatal diabetes (29), it seems likely the diabetes may later relapse and patients should be monitored for this possibility. sulfonylureas are an effective therapy for most patients with neonatal diabetes caused by katp channel mutations and are now the treatment of choice for this disease (24). we observed that tolbutamide blocked hete1506d and hete1506 g currents as much as wild - type katp channels, which explains why our patients could be successfully treated with glibenclamide. so why does the mutation of e1506 to k abolish resting whole - cell katp currents, whereas the mutation to e and g enhances them ? we observed that the neonatal diabetic mutant channels are substantially less sensitive to atp inhibition if they have been previously exposed to physiological atp concentrations of 110 mmol / l (37). in contrast, this effect was very small for wild - type channels and absent for e1506k channels. these results are consistent with the differences in the resting whole - cell current amplitude we measured in intact oocytes and suggest that neonatal diabetic channels have larger resting currents because they are less sensitive to atp. metabolic inhibition will result in a fall in atp and an increase in mgadp, both of which will affect the response of the katp channel. both reduced atp inhibition at kir6.2 and increased mgadp activation at sur1 will contribute to the increase in wild - type current and, to a lesser extent, the heterozygous channels. the impaired response of the home1506k channels may be explained by their markedly reduced mgadp activation, as previously suggested (27). we suggest the small increase in the home1506d and home1506 g currents is because their reduced mgadp activation is outweighed by their greatly reduced atp sensitivity, allowing the channels to open in response to falling atp levels. because atp sensitivity did not change in the absence of mg, it appears that the mutations do not reduce nucleotide block at kir6.2. e1506 lies within nbs2 of sur1, close to the -phosphate of atp and its associated mg atom (38). the equivalent residue in other abc transporters is known to be involved in nucleotide binding and atpase hydrolysis (32) and its mutation to aspartate dramatically reduces atpase activity and the initial p (pi) release in abc proteins (39,40). thus mutation of e1506 in sur1 is likely to alter the conformation that mg nucleotides adopt in the nbs and thereby impair 1) nucleotide binding, and/or 2) atpase activity, and/or 3) the mechanism by which mgadp occupation at nbs2 is translated into channel opening. it seems possible that the e1506 mutations trap nbs2 in different conformational states of the atpase cycle (fig. 7c) that either promote channel activity (e, g) or do not (k). all three mutations markedly reduce mgadp activation of the channel, arguing that occupancy of this state is reduced and/or the translation of the mgadp - bound state into channel activity is impaired. mutation of the equivalent residue in mrp1 to aspartate does not alter mgadp binding (32), which suggests that sur1-e1506d might bind mgadp but that binding no longer leads to channel activation. all mutations support mgatp activation of the channel, as indicated by the use of a kir6.2 atp - insensitive mutation. it is not possible to distinguish whether the channel is activated by occupancy of the mgatp - bound state or the mgadp + pi state, or both (fig. nevertheless, the ability of mgatp, but not mgadp, to stimulate mutant channels almost as effectively as wild - type channels argues that one or both of these states can do so. the slower off - rate of mgatp seen with the neonatal diabetes mutations (especially e1506d) may indicate that the channel becomes trapped in a particular state of the reaction cycle that is associated with increased channel activity ; for example, the mgatp - bound or mgadp + pi state. the off - rate of mgadp was much faster than that of mgatp for the e1506d channels, and mgadp had little stimulatory effect, which supports arguments that it can not be the mgadp - bound state. the most striking difference between the e1506k and e1506g / e1506d channels is that shown in figs. 6 and 7 : pre - exposure to millimolar concentrations of mgatp desensitizes the channel to subsequent inhibition by a lower atp concentration. this can not be due to an effect of mgatp on kinases, for example, because this should be similar for all channels. first, exposure to a saturating atp concentration for an extended period may force the channels into a state in which nbs1 and nbs2 on all four subunits are both occupied by nucleotide, and this may lead to greater channel activation. second, it may relate to the slower off - rate of mgatp of neonatal diabetes channels, which could maintain the channel in an activated state for an extended period, so impairing atp block. as shown previously, mg - nucleotide activation not only enhances channel activity per se but also reduces atp inhibition at kir6.2 (41). in the -cell, where atp levels are normally within the millimolar range, so why does the mutation of e1506 to k abolish resting whole - cell katp currents, whereas the mutation to e and g enhances them ? we observed that the neonatal diabetic mutant channels are substantially less sensitive to atp inhibition if they have been previously exposed to physiological atp concentrations of 110 mmol / l (37). in contrast, this effect was very small for wild - type channels and absent for e1506k channels. these results are consistent with the differences in the resting whole - cell current amplitude we measured in intact oocytes and suggest that neonatal diabetic channels have larger resting currents because they are less sensitive to atp. metabolic inhibition will result in a fall in atp and an increase in mgadp, both of which will affect the response of the katp channel. both reduced atp inhibition at kir6.2 and increased mgadp activation at sur1 will contribute to the increase in wild - type current and, to a lesser extent, the heterozygous channels. the impaired response of the home1506k channels may be explained by their markedly reduced mgadp activation, as previously suggested (27). we suggest the small increase in the home1506d and home1506 g currents is because their reduced mgadp activation is outweighed by their greatly reduced atp sensitivity, allowing the channels to open in response to falling atp levels. because atp sensitivity did not change in the absence of mg, it appears that the mutations do not reduce nucleotide block at kir6.2. e1506 lies within nbs2 of sur1, close to the -phosphate of atp and its associated mg atom (38). the equivalent residue in other abc transporters is known to be involved in nucleotide binding and atpase hydrolysis (32) and its mutation to aspartate dramatically reduces atpase activity and the initial p (pi) release in abc proteins (39,40). thus mutation of e1506 in sur1 is likely to alter the conformation that mg nucleotides adopt in the nbs and thereby impair 1) nucleotide binding, and/or 2) atpase activity, and/or 3) the mechanism by which mgadp occupation at nbs2 is translated into channel opening. it seems possible that the e1506 mutations trap nbs2 in different conformational states of the atpase cycle (fig. 7c) that either promote channel activity (e, g) or do not (k). all three mutations markedly reduce mgadp activation of the channel, arguing that occupancy of this state is reduced and/or the translation of the mgadp - bound state into channel activity is impaired. mutation of the equivalent residue in mrp1 to aspartate does not alter mgadp binding (32), which suggests that sur1-e1506d might bind mgadp but that binding no longer leads to channel activation. all mutations support mgatp activation of the channel, as indicated by the use of a kir6.2 atp - insensitive mutation. it is not possible to distinguish whether the channel is activated by occupancy of the mgatp - bound state or the mgadp + pi state, or both (fig. nevertheless, the ability of mgatp, but not mgadp, to stimulate mutant channels almost as effectively as wild - type channels argues that one or both of these states can do so. the slower off - rate of mgatp seen with the neonatal diabetes mutations (especially e1506d) may indicate that the channel becomes trapped in a particular state of the reaction cycle that is associated with increased channel activity ; for example, the mgatp - bound or mgadp + pi state. the off - rate of mgadp was much faster than that of mgatp for the e1506d channels, and mgadp had little stimulatory effect, which supports arguments that it can not be the mgadp - bound state. the most striking difference between the e1506k and e1506g / e1506d channels is that shown in figs. 6 and 7 : pre - exposure to millimolar concentrations of mgatp desensitizes the channel to subsequent inhibition by a lower atp concentration. this can not be due to an effect of mgatp on kinases, for example, because this should be similar for all channels. first, exposure to a saturating atp concentration for an extended period may force the channels into a state in which nbs1 and nbs2 on all four subunits are both occupied by nucleotide, and this may lead to greater channel activation. second, it may relate to the slower off - rate of mgatp of neonatal diabetes channels, which could maintain the channel in an activated state for an extended period, so impairing atp block. as shown previously, mg - nucleotide activation not only enhances channel activity per se but also reduces atp inhibition at kir6.2 (41). in the -cell, where atp levels are normally within the millimolar range, this mechanism would result in reduced atp inhibition and thus larger resting katp currents. | objectivetwo novel mutations (e1506d, e1506 g) in the nucleotide - binding domain 2 (nbd2) of the atp - sensitive k+ channel (katp channel) sulfonylurea receptor 1 (sur1) subunit were detected heterozygously in patients with neonatal diabetes. a mutation at the same residue (e1506k) was previously shown to cause congenital hyperinsulinemia. we sought to understand why mutations at the same residue can cause either neonatal diabetes or hyperinsulinemia.research design and methodsneonatal diabetic patients were sequenced for mutations in abcc8 (sur1) and kcnj11 (kir6.2). wild - type and mutant katp channels were expressed in xenopus laevis oocytes and studied with electrophysiological methods.resultsoocytes expressing neonatal diabetes mutant channels had larger resting whole - cell katp currents than wild - type, consistent with the patients diabetes. conversely, no e1506k currents were recorded at rest or after metabolic inhibition, as expected for a mutation causing hyperinsulinemia. katp channels are activated by mg - nucleotides (via sur1) and blocked by atp (via kir6.2). all mutations decreased channel activation by mgadp but had little effect on mgatp activation, as assessed using an atp - insensitive kir6.2 subunit. importantly, using wild - type kir6.2, a 30-s preconditioning exposure to physiological mgatp concentrations (> 300 mol / l) caused a marked reduction in the atp sensitivity of neonatal diabetic channels, a small decrease in that of wild - type channels, and no change for e1506k channels. this difference in mgatp inhibition may explain the difference in resting whole - cell currents found for the neonatal diabetes and hyperinsulinemia mutations.conclusionsmutations in the same residue can cause either hyperinsulinemia or neonatal diabetes. differentially altered nucleotide regulation by nbd2 of sur1 can explain the respective clinical phenotypes. |
we report the case of an independent 78-year - old man with low back pain and mild lower limb weakness in whom iatrogenic meningitis and associated bacteremia developed after a computed tomography myelogram. the procedure was performed on june 21, 2013, on an outpatient basis in the radiology department of geelong hospital, a teaching tertiary hospital. the patient s medical history included atrial fibrillation that was managed by a permanent pacemaker, which precluded the use of magnetic resonance imaging. other conditions in his medical history included hypertension, gout, rash after penicillin exposure, and moderate chronic obstructive pulmonary disease that did not require long - term prednisone. the patient underwent fluoroscopy - guided spinal injection of 10 ml of iohexol 300 mgi / ml contrast medium from a single - dose vial prepared by a nurse in accordance with centers for disease control and prevention guidelines (10). the procedure was performed at the level of lumbar disc space 45 by using a 22-gauge spinal needle. aseptic measures included the use of sterile gloves, gown, drapes, and adequate skin antisepsis by the proceduralist. the opening pressure was normal ; the patient showed no signs of complications immediately post - procedure and was discharged after 4 hours of observation. the patient came to the emergency department within 18 hours of the procedure after onset of confusion, severe headache, neck pain, nausea, vomiting, and fever. the patient had a deteriorating conscious state ; the examination showed no additional remarkable findings. the patient s condition was investigated by using a septic screen, including blood cultures and a diagnostic lumbar puncture. subsequently, treatment with intravenous ceftazidime, vancomycin, and dexamethasone were commenced for presumed iatrogenic meningitis. the initial investigations showed a leukocyte count of 16.110/l and a c - reactive protein level of 9.8 mg / l that increased to 185 mg / l within 24 hours of the patient s return. the diagnostic lumbar puncture revealed turbid cerebrospinal fluid consisting of 4,260 10/l leukocytes, 99% polymorphonuclear leukocytes, 270 10/l erythrocytes, and levels of 3.0 g / l protein, and 1.7 mmol / l glucose. initial microscopic examination of cerebrospinal fluid revealed a gram - positive coccus ; on review, intracellular diplococci were identified, suggesting an undercolored specimen that likely represented neisseria species. ceftazidime was discontinued and intravenous ceftriaxone, 2 g twice daily, was initiated ; vancomycin was stopped on confirmation of n. sicca / subflava infection. a single set of blood cultures initiated on admission were also positive for this bacterial species, but subsequent blood cultures were negative. the final isolates of n. sicca / subflava from blood culture and cerebrospinal fluid were penicillin resistant but ceftriaxone sensitive. the patient required 24 hours of management in the intensive care unit because of profound confusion and severe agitation. he improved substantially during a period of 7 days in the hospital and was transitioned from intravenous ceftriaxone to a 7-day course of oral ciprofloxacin, 500 mg twice daily, to be completed after discharge. on discharge, the proceduralist who performed the spinal injection denied upper respiratory tract symptoms, but admitted to not using a face mask because of unawareness of the hospital lumbar puncture protocol mandating face mask use when conducting all lumbar punctures. a nasopharyngeal swab specimen confirmed that the proceduralist was a carrier of n. sicca / subflava who had an identical antibacterial drug resistance pattern to that identified in the case - patient. molecular typing of the organism could not be performed because the patient s isolate had been discarded. review by the infection control team identified the low level of awareness of and adherence to the hospital protocol for wearing face masks as a contributory factor. we describe the clinical course of iatrogenic meningitis caused by n. sicca / subflava with associated bacteremia after a spinal injection procedure. the suspected mechanism of transmission in this case is contamination of the sterile field or equipment by oropharyngeal secretions caused by nonuse of a face mask by a carrier of this organism. the prolonged and technically difficult nature of the procedure likely contributed to contamination by increasing exposure. this hypothesis is supported by the isolation of n. sicca / subflava with an identical antimicrobial resistance pattern in a swab sample from the proceduralist s nasopharynx. an alternative mechanism could involve oropharyngeal secretions from assistant staff or contamination of the contrast medium for spinal injection, although the latter is less likely because it was prepared in accordance with guidelines. in this case, nonadherence to face mask use standards resulted from lack of awareness by the clinician. to improve clinician awareness after this event the protocol mandated the use of face masks for all lumbar punctures, and it was disseminated to all clinical areas where lumbar punctures were performed. we believe that the best method for promotion of face mask use is making face masks available in preparatory areas and procedure rooms and requiring that all lumbar punctures are performed with the use of a face mask. this could include documentation of face mask use for all lumbar punctures and intermittent auditing. in conclusion, n. sicca / subflava, an organism that is harmless in the human oropharynx, can cause invasive infection in immunocompetent adults when introduced directly into the subarachnoid space. prevention is essential ; thus, wearing of face masks should be mandatory for all personnel present during lumbar punctures and all other sterile procedures, and compliance should be monitored. | we report a case of invasive neisseria sicca / subflava meningitis after a spinal injection procedure during which a face mask was not worn by the proceduralist. the report highlights the importance of awareness of, and adherence to, guidelines for protective face mask use during procedures that require sterile conditions. |
as with left main coronary artery diseases, patients with coronary ostial stenosis are at high risk of premature death, which relates directly to the extent of the area of myocardium placed in jeopardy., the incidence of ostial stenosis has varied between 0.13 and 2.7%, and in the majority of cases there is a coexisting disease in the multiple coronary vessels. it has been assumed to be atherosclerotic in origin and excludes the coexisting homozygous familial hypercholesterolemia, otherwise normal coronary vessels in patients with syphilis or other form of aortitis, congenital anomaly and iatrogenic ostial stenosis as a complication of coronary angiography or after coronary perfusion at the time of cardiac surgery. the clinical and angiographic profile of isolated coronary ostial stenosis is unique and at variance with that normally expected in patients with atherosclerotic coronary disease. a 50-year - old postmenopausal woman was admitted to the hospital because of cresendo angina. she was well except for tuberculous pleurisy experienced seven years earlier, when the onset of chronic stable angina pectoris took place, which, since then, responded to nitroglycerin. during the year or two before her admission, pain radiating to both shoulders and upper arms frequently accompanied the angina episodes. after admission, the angina was controlled by bed rest and intensive medical treatment with beta blocking and calcium channel blocking drugs and sublingual nitrates. there was no history of hypertention, cigarette smoking, use of alcohol, or birth control pills. upon admission, however, the head and neck were normal ; the jugular vein was not distended. the hematocrit was 37% ; the white - cell count was 9,900 per cubic millimeter with 61% neutrophils, 6% band form, 35% lymphocytes and 2% basophils. the prothrombin time was 10.3 seconds with 100% of control ; the bleeding time was 2.00 minutes ; the clotting time was 7.00 minutes. the sgot was 15 u, the ldh 120 u and the cpk 41 u per liter. the total cholesterol was 250 mg, the triglyceride 99 mg, and the hdl - cholesterol 59 mg per deciliter. the electrocardiogram was normal at rest and showed slight depression of the st segments and inverted t waves in leads i, avl and v4 through v6 during the angina episodes (fig. a chest x - ray showed that the lungs were clear and the heart size was normal ; the pulmonary vascular pattern was normal. the pulmonary artery pressure was 50/20 mmhg and pcwp 20 mmhg ; the aortic pressure was 165/85 mmhg and the lvedp 15 mmhg ; the cardiac output using the fick oxygen method and the ejection fraction were 7.831 liter perminute and 0.75, respectively. on left coronary angiography, a profound decrease in distal coronary pressure after coronary engagement with angina and the appearance of st - t wave changes in monitoring, the electrocardiogram were noticed (fig. 2). there was no abnormalities on the left coronary angiography on the routine lao and rao projection. but a shallow lao projection with a mild craniocaudal tilt of the left coronary angiography demonstrated a critical stenosis of the left main coronary ostium (fig. the patient underwent coronary bypass graft surgery and has been doing well without occurrence of angina since the surgery. isolated coronary ostial stenosis appears to be a rare lesion and in one series occurred with an incidence of 0.2% in a population of patients with coronary heart disease defined by coronary angiography. it occurs predominantly in women, usually before menopause (mean age 51 years). despite previous reports which have suggested a more frequent involvement of a right coronary ostium, recent analysis shows no significant differences in the distribution among left, right and bilateral ostial stenosis. it has been assumed that atherosclerosis, particularly early atheroma, is the most likely cause of this lesion. other investigations have suggested a congenital arterial hypoplasia complicated by progressive thickening of the aortic intima by advancing age or an inflammatory basis. histologic studies of nondiseased coronary ostia have suggested the presence of a circumferential sphincter - like muscle in the right coronary orifice of many patients, and it has been proposed that this may offer resistance to blood flow. occurring predominantly in women, usually before menopause, it is unknoun whether humoral factors are important. it is unclear whether the apparent rarity of this condition represents the true state of affairs or whether it is in part a reflection of the failure to report or recognize these cases at angiography. it is possible, using a preformed catheter, that the ostial lesion may be crossed during coronary engagement and remain undetected during subsequent cineangiography. thompson has suggested that a series of events should alert the cardiologist to the possibility of an ostial stenosis, including : 1) difficulty in cannulation of the coronary ostium ; 2) a profound decrease in distal coronary pressure after coronary engagement with or without angina or the appearance of st segment change in the monitoring electrocardiogram ; and 3) failure to observe return of contrast medium into the sinus of valsalva after intracoronary injection. the use of an amplatz catheter, approaching the coronary ostium from below, may help to prevent catheter impaction in this circumstance. nonselective injection into the sinus of valsalva usually provides inadequate definition of coronary ostium and, as reported previously, the left coronary ostium is best visualized in the shallow lao projection (15 to 25) with approximately 20 of craniocaudal tilet. once a left ostial lesion has been demonstrated, only a limited number of other views are advisable, including an rao projection. instant video playback facilities are essential in this regard. nitroglycerin administered immediately prior to coronary angiography reduces the possibility of spontaneous as well as catheter induced spasm. clinically, the observed severe symptoms of nyha functional class iii and iv is significant clue, and strongly positive tests in the early stage, frequent in the presence of left main coronary ostial stenosis, should alert the clinician to the possibility of left main coronary artery disease. suspecting the presence of left main coronary ostial stenosis prior to catheterization is important in reducing the risk of coronary angiography and improving the chances of correct diagnosis. angiography entails some risk because the catheter may readily occlude the stenotic ostium, resulting in a fall in pressure at the catheter tip, chest pain, dyspnea, diaphoresis, and a fall in systemic pressure. frequently, the catheter and ostium are not viewed in a plane suitable for demonstration of the ostial stenosis. unless the angiographer is aware of the possibility of ostial occlusion by the catheter, this may occur and lead to hypotension, arrhythmia, and cardiac arrest. when the junction of the catheter and ostium are viewed in the proper obliquity, the stenosis of the left main coronary artery can be observed. although the ostial stenosis can be appreciated visually, the diagnosis is frequently made by pressure changes and by failure of the contrast medium to be flushed from the coronary artery until the occluding catheter is removed from the ostium. by virtue of the extensive areas of myocardium placed in jeopardy, patients with coronary ostial stenosis, particularly of the left coronary artery, are at high risk of myocardial infarction and premature death. whether the hemodynamic consequences of this lesion are different from those of left main coronary artery disease is entirely unknown. there is little doubt that coronary bypass graft is indicated for symptomatic left ostial stenosis, as left main coronary disease. optimal therapy consists of coronary bypass to the lad and one or more branches of the circumflex. coronary bypass surgery does not carry a higher mortality, and further longitudinal studies will be required to determine the influence on long - term prognosis. | a patient with isolated left coronary ostial stenosis proved by coronary angiography is presented. isolated left coronary ostial stenosis is a rare condition of unknown etiology.unique clinical and angiographic profiles are discussed in detail with a review of the literature, along with a suggestion of a natural history distinct from that usually seen in atherosclerotic coronary diseases. |
disease research in pathology informatics requires archiving, retrieving, organizing, sharing, and analyzing diverse pathology - related data sources. the most important data source for pathologists is the data collected by anatomic and clinical pathologists (e.g., blood tests, surgical pathology reports, autopsy reports, annotated images, and specialized studies). it is a sad irony that the data collected by pathologists is seldom available for serious scientific inquiry. many pathologists can not freely access the full set of anatomic and clinical data collected within their own departments, though de - identified pathology data is exempted from regulation by hipaa and the common rule. any pathology department could, without violating federal laws, de - identify and distribute their archived anatomic and clinical pathology datasets to the scientific community. for reasons economic, legalistic, and psychologic, no pathology departments have, as yet, distributed de - identified collections of their collected datasets to the public. as a result, there is virtually no scientific research currently being conducted on large, multi - institutional collections of pathology data. moreover, the primary data for research done on collections of single - institution data is seldom released to the public. when research is conducted on pathology data, and the data is withheld from the public, there is no way to validate the conclusions. hence, the u.s. national academy of sciences, along with the editors of many scientific journals, have established a policy requiring authors to release the primary data supporting their conclusions. because pathology departments have not released their de - identified research records to the public, record - based pathology informatics research can not be published in journals that conform to the national academy of sciences recommendations. in the rare instance where an institution has published scientific results based on global analyses of their own datasets, the raw data, upon which those results are based, scientific results have no value unless the results are backed by data that can be openly examined. where pathology departments have failed, the u.s. enormous sources of individualized but de - identified death records and cancer records are available at no cost to medical researchers. eighty - five million de - identified death records are available from the cdc (center for disease control and prevention). each record contains basic demographics on the decedent (age, race, gender, place of death), the cause of death, and (if provided in the death record) the underlying causes of death and significant additional medical conditions. each annual data file provides about 2 million byte - indexed sequential - line records. this means that there is one death record per line, and each line contains coded data indexed for ranges of bytes that are designated by a data dictionary. for example, the icd-10 code of the underlying cause of death, for deaths occurring in 1999, are found in bytes 142 - 145 of each record. this may seem like an awkward way of organizing data, when you consider the ease with which modern specifications (such as rdf) can encapsulate data with metadata. nonetheless, the byte - indexed sequential files can be parsed very efficiently with just a few lines of code. though pathologists prefer autopsy data over death certificate data, autopsies are performed on a very small fraction of decedents. despite early efforts to standardize and collect autopsy data, pathologists have not succeeded in sharing their autopsy data in a national database. consequently, death certificates are the most important source of mortality data available to medical researchers. seer offers a public use dataset, containing de - identified records on about 4 million cancers that have occurred since 1973. each seer record is a single cancer case. with about 4 million carefully curated cases, scientists can draw certain types of inferences that could not possibly be made with the data accumulated at a single medical institution. to get the seer public use data files, you must first complete a data access request available at : seer.cancer.gov/data/request.html seer sends you a username and password that you will need to access the data files. each seer record is a line on a data file, and consists of 264 alphanumeric characters. byte data includes the patient 's race, gender, age at diagnosis, primary tumor site, diagnosis, and information related to tumor size and occurrence of metastases. a data dictionary provides the byte location of the various field values contained in each record. in the past several years, i have written hundreds of blog entries explaining how open source materials (i.e., data, algorithms, nomenclature, metadata specifications, and software) can be used to collect, organize, integrate, and analyze pathology - related information. the blogs are available at : julesberman.blogspot.com a blog tutorial for seer data files appeared on november 14, 2008. a blog tutorial on the cdc mortality files appeared on december 2, 2008. all of the 300 + blogs entries for the blog site can be accessed through a linked archive web page at : http://www.julesberman.info/blog_in.htm blog entries related to the cdc and seer data include topics such as data mashups (e.g., mapping the geographic locations of disease occurrences), age distributions (e.g., following the average age of occurrence of diseases that progress through diagnostic categories over time), and trends in disease incidence (e.g., measuring the annual incidence of genetically screened diseases). there are blog entries of general interest to those working with pathology datasets, including entries on de - identification methods, image manipulation and annotation, data specification methods, and various computational algorithms. | the day has not arrived when pathology departments freely distribute their collected anatomic and clinical data for research purposes. nonetheless, several valuable public domain data sets are currently available, from the u.s. government. two public data sets of special interest to pathologists are the seer (the u.s. national cancer institute 's surveillance, epidemiology and end results program) public use data files, and the cdc (center for disease control and prevention) mortality files. the seer files contain about 4 million de - identified cancer records, dating from 1973. the cdc mortality files contain approximately 85 million de - identified death records, dating from 1968. this editorial briefly describes both data sources, how they can be obtained, and how they may be used for pathology research. |
ankylosing spondylitis is a chronic, autoimmune and progressive spondyloarthropathy affecting the spine and sacroiliac joints. involvement of the cervical joints, temporomandibular joints and cricoarytenoid joints in these patients may cause difficulty during intubation. although there are various options available for securing the airway in these patients, ranging from awake fibreoptic intubation to surgical tracheostomy, the management depends on the patient, the clinical setting and the skills of the anaesthesiologist. awake fibreoptic intubation is the gold standard for known difficult airways, but patient refusal is a limitation for its use. because the literature is scanty about the use of airtraq in the airway management of patients with severe ankylosing spondylitis, these two reports of patients planned for general anaesthesia will reveal the further scope of airway management. the airtraq is a recently introduced optical airway device to facilitate tracheal intubation in patients with both normal and difficult airways. the device provides a high - quality view of the glottis without the need to align the oral, pharyngeal and tracheal axes. one channel acts as a conduit for passing the endotracheal tube (ett) while the other channel consists of an optical system that transfers the image from the illuminated tip to a proximal viewfinder. the airtraq is anatomically shaped and standard ett of all sizes can be used using different sizes of airtraq. we describe two cases with severe anylosing spondylitis who were successfully intubated using an airtraq laryngoscope. a 40 year - old male, a known case of ankylosing spondylitis for 15 years, was scheduled for right total hip replacement for avascular necrosis of the head of femur. on pre - anaesthetic examination, the patient had thoracolumbar kyphosis with restriction of movements in the dorsolumbar and lumbosacral spine. the patient was unable to lie supine and required pillows beneath the head and neck for support. x - ray lumbar spine revealed squaring of vertebra, calcification of anterior and posterior longitudinal ligaments and reduced interspinous spaces and fusion of the posterior elements at all levels. airway assessment revealed a restricted mouth opening with interincisor gap of 1.9 cm and mallampati grade iv. he was unable to extend his neck, with a steromental distance of 10.2 cm and thyromental distance of 5.4 cm. the patient was planned for general anaesthesia as the patient did not give consent for awake fibreoptic intubation. the patient was explained the possibility of difficult airway and consent for possible surgical tracheostomy was taken. on the day of surgery, the patient was premedicated with intravenous (i.v.) midazolam 2 mg and intramuscular (i.m.) the patient was made to lie supine with the head supported with pillows and all the standard monitors were applied. and fentanyl 1 g / kg. after confirming adequate bag mask ventilation, neuromuscular relaxation as the airtraq handle is comparatively larger and the patient neck movement was severely compromised, the table was adjusted to the head - down position with flexion of both the knees to facilitate the introduction of airtraq in the oral cavity. the blade of the airtraq laryngoscope was introduced into the oral cavity in the midline over the base of the tongue and the tip was positioned in the vallecula. the trachea was intubated with a size 8.0 mm tracheal tube in the first attempt after adequate visualisation of the vocal cords, which required minor adjustments of airtraq. anaesthesia was maintained with nitrous oxide (66%) and sevoflurane (12%) in oxygen and vecuronium bromide. the intraoperative course was uneventful and at the end of surgery neuromuscular blockade was reversed with i.v. the patient was extubated and shifted to the post - anaesthesia care unit. a 65-year - old female patient, a known case of ankylosing spondylitis for 25 years, airway examination revealed a restricted mouth opening with interincisor gap of 1.8 cm and mallampatti grade iii. indirect laryngoscopy could not be performed due to restricted mouth opening and limitation of neck mobility. x - ray cervical spine showed gross osteopenia with complete fusion of posterior elements with end - plate sclerosis and decreased joint space at the c4-c5 and c5-c6 levels. patient was planned for general anaesthesia as she did not give consent for awake fibreoptic intubation. on the day of surgery, after written consent, the patient was premedicated with i.v. glycopyrrolate 0.2 mg. the patient was made to lie supine with the head supported by four pillows and all the standard monitors were applied. the blade of the airtraq laryngoscope was introduced into the oral cavity in the midline over the base of the tongue and the tip was positioned in the vallecula. the trachea was intubated with a size 7.0 mm tracheal tube in the first attempt after centralizing the glottic opening in the view field, which required minor adjustments of airtraq and side - to - side wrist movement. anaesthesia was maintained with 66% nitrous oxide in oxygen and 1% sevoflurane and vecuronium bromide. surgery was uneventful and at the end of surgery, neuromuscular blockade was reversed with i.v. the patient had a smooth recovery and was shifted to the post - anaesthesia care unit for further management. a 40 year - old male, a known case of ankylosing spondylitis for 15 years, was scheduled for right total hip replacement for avascular necrosis of the head of femur. on pre - anaesthetic examination, the patient had thoracolumbar kyphosis with restriction of movements in the dorsolumbar and lumbosacral spine. the patient was unable to lie supine and required pillows beneath the head and neck for support. x - ray lumbar spine revealed squaring of vertebra, calcification of anterior and posterior longitudinal ligaments and reduced interspinous spaces and fusion of the posterior elements at all levels. airway assessment revealed a restricted mouth opening with interincisor gap of 1.9 cm and mallampati grade iv. he was unable to extend his neck, with a steromental distance of 10.2 cm and thyromental distance of 5.4 cm. the patient was planned for general anaesthesia as the patient did not give consent for awake fibreoptic intubation. the patient was explained the possibility of difficult airway and consent for possible surgical tracheostomy was taken. on the day of surgery, the patient was premedicated with intravenous (i.v.) midazolam 2 mg and intramuscular (i.m.) the patient was made to lie supine with the head supported with pillows and all the standard monitors were applied. and fentanyl 1 g / kg. after confirming adequate bag mask ventilation, neuromuscular relaxation as the airtraq handle is comparatively larger and the patient neck movement was severely compromised, the table was adjusted to the head - down position with flexion of both the knees to facilitate the introduction of airtraq in the oral cavity. the blade of the airtraq laryngoscope was introduced into the oral cavity in the midline over the base of the tongue and the tip was positioned in the vallecula. the trachea was intubated with a size 8.0 mm tracheal tube in the first attempt after adequate visualisation of the vocal cords, which required minor adjustments of airtraq. anaesthesia was maintained with nitrous oxide (66%) and sevoflurane (12%) in oxygen and vecuronium bromide. the intraoperative course was uneventful and at the end of surgery neuromuscular blockade was reversed with i.v. a 65-year - old female patient, a known case of ankylosing spondylitis for 25 years, was scheduled for subtotal thyroidectomy for colloid goitre. airway examination revealed a restricted mouth opening with interincisor gap of 1.8 cm and mallampatti grade iii. indirect laryngoscopy could not be performed due to restricted mouth opening and limitation of neck mobility. x - ray cervical spine showed gross osteopenia with complete fusion of posterior elements with end - plate sclerosis and decreased joint space at the c4-c5 and c5-c6 levels. patient was planned for general anaesthesia as she did not give consent for awake fibreoptic intubation. on the day of surgery, after written consent, the patient was premedicated with i.v. the patient was made to lie supine with the head supported by four pillows and all the standard monitors were applied. the blade of the airtraq laryngoscope was introduced into the oral cavity in the midline over the base of the tongue and the tip was positioned in the vallecula. the trachea was intubated with a size 7.0 mm tracheal tube in the first attempt after centralizing the glottic opening in the view field, which required minor adjustments of airtraq and side - to - side wrist movement. anaesthesia was maintained with 66% nitrous oxide in oxygen and 1% sevoflurane and vecuronium bromide. surgery was uneventful and at the end of surgery, neuromuscular blockade was reversed with i.v. the patient had a smooth recovery and was shifted to the post - anaesthesia care unit for further management. ankylosing spondylitis affects about 1% of men and 0.5% of women, with a peak age of onset between 20 and 30 years. the cervical spine is involved late in the disease leading to restriction of the neck movement and head rotation causing non - alignment of the oral, pharyngeal and laryngeal axes. moreover, involvement of the atlanto - occipital joint, temporomandibular joint and cricoarytenoid joints leads to a potentially difficult airway situation. various options are presently available for intubation in difficult airway, but these options get reduced in patients with restricted neck movements and limited mouth opening. awake fibreoptic intubation is considered the safest option ; however, some patients may refuse being awake and prefer general anaesthesia during intubation. we initially planned for awake fibreoptic intubation in our cases, but the patients were apprehensive about the procedure and refused to remain awake. lai. reported glidescope to be a useful device for visualising the larynx and facilitating nasotracheal intubation in patients with ankylosing spondylitis. in their study, glidescope improved visualisation of larynx in 11 patients and intubation was successful in eight patients. airtraq laryngoscope is a newly introduced single - use intubation device, simple to use and has got an easy learning curve that does not require hyperextension of the neck hence is useful for patients with cervical spine injury also. it is inserted into the midline in the oral cavity loaded with polyvinyl chloride tracheal tube into the valeculla and after visualizing the glottic opening the airtraq blade is adjusted with a fine wrist movement to centralize the glottic opening over the eyepiece of the airtraq. it does not obstruct the endoscopic view of the vocal cord during laryngoscopy because of its inbuilt conduit for the tracheal tube. studies have reported the effectiveness and utility of the airtraq for tracheal intubation in patients with cervical spine immobilization and in morbidly obese patients. but, this device has got the limitation of inbuilt conduit meant for tracheal tube that can not be manipulated in the conduit. direct manipulation of the tube is not possible and the entire device has to be tilted and oriented with the tracheal tube in situ. also, it can not be introduced into a mouth opening less than 16 mm for size 2 airtraq and 18 mm for size 3 airtraq. however, there is a relative scarcity of the literature on the use of this device for intubation in patients of ankylosing spodylitis. in a case series of four patients with difficult airway reported successful awake intubation using airtraq laryngoscope. also used airtraq successfully as rescue device following failed awake fibreoptic intubation in a patient with severe ankylosing spondylitis. we used airtraq successfully for intubation in our patient keeping in view restricted neck movements and non - alignment of the three axes, i.e. oral, pharyngeal and laryngeal. neuroaxial anaesthesia was not planned in the first patient because of limited joint spaces and improper positioning of the patient. neuroaxial anaesthesia in such patients may be technically difficult although not impossible due to limited joint mobility and reduced joint spaces. we therefore conclude that intubation with airtraq is a good alternative for elective intubation in patients of ankylosing spondylitis who refuse awake fibreoptic intubation or if functional fibreoptic bronchoscope is not available. | airway management in patients of ankylosing spondylitis remains a challenge for anaesthesiologists. many new airway devices have been used for securing airway in these patients. the airtraq optical laryngoscope is one of the new rigid laryngoscopes with a proximal view finder that reflects an image transferred from the distal tip of the blade through a series of lenses, prisms and mirrors. we report two cases of ankylosing spondylitis who were scheduled for total hip replacement and subtotal thyroidectomy and were successfully intubated using the airtraq laryngoscope. |
all subjects were participants in the screening cohort of the dpt-1 study (5) who were screened for the presence of icas between february 1994 and october 2002 for potential study accrual to delay or prevent type 1 diabetes. the current study group was comprised of first- and second - degree relatives of patients with type 1 diabetes (318 years of age) who screened negative for all daas (ica, ica512, gad65, and miaa) at their first screening and returned for a rescreening. miaa was measured on all subjects at their initial screening and used as inclusionary criteria to determine autoantibody - free status ; thereafter, miaa was only measured on a small proportion of the daa - negative subjects at rescreening. autoantibody - negative subjects were rescreened at subsequent visits for ica, ica512, and gad65. for all other daas, those aged 310 years were rescreened annually, and those aged > 10 years were screened biennially. ica values were determined using the standard indirect immunofluorescence method using cryo - cut sections of frozen sections of human pancreas at the dpt-1 ica core laboratory (gainesville, fl, and new orleans, la). the ica assays had a specificity of 100% with a sensitivity of 74.4% (6). gad65 and ica512 were determined at the barbara davis center in denver, colorado, and measured simultaneously by combined gad65 and ica512 radioassay as previously described (full - length gad65 and ica512bdc cdna clones) (7). the assay was performed in 96-well filtration plates with autoantibody - bound [h]gad65 and [s]ica512 precipitated with protein a - sepharose. the cut points were set at indices of 0.032 (gad65) and 0.049 (ica512a). this assay had a specificity of 99% and a sensitivity of 83.7% in subjects 30 years of age with new - onset type 1 diabetes in the ids combinatorial autoantibody workshop (6). iaa values, using the microvolume requiring assay, were determined at the barbara davis center or the joslin diabetes center (boston, ma) (8). the sensitivity for the miaa assay was 74%/56%, and the specificity was 90%/98% for the denver and boston laboratories. the correlation coefficient between both laboratories was r = 0.90, p 10 years of age (p = 0.76). median ages at seroconversion (years [q1q3 ]) were similar with the exception that children seroconverted to gad65 at a significantly younger age than to ica (p = 0.02) (gad65, 9.5 years [7.112.6 ] ; ica, 10.2 years [8.213.7 ] ; and ica512, 9.9 years [7.313.5 ]). for those individuals who developed multiple autoantibodies, gad65 seroconversion generally occurred earlier than ica or ica512, with ica seroconversion usually occurring after ica512. if a child tested positive for two daas at the same testing, it was likely a combination of gad65 and ica512, as the timing of each of these daas generally preceded the occurrence of ica, giving rise to two positive autoantibodies. the 2-year risk for seroconversion by age showed significant decreases (p 10 years of age were rescreened biennially. therefore, timing to seroconversion for those older than 10 years of age could occur anytime during a 2-year window. this difference in the screening interval for those > 10 years of age could potentially underestimate the risk, where the time to daa seroconversion could be a year earlier than observed. however, after assessment of the impact of this potential bias by adjusting for the screening cycle difference, age continued to be significantly associated with daa seroconversion independent of the screening cycle. also, it is likely the 2-year risks of daa seroconversion may be underestimated because of the median age at initial screening in this cohort (10 years) ; however, this provided additional support to initiate screening strategies earlier in life. additionally, iaa seroconversion was not evaluated in this study because of limited measures on only a very small proportion of those rescreened. this limitation may be apparent in assessing risk for type 1 diabetes development, since iaas have been shown to be a significant indicator of risk, as have znt8 autoantibodies. lastly, this study was not able to assess risk for type 1 diabetes because of limitations in type 1 diabetes development follow - up on the screened population in the dpt-1. although many studies have assessed the presence of autoimmunity in type 1 diabetic high - risk subjects, the data have been limited in assessing screening strategies for type 1 diabetes autoimmunity. understanding the etiology of the preclinical period of diabetes by determining the time of autoantibody seroconversion may assist in identifying potential environmental triggers associated with the etiology of islet autoimmunity. this study provides evidence that the rate of autoantibody seroconversion declines with age, varies throughout childhood and adolescence, and extends into early adulthood. those in early childhood have the highest 2-year risk for any daa seroconversion (2.7% at 3 years), while the risk declines further from 1.8% at 10 years to 1.2% for those over 16 years of age. these findings suggest starting screening in early childhood for those at increased risk (i.e., those with a relative with type 1 diabetes) and screening annually for all daas (including znt8 autoantibodies and iaas not measured in this study) through early adolescence to capture the majority of those at risk for diabetes autoimmunity. | objectivealthough type 1 diabetes autoimmunity frequently begins in childhood, little is known about the relationship between age and autoimmunity development. our aim was to determine the timing of seroconversion to diabetes - associated autoantibody (daa) positivity and risk in first- and second - degree relatives of patients with type 1 diabetes.research design and methodsstudy subjects were identified through the diabetes prevention trial - type 1 (dpt-1). children 318 years of age (n = 42,447) were screened for daas ; 1,454 were ica positive (10 jdf units), 1,758 were gad65 positive, and 899 were ica512 positive at the time of initial screening. subjects who were initially antibody negative (n = 39,212) were recalled for rescreening, and 11,813 returned for rescreening.resultsdaa seroconversion occurred in 469 (4%) children ; 258 seroconverted to ica, 234 to gad65, and 99 to ica512. the median time to seroconversion was 2 years. the 2-year risk for daas was highest in early childhood. for each 1-year increase in age in this cohort, the risk of any autoantibody seroconversion (hr 0.95, 95% ci 0.920.97) decreased by 5%, and for any two autoantibodies risk decreased by 13% (0.87, 0.820.93).conclusionsrisk of autoantibody seroconversion among children followed in dpt-1 is age dependent. younger children have the highest risk for daas, with the majority of children seroconverting by 13 years of age (75%). this suggests that annual screenings should be started in early childhood and continued through early adolescence to identify the majority of subjects at risk for type 1 diabetes and eligible for prevention trials. |
the prevalence of clinical depression and presence of elevated depressive symptoms are higher among persons with diabetes compared to the general population. this association may be bidirectional. newly diagnosed diabetics suffer not only from somatic symptoms of the disease, but also from indisposition due to lifestyle alteration, which can possibly cause depressive emotions in the initial stage of diagnosis. negative emotions can cause stress in diabetic patients, while a series of adverse hormones increase, they reduce the sensitivity of insulin and induce insulin resistance. negative emotions can adversely affect glucose control or the quality of life and should be detected early to be intervened. the present study was conducted with the aim of studying the association of depression with diabetes and its complications in newly diagnosed type 2 diabetics and to compare its prevalence with age- and sex - matched healthy controls. this single point cross - sectional case control study was conducted at a tertiary care hospital from august 2010 to july 2011. cases were selected as per the american diabetes association (ada) 2007 guidelines and were newly diagnosed (3 months since diagnosis) subjects who gave consent for the study. diabetic patients with other predisposing factors for depression such as other chronic diseases, disability, other major psychiatric illness, those with recent stressful and psychological causes of depression, and patients with gross cognitive deficit were excluded from the study. iv tr criteria and severity was assessed using the beck depression inventory (bdi). those having difficulty in understanding the english language were given assistance in the form of literal translation of bdi in the local language, without modifying the basic tool. a bdi score of 11 16 was taken as mild ; 17 30 as moderate, and more than 30 as severe depression. the control subjects were from the same socioeconomic background, to minimize possibility of any bias. a complete physical and mental examination was done and they were assessed on the aforementioned parameters in the same way as was done for the patient group. cases and controls were investigated for their diabetic status ; cases were also investigated for complications of diabetes and then bdi was applied. chi square test and two sample proportion tests were used to calculate the p value. mean age of cases was 47.67 9.78 years and mean age of controls was 46.85 10.08 years. among the cases 65% were males and 35% were females. male : female ratio was 1.86 : 1. among the controls 61.66% were males and 38.33% were females with a male : female ratio of 1.61 : 1. on statistical analysis it was found that both groups were age- and sex - matched (p = 0.6519 for age and 0.70 for gender distribution). similarly they were matched regarding other demographic data like socioeconomic status (p = 0.99), marital status (p = 0.57), and religion (p = 0.64) [table 1 ]. demographic characteristics of the study population out of the 60 newly diagnosed diabetics, depression was found in 43.34%, while it was present in only 13.33% out of 60 age- and sex - matched healthy controls (p = 0.002) [table 2 ]. status of depression in cases and controls mild - to - moderate depression was present in 69.33% of the cases and 87.5% of the controls. severe depression was present in 30.77% of the cases and 12.5% of the controls. among the cases, 42.85% of the females and 43.58% of the males had depression. similarly, among the controls, 13.04% of the females and 13.51% of the males had depression. thus, in our study there was no significant difference among the cases and controls regarding the gender distribution of depression. mg / dl while the mean ppbg value was 230.1 47.43 mg / dl. the average hba1c of the cases was 8.26 1.79%. among those diabetics who were found to be depressed, the average values of fbg, ppbg, and hba1c were 177.80 47.15 mg / dl, 244.50 57.56 mg / dl, and 8.56 1.66%, respectively, while among the non - depressed diabetics, the same values were 149.5 26.85, 219.09 34.97 mg / dl, and 8.04 1.88%, respectively [table 3 ]. although the levels of all the three parameters (fbg, ppbg, and hba1c) were higher in the depressed individuals as compared to the non - depressed individuals, the difference was significant only for fbg (p = 0.0048) and ppbg (p = 0.0386), but not for hba1c (p = 0.26). glycemic status of newly diagnosed diabetics the presence of diabetic complications among those with depression versus those without depression is shown in table 4. thus, a majority of diabetics had evidence of nephropathy even at diagnosis ; 80.76% diabetics having depression had microalbuminuria at diagnosis, while only 47.05% of the non - depressed diabetics had microalbuminuria (p = 0.0078). thus, positive urine microalbumin was significantly associated with depression in newly diagnosed type 2 diabetics. presence of complications in depressed versus non - depressed newly diagnosed diabetics serum creatinine was raised in 13.33% of the cases, among which 26.92% of the cases having depression had raised serum creatinine, while only 2.94% of the cases who had no depression had raised creatinine (p = 0.0068). dyslipidemia was present in 65% of the cases ; 65.38% of the cases having depression had dyslipidemia, while 64.70% of the cases who were without depression had dyslipidemia (p = 0.9564). abnormal retinal findings were present in 15% of the cases. among the depressed diabetics 19.23% had abnormal fundus examination, while among those who did not have depression thus, diabetic retinopathy was not significantly associated with depression in newly diagnosed type 2 diabetics. electrocardiographic abnormalities such as st - t changes were present in 16.67% of the cases. among depressed diabetics, 19.23% had abnormal electrocardiograms (ecg), while among those not having depression, 14.70% had ecg abnormalities (p = 0.6408). in the present study the prevalence of depression in newly diagnosed cases of t2 dm was 43.34%, while in normal healthy controls it was 13.33%. most studies looking for such an association have been done in diabetics, irrespective of the duration since diagnosis. found evidence of depressive symptoms in 29% of the males and 30.5% of the females, with newly diagnosed diabetes. perveen. also found significantly higher prevalence of depression among diabetics compared to non - diabetics, while yu. found that 28% newly diagnosed diabetics in their study had depression. evaluated 300 diabetic patients for depression, irrespective of the duration of diabetes, and found that 41% of the diabetics had depression. in a study by sotiropoulos found that depression prevalence was 14.7% among those with diabetes as opposed to 4.9% among those without diabetes, in a rural area of pakistan. conducted a hospital - based prospective study and found major depression in 71.8% of a sample of 206 iranian patients with diabetes. ali. found that the prevalence of depression was significantly higher among patients with t2 dm (17.6%) compared to those without diabetes (9.8%). various indian studies have reported prevalence rates of depression that vary from 9 83% in primary care practices.[913 ] there are few population - based studies from india. the chennai urban rural epidemiology study, which is the largest population - based study from india, to report on prevalence of the depression, showed that among urban south indians, the prevalence of depression was 15.1%. the presence of depression in 13% of the healthy controls in the present study correlated with the above - mentioned studies. the prevalence of depression in 43% of the newly diagnosed diabetics correlated with the meta - analyses of several studies done by anderson., who reported the prevalence of depression in diabetes ranging from 8 to 61%. similarly all the above - mentioned studies concluded that depression was significantly associated with diabetes. however, a study by brown and colleagues, which also considered newly diagnosed diabetics in their study, did not find any relation between the two. in the present study it was also found that the glycemic status was worse among those having depression compared to non - depressed diabetics., who also found that the mean fbg and hba1c among depressed diabetics was significantly higher than in non - depressed diabetics. larijani. also correlated hyperglycemia with depression. in the present study, although the level of all the three parameters fbg, ppbg, and hba1c were higher in depressed individuals, as compared to non - depressed individuals, the difference was significant only for fbg and ppbg, but not for hba1c. various studies have also noted an association between diabetic complications and depression. in the present study it was also found that among patients of newly diagnosed t2 dm, the majority of those having nephropathy (screened by microalbuminuria and serum creatinine) also had significantly associated depression (p = 0.0078 for microalbuminuria and p = 0.0068 for serum creatinine). however, the relation between dyslipidemia, retinopathy, or ecg abnormalities and depression was not significant. yu. also found a significant association between microalbuminuria and depression in newly diagnosed diabetics, whereas, raval. found that significant risk factors associated with depression in type 2 diabetes included age, socioeconomic status, waist circumference, neuropathy, nephropathy, microvascular and macrovascular complications, diabetic foot, peripheral vascular disease (pvd), and a greater pill burden. however no significant risk was attributed to gender, hba1c, dyslipidemia, retinopathy, blood pressure, or bmi. one of the major strengths of our study is its matched case control design, which controlled two fundamental confounders of age and sex, at the design stage. cases were recruited on the basis of diabetic status measured by objective laboratory values, which avoided selection biases. both cases and controls were selected from the same source population, so they were representative of the source population with respect to depression within age- and sex - matched strata. finally, the screening tool used for depression exclusively screened out depression, thus avoiding false - positive results due to anxiety and somatic symptoms. limitations of the study : because of the stringent selection criteria, the sample size was small, and hence, the results were subjected to type ii error and they could not be generalized. the study was a cross - sectional study, and to further assess the effects of the diabetic disease process over mental status, longitudinal studies were needed. in this study, the differences noted compared to the previous studies might be because of the small sample size. in newly diagnosed type 2 diabetics, the prevalence of depression was significantly higher as compared to age- and sex - matched healthy controls. the presence of depression correlated with the level of hyperglycemia at presentation, as measured by the fbg and ppbg values. the prevalence of other complications of diabetes (retinopathy, dyslipidemia, or ecg abnormalities) was found to be higher among those with depression. therefore, the association between t2 dm and depression was epidemiologically associated even in our subpopulation, highlighting the integrated management protocols between various specialities and psychiatric management helping the overall prognosis. screening for depression by a self - scored questionnaire may be easily carried out, even in a busy clinic. treating depression with psychotherapy, medication, or a combination of these treatments may improve the patients well - being and ability to manage symptoms of both diseases, thus, improving the quality of their lives as well as providing disease - free longevity. | introduction : the prevalence of major depressive disorders is higher among persons with diabetes compared to the general population. these associations may be related to the increased risk of depressive symptoms in individuals with diabetes, increased risk of type 2 diabetes (t2 dm) in individuals with depressive symptoms, or both.objectives:to study the association of depression with diabetes and its complications in newly diagnosed type 2 diabetes in eastern up.material and methods : sixty cases and an equal number of age- and sex - matched controls were assessed for depression, diabetes complications, and a demography profile.results:depression was found in 26 (43.34%) cases, while it was present in only eight (13.33%) controls (p = 0.002). depression correlated with the level of hyperglycemia at presentation, as measured by fasting and postprandial blood glucose (fbg and ppbg) values. the presence of diabetic nephropathy was significantly associated with depression, while the prevalence of other complications of diabetes (retinopathy and dyslipidemia), although higher among those with depression, was not statistically significant. the level of hba1c was 8.56 1.66 in the depressed versus 8.04 1.88 in the non - depressed diabetics (p = 0.26).conclusions : the association of depression with diabetes and its complications in newly diagnosed type 2 diabetics was highlighted in our subpopulation and emphasized the need for integrated health services. the prevalence of depression was higher among them compared to controls. the chances of becoming depressed increased as the diabetes complications worsened. |
although great advances have been made in the field of oral health in the world, dental caries remains a significant problem world - wide. fluoride - releasing restorative materials applied in dentistry, with regular release of small amounts of fluoride in the oral cavity, act as caries preventive systems. among these restorative materials, glass ionomers (gis) release higher amount of fluoride and composite resins containing fluoride have lower fluoride release. gis have the ability to release fluoride and this fluoride release is considered as one of the main advantages of gis. based on antimicrobial activity of gi, these restorative materials prevent the occurrence of secondary caries. furthermore, studies have shown that gis, in addition to fluoride ion release properties, have the potential to reuptake fluoride ion and that fluoride recharge can be more important than fluoride release. laboratory studies have indicated that gis can uptake from various sources such as toothpastes and mouthwashes. even some of the old versions of gis have the ability to recharge and release fluoride and as long as the restoration is in place, it maintains anti - caries properties. presence of fluoride in the oral environment assures long - lasting fluoride release, since fluoride is attached to gi by chemical bonds and then progressively released. considering the aforementioned information, the effect of type of gis and fluoride therapy on the fluoride release is somewhat unclear. therefore, the aim of this study was to determine and compare the fluoride release of three different gis (fuji ix, chem flex and fuji ii) in deionized water with different recharge sources of fluoride, acidulated phosphate fluoride (apf) and sodium fluoride (naf) gels. in this in vitro study, the amount of fluoride release was evaluated for three gis, fuji ix, chem flex and fuji ii after exposure to naf 2% and apf 1.23% gels, taking into account type i error (= 0.05) and type ii error (= 0.1, power = 0.9) as well as using the results of a previous study, the estimated standard deviation and the mean values of fluoride released from each type of gi. the number of samples in every subgroup was evaluated 3 and for more accuracy it was considered 5. hence, the total number of samples in all gi groups was considered 15. fifteen disc - shaped samples with 6 mm diameter and 2 mm thickness were prepared using a frame made of perspex according to the manufacturer 's instructions. to immerse samples in the deionized water, a piece of dental floss without fluoride the surfaces of samples were put under steady pressure with a transparent matrix and glass slide during polymerization. then, all samples were separately immersed in the plastic bottles containing 10 ml deionized water and were first kept in humid and at 37c temperature for an hour. after that, all samples were separately transferred to a new plastic bottle containing 10 ml deionized water and replaced in humid environment and at 37c for 24 h. during the 23 days trial period, the deionized water was daily changed by moving each sample to a new bottle containing 10 ml fresh solution. fluoride release was measured at days 1,4,7,10 and 13 using the potentiometry method by fluoride specific electrode (qse 333) and reference electrode (jenway ag - agcl), which were attached and connected to a ph - ion digital device (ph - ion:025867 micro 2 ; edt instrument, england). after preparation, the 15 discs of each experimental gis were randomly divided into 3 groups of 5 discs. one group was assigned as control and after wiping up the samples by a filter paper, they were re - immersed in a new solution of 10 ml deionized water. in the second group, having been wiped up the same way, the samples were exposed to naf gel 2% (dentsply, latin america) for 4 min and dried again with filter paper and replaced in a new solution of 10 ml deionized water. for the third group, the samples were dried by the filter paper and exposed to apf gel 1.23% (cina bartar co., iran) for 4 min, wiped up again with filter paper and replaced in a new solution of 10 ml deionized water. following this, all sample bottles were put back in the humid environment at 37c temperature for 24 h and the deionized water was daily renewed for the remaining period of experiment. in order to measure the amount of released fluoride, tisab ii solution was added to produce an appropriate ph for fluoride electrode activity and ion strength regulation. then, the solution was mixed by a magnetic stirrer and the amount of fluoride was determined using a specific fluoride electrode. the millivoltage of the solutions was obtained by the device and compared to millivoltages of the standard solutions after calibration curve was drawn. after the first stage in the second period, the re - release of fluoride from the samples after exposure to naf and apf gel was measured in the same way on days 14, 17, 20 and 23. one - way anova and tukey tests were applied in order to analyze the data. the amounts of fluoride released from fuji ii, fuji ix and chem flex after exposure to naf and apf gels are respectively shown in figures 13 and table 1. fluoride released from fuji ii while exposed to acidulated phosphate fluoride and sodium fluoride fluoride released from fuji ix while exposed to acidulated phosphate fluoride and sodium fluoride fluoride released from chem flex while exposed to acidulated phosphate fluoride and sodium fluoride the results for fluoride release in the tested groups in different days the results of one - way anova showed a significant difference between daily values of fluoride released from the three gis before exposure to fluoride. however, the finding obtained from tukey test indicated that the three types of gis (fuji ii, fuji ix and chem flex) significantly released more fluoride on the 1 day than the 4 day. further, the results of tukey test for the mean difference, with 95% confidence interval, revealed that the fluoride levels released from chem flex and fuji ix were significantly higher than that of fuji ii (p < 0.05). also, the re - release of fluoride from all gis which were exposed to apf gel was higher than gis samples that were exposed to naf gel. in general, exposure to fluoride gels increased the release of fluoride from samples compared with those that were not exposed. in previous studies, various solutions such as deionized water and artificial saliva were used to measure fluoride release. believed that in natural conditions, the pellicle and plaque on teeth affected the release of fluoride from gi. therefore, we can not mimic the effect of plaque and pellicle, which act as a membrane against fluoride infusion by artificial saliva, since artificial saliva is not an ideal environment to measure fluoride release from gi. in this study, deionized water was used as a storage media and fluoride ion - selective electrode was used to measure the amount of released fluoride. electrode is only capable of measuring the fluoride ions in solution ; therefore, it is necessary to use a buffer solution that regulates the ph and ionic strength of the solution. in most studies, it also makes ph and ionic strength of the solution be well adjusted to ensure the best conditions to measure fluoride. in this study, as diaz - arnold. study to avoid saturated solution due to equilibrium phenomena and to obtain more accurate results, the solutions were replaced every 24 h and the amount of released fluoride was measured at specified days. this study was conducted to measure the release of fluoride in two stages : first, measuring fluoride release after preparation of chem flex, fuji ii and fuji ix samples and storing them in deionized water and second, after recharging the samples through exposure to naf and apf gels. the results showed that at the first stage, the three gis had the greatest amount of fluoride release in the 1 day which significantly decreased in the following days and were pan after 7 - 10 days. these findings were in accordance with the results of the studies conducted in this field. according to yap., high concentrations of fluoride release in the 1 day are due to significant differences in fluoride concentration of gis in comparison with that of deionized water. it should be re - emphasized that the fluoride release is a diffusion controlled phenomenon. based on the amount of fluoride released in the first stage, a similar pattern was found for all samples ; however, fuji ix had the highest amount of fluoride release. fluoride release of chem flex was the same as the fuji ix with little variation, but the fluoride released from fuji ii was the least with a significant difference. results obtained in this study were in line with the findings of the studies conducted by el mallakh and sarkar and swartz. forsten in his study has reported that fluoride released from gis equilibrated within a time and after 2 months the amount of fluoride release was minimal in most cases. dhull and nandlal during a period of 30 months, reviewed the fluoride released from gi and reported that the release took place in two phases, the first phase was short and quick and the second phase was long with little changes. in the initial step of release, the initial release of fluoride is desirable because it reduces the remaining viable bacteria on the border of caries lesion and stimulates enamel and dentin remineralization. billington. in their study have reported that the fluoride release rate depends on the solution ph and in an acidic solution, the amount of fluoride released from gis is higher. it is expected that if a higher amount of fluoride is used in gis composition, a greater fluoride will be released. in the present study, evaluation of the daily values of fluoride release revealed that the three tested gis reached a relatively constant level of fluoride release after the 1 week. these results are in agreement with the findings of wiegand. in that the graphs of fluoride release from gis were pan after around the 10 day. ilie and hickel showed that gis have the capacity of uptaking fluoride from toothpaste and releasing this fluoride again. the three gis were exposed to apf and naf gels for 4 min ; they were then placed in deionized water and the released fluoride was measured again. since the recommended duration for fluoride therapy is 4 min, to achieve closer clinical conditions, the samples were exposed to fluoride gels for 4 min. it was shown that the three types of gi were able to uptake and release fluoride, which is consistent with the results of other studies. alvarez. demonstrated that the amount of released fluoride will never achieve its initial amount, but daily exposure to fluoride will enhance the release of fluoride from these restorative materials. the amount of released fluoride after exposure to fluoride gel is dependent on soluble fluoride concentration. therefore, it seems that the fluoride taken up after recharging occupies the sites, which have been previously occupied by fluoride, before it is released. higher porosity will allow deeper diffusion of the recharging agent into the sample, leading to a higher amount of fluoride storage and release. due to the high viscosity of the apf gel, it may be trapped in the pores and cracks of the specimens and consequently release fluoride ions as the gel dissolves in the artificial saliva in which it was placed. also, the amount of fluoride - release increases when the acidic solution of 1.23% apf is used in comparison to the neutral solution of 2% naf. seppa stated that the exposure of old gis to fluoride gels would amplify the antibacterial activity of gis due to increase of fluoride release. in the present study, the highest amount of fluoride was released in the first 2 days after using the gels. the highest amount of fluoride release for the three gis was observed after exposure to apf gel that might be due to solubility. have reported that apf gel significantly reduces the hardness of the gis and can have an impact on the longevity of restorations. although the amount of fluoride released from the three gis after exposure to apf gel was more than that of naf gel, the apf gel reduced the surface hardness of gis and increased their solubility, therefore the increase that was observed in fluoride release was not associated with a true chemical recharging. in this study, three gis were exposed to naf gel for 4 min, fluoride was recharged and the gis slowly released fluoride, but freedman and diefenderfer did not observe any significant degradation on the surface of material after exposure of gis to naf gel. chem flex with a slight difference and fuji ii ranked second and third, respectively. the amount of fluoride released after exposure to apf gel in the three groups was the highest. this article illustrated the importance of gis restorations in pediatric dentistry because of their ability to release fluoride, especially after exposure to different fluoride gels. | background : glass ionomer (gi) restorations exposed to fluoride have the ability to slowly release fluoride. therefore, the aim of this study was to investigate fluoride release from three gis before and after exposure to sodium fluoride (naf) and acidulated phosphate fluoride (apf).materials and methods : fifteen disc - shaped samples (6 mm in diameter and 2 mm in thickness) from three gis (fuji ii, fuji ix, chem flex) were made and suspended in a polypropylene recipient containing 10 ml distilled water and stored at 37c. at the 13th day, the samples of each gi were randomly divided into 3 groups. groups 1 and 2 were exposed to naf and apf gels for 4 min and group 3 served as control in distilled water. the fluoride released was measured at day 1, 4, 10, 13, 14, 17, 20 and 23 by potentiometer. data were analyzed by one - way anova and tukey test. p < 0.05 was considered as significant.results:fluoride release was highest after 24 h for the tested gis, but fuji ii demonstrated the least amount. fuji ix showed the highest fluoride release followed by chem flex. exposure to fluoride gels significantly increased fluoride release for all materials (p < 0.05). the amount of fluoride release for the three gis was significantly higher in apf groups during the test period.conclusion:highly viscous conventional gis (fuji ix and chem flex) released higher quantity of fluoride. |
although considered common in the environment, the suspected endocrine - disruptor16 bisphenol a (bpa) has been associated with larger urban areas in more developed countries where the manufacture, distribution, and use of epoxy resins and polycarbonate plastics is concentrated.712 recent studies of more urbanized areas have detected the compound in environmental samples of wastewater treatment effluent,7,8,10,13 urban runoff,10 combined sewer overflows,10 and atmospheric aerosols from the burning of municipal waste.9 this emphasizes the range of pathways by which bpa may enter the environment, causing a large proportion of the population to be at risk for exposure. because the release of bpa into the environment has been associated with more urban, industrialized activities in more developed countries, it is thought to be less of a concern for populations in more rural areas of less developed countries. as part of a larger population - based study, the modeling the epidemiologic transition study (mets)14 was conducted to investigate epidemiologic determinants of obesity, diabetes, and cardiovascular disease across a number of developed and less developed countries. a small pilot study was conducted to determine if bpa could be detected in a subset of mets study participants and their local water samples. results of this study may help clarify the role that this potential endocrine disruptor plays in the incidence of targeted diseases throughout the mets participant countries. in 2010, samples from 109 participants from three mets sites, including rural ghana (nkwantakese), urban jamaica (kingston), and urban united states (maywood, il within the chicago metropolitan area), were analyzed for bpa. these study sites were chosen because they represent a wide range of socioeconomic development levels as defined by the united nations human development index (hdi) 2010 (http://hdr.undp.org/en/statistics/). the selection of study participants and urine collection was in accordance with mets9 protocols and procedures. analyses of urine samples were performed by nms labs (willow grove, pa, usa) using gas chromatography (gc) with detection by mass spectrometry (ms) in the selected ion - monitoring mode. urinary bpa was standardized to creatinine to account for urine concentration. to examine nearby drinking and environmental water sources as a potential pathway of bpa exposure,1519 a total of 39 (250 ml) water samples 9 water samples were collected : 3 were drinking water taken from taps within homes, 2 were local river water, and 4 were bottled drinking water. in kingston, 18 water samples were collected : 6 were drinking water taken from taps within different homes, 8 were taken from local streams and reservoirs (4 each), and 4 were from bottled drinking water. in ghana, 12 water samples were collected : 10 were taken from local bore - hole water pumps used for drinking water by villagers and 2 were taken from local streams. all samples were collected and processed using materials that were verified by the manufacturer to contain zero bpa, and standards were used to confirm zero contamination. water samples were analyzed using ultra - high performance liquid chromatography (hplc) with detection by triple quadruple mass spectrometry. a more detailed description of mass spectrometry analyses can be found in supplementary file 1. urinary bpa was assessed for 109 adults (n = 38, 35, and 36 for the maywood, nakwantakese, and kingston sites, respectively). participants were 55.3%, 42.9%, and 47.1% male for maywood, nkwantakese, and kingston, respectively, with mean (sd) ages of 31.9 (5.2), 36.7 (6.4), and 34.1 (5.8) years. of the participants, 35 (92.1%), 26 (74.3%), and 33 (91.7%) showed detectable levels (minimum detection limit = 0.013 ng / ml) of urinary bpa in maywood, nkwantakese, and kingston, respectively (table 1). the mean (sd) urinary bpa concentration was 2.47 ng / ml (5.0), 2.19 ng / ml (3.7), and 2.11 ng / ml (2.2) for maywood, nakwantakese, and kingston, respectively. the range of detected urinary bpa was 1.1730.30 ng/ ml, 1.0618.95 ng / ml, and 1.088.98 ng / ml for maywood, nkwantakese, and kingston, respectively. bpa was detected in 20 of the 39 water samples analyzed (minimum detection limit = 0.01 ng / ml) ; 5 of 9 samples from maywood, 7 of 12 nkwantakese, and 4 of 14 from kingston. table 2 shows the levels of bpa detected in different water samples across the three study sites. in maywood, bpa was detected in drinking water (1 of 3 samples, 0.01 ng / ml), river water (2 of 2 samples, mean 0.12 ng / ml), and bottled water (2 of 4 samples, 0.003 ng/ ml). for nkwantakese (ghana), bpa was detected in drinking water (7 of 10 samples, mean 0.008 ng / ml), but not in stream water (0 of 2 samples). in kingston, bpa was detected in drinking water (2 of 6 samples, mean 0.003 ng / ml), stream and reservoir water (2 of 8 samples, mean 0.016 ng / ml), and bottled water (4 of 4 samples, mean 0.008 ng / ml). urinary bpa was detected in urine samples of participants living at all three sites examined. a total of 106 of the 109 study participants showed detectable bpa in urine samples analyzed. twenty of the 39 water samples taken contained detectable levels of bpa. despite their association with more industrialized areas in developed countries, the levels of compound detected in the urine and water samples from rural nkwantakese were comparable to those of the much more urbanized cities of kingston and maywood. it is possible that some of bpa pollution found in this village may have resulted from industrial and manufacturing activities from the closest city of kumasi (approximately 25 km away) or from upstream. while the sources of bpa in ghana and jamaica were not identified, the use of plastics, epoxies, and insulation (common sources of bpa) is ubiquitous. the fact that bpa was detected in drinking water in all three sites also indicates a potential route of exposure ; however, the presence of urinary bpa suggests that food is a likely exposure point as well. the results of this study suggest that even in rural areas of a less developed country, bpa is common and that future studies of the compound should include areas less commonly associated with bpa exposure and pollution. | the suspected endocrine disruptor bisphenol a (bpa) is associated with the manufacture, distribution, and use of epoxy resins and polycarbonate plastics ; thus, studies of this compound have focused primarily on urban areas in developed countries. this small study investigating urinary bpa of 109 people was conducted in the urban united states, urban jamaica, and rural ghana. additionally, local drinking and surface water samples were collected and analyzed from areas near study participants. levels of bpa in both urine and water were comparable among all three sites. thus, future studies of bpa should consider expanding investigations to rural areas not typically associated with the compound. |
joint contracture occurs after a long period of fixation and/or immobilization due to fracture or impaired consciousness. the influence of the skin on joint contracture has been determined in several studies based on the change in range of motion (rom) between the first measurement after cast removal and the second measurement after skin resection1,2,3,4. studies investigating multiple measurements of rom in joint contracture have reported that a single joint movement can improve the rom5, 6. therefore, the authors of these previous studies suggested that joint contracture is influenced by both joint movement and skin incision. however, no studies have measured rom twice without skin resection to determine the changes in rom. in the present study, our purpose was to determine the effect of the joint movement through rom measurements performed twice after removal of the cast, and the effect of the skin when rom was measured once after the cast removal and once again after skin resection of rats with plantar flexion contractures. twelve, 8-week - old female, wistar rats with an initial body weight of 189.1 5.3 g were used. the animals were housed in a temperature - controlled room at 23 c with a 12-hour light - dark cycle. the experiment was conducted in accordance with our university s guidelines for animal experimentation (no. the rats were randomly divided into 2 groups. under pentobarbital sodium anesthesia (40 mg / kg b.wt), the right ankle joint of each rat was immobilized in full plantar flexion in a cast from the toes to above the knee joint. in addition, the cast was covered with a stainless steel net to prevent breakage and falling. when the toes became edematous due to fixation, the cast was replaced. in the resection group (n = 6), the skin of the right ankle was removed surgically, but it was not removed in the non - resection group (n = 6). before the experiment and after 2 weeks, dorsiflexion of the ankle joint was measured. at the end of the immobilization period, the rats were sacrificed with pentobarbital sodium anesthesia (40 mg / kg b.wt), and blood was removed by cutting the abdominal aorta. in the resection group, the first measurement of the dorsiflexion angle was obtained after the cast was removed, followed by the second measurement after skin resection. skin resection was carefully done without affecting dorsiflexion of the ankle joint. in the non - resection group, both measurements of the dorsiflexion angle were obtained in the same manner after the cast was removed. when measuring dorsiflexion, the rat was positioned on its side with the hip and knee fixed in flexion. a force of 0.3 n was applied perpendicularly to the sole of the foot using a tension meter7 (kyowa co., lts-1ka). recorded videos were downloaded to a computer to extract still images of dorsiflexion at the moment of application of the 0.3 n force. the rom of dorsiflexion was measured from the still images using computer software (imagej version 1.44p ; usa). the rom of dorsiflexion was defined as the angle obtained from a line parallel to the longitudinal axis of the fibula and a line parallel to the bottom of the heel to eliminate forefoot movement from the measurement. dorsiflexion was measured 3 times, and the mean value was used in the analysis. the wilcoxon test was used to compare the change in rom between the first and second measurements. the mann - whitney u test was used to compare the differences in measurements between the two groups. the changes in rom of ankle dorsiflexion (in degrees)groupafter two weeksfirst measurementsecond measurementfirst secondresection (n=6)113.19.9103.911.59.272.8non - resection (n=6)105.74.7102.13.83.641.0values are means sd. a : significant decrease compared with first measurement (p<0.05). b : significant difference between resection and non - resection group (p<0.01). the rom measurements decreased significantly in both groups between the first and second measurements. compared with the non - resection group, the resection group showed a significant increase in rom between the first and second measurements. values are means sd. a : significant decrease compared with first measurement (p<0.05). skin contracture occurs after damage from burns or wounds, such as hypertrophic scarring due to damage beyond the basement membrane separating the epidermis and dermis9. contracture of the skin and soft tissue that occurs after joint immobilization has been investigated in previous animal experiments. in these studies, the cause and contribution rate of soft tissue contracture were clarified by measuring the rom of the joints in general. ichihashi.3 fixed rats knees in full flexion for 30 days and measured the rom of knee extension after removal of the cast, and resection of the skin and muscles. okamoto.4 fixed rats in full plantar flexion for 1, 2, 4, 8, and 12 weeks and reported that the rom of plantar flexion improved after cast removal and resection of the skin and muscles for all time periods of immobility. these previous studies indicate that skin affects contracture formation resulting from joint immobilization. in our study, joint contractures occurred in all rats after joint fixation. the method of immobilization used in this study was carried out in accordance with that reported by ono. our results show that the rom of dorsiflexion increased between the first and second measurements in both groups. this indicates that the rom increase observed in the second measurement in the non - resection group may be due to the stretching of the skin, because the measurements were carried out using the same torque. furthermore, the increased rom in the second measurement in the resection group was considered to be the sum of the stretching effect from moving the joint during the measurements and the skin resection. the change in the rom between the first and second measurements was greater in the resection group than in the non - resection group. the second rom measurement in the resection group improved 9.27 2.8 degrees from the first measurement, an improvement of approximately 12% of the full rom limitation. in the non - resection group, the rom improved 3.64 1.0 degrees, an improvement of approximately 5% of the full rom limitation. therefore, we estimate the skin effect was approximately 7%, the result of subtracting the joint movement effect (5%) from the change in rom (12%) in the resection group. skin contracture after burns and wounds is reported to be caused by excess proliferation of fibroblasts11, abnormal generation of collagen fibers12, and contraction of the wound13, followed by scar contracture. the results of our study and previous studies demonstrate that skin has a critical role in the formation and treatment of joint contracture, because we found that skin resection improved rom after joint immobilization. stretching of the skin by an open - window procedure14, 15 may be effective when immobilization treatment is used after fracture. in the future, it will be necessary to examine how the skin itself changes during joint immobilization. | the effect of skin resection on joint contracture was determined by comparing the first measurement of range of motion after cast removal and the second measurement after the skin resection. this study aimed to verify that both the joint movement during the measurement and skin affect range of motion. [subjects ] twelve female wistar rats were used. [methods ] the right hind limb ankle of each rat was immobilized in complete plantar flexion in a cast. in the resection group (n = 6), the skin of the right hind limb ankle was removed surgically, but not in the non - resection group (n = 6). in the resection group, the first measurement of the dorsiflexion angle was obtained after the cast was removed, and the second measurement was obtained after skin resection. in the non - resection group, both measurements of the dorsiflexion angle were obtained soon after the cast was removed. [results ] compared with the non - resection group, the resection group showed a significant increase between the first and second measurements of range of motion. [conclusion ] these results show that range of motion is substantially affected by skin, in addition to joint movement, during measurement. |
functional glycomics is an emerging field of science, aiming to create a cell - by - cell catalogue of glycosyltransferase expression and detected glycan structures in relation to health and diseases (13). however, modern analytical methods such as mass spectrometry and nmr have afforded the ability to elucidate most structural details at the concentration levels required for glycomics (4,5). several national and international initiatives aiming to decipher the biological function of carbohydrates have emerged for the recent years (68). in a similar fashion to the finished human genome project which determined the sequences of the chemical base pairs that make up a human dna most of these glycomics projects intend to make their data freely accessible under an open access philosophy. unfortunately, the exchange of data between different glyco - related databases is seriously hampered by the dearth of generally accepted digital exchange formats and standardized structural and biological descriptions (9). similar to the genomics and proteomics field, a description of glycan structures would be an appropriate way to establish an efficient connection of glyco - related information resources. however, glycan sequences can not be described by a simple linear one - letter code as each pair of monosaccharides can be linked in several ways and branched structures can be formed. the glycosciences.de portal (7) demonstrates that data originating from various resources can be efficiently integrated using a linear notation for unique description of carbohydrate sequences (linucs) (10). the extended alphanumeric iupac description and glycosidic linking information are applied to build up a hierarchy of the various branches starting from the reducing end of the oligosaccharide chain, which is then converted into a linear representation. however the commercially available glycosuitedb (11) uses the so - called condensed form of the iupac description to create a linear representation, where four rules are applied to obtain a unique linear code. the glycan database of the us consortium for functional glycomics (6) uses the so - called linear code (12), using a one or two character - based representation of saccharide units and linkages. the ordering of glycan branches is established using a special lookup table where the hierarchy of monosaccharide structures is defined. the kegg carbohydrate matcher (kcam) (8,13) uses a connection table based graph representation to encode carbohydrate structures, where monosaccharides are represented by nodes and glycosidic bonds as edges. glycosciences.de, glycosuite, cfg glycan database and kegg - glycan concentrate on glycan structures found in mammalian species. in contrast, the mission of the russian bacterial carbohydrate structure database (bcsdb) [(14), for urls see appendix ] is to provide all published glycan structures found in bacteria. since the monosaccharide namespace as well as the type of linkages found in bacterial polysaccharides differ considerably from those found in mammals, bcsdb uses an internal representation of glycans, which diverges from those used to describe structures found in mammals. looking at various existing carbohydrate databases accessible through the internet, it is obvious that diverse ways to encode and store complex carbohydrates are in use. however, users who would like to access all publicly available glyco - related data spread over many databases have not only to cope with varying graphical and non - graphical interfaces to input glycan structures, but also must be aware that the definition of building blocks and topologies may be different. each database has developed its own set of rules to solve some problematic encoding situations such as treatment of monovalent substituents, phosphates, sulphates, repeat units, unknown linkages and other uncertain structural features of glycan structures. it is, of course, an attractive vision [expressed during the joint meeting of the japanese and american consortia for glycomics (15) ] to have a single user interface which will provide access to all relevant world - wide distributed resources without any technical and administrative barrier. a prerequisite for an efficient exchange of data is the agreement to a generally accepted exchange format as well as a common application programming interface. consequently, several proposals for an xml - based description of glycan structures have already been published (16,17). to avoid any further confusion about xml descriptions of glycans, the seven larger initiatives in this field [cfg, bcsdb, glycosciences.de, eurocarbdb, kegg, hgpi and ccrc (for abbreviations see appendix) ] agreed to further develop the xml description for the encoding of glycan structures on the basis of the already existing glycan data exchange (glyde) (17). the progress discussion is open to all interested scientists and takes place at the forum pages of the eurocarbdb project. concerning the technical realization of the online connection between existing databases, it seems that the simple object access protocol (soap) is now the broadly accepted procedure for automated communication between web - applications. being designed to communicate via the internet, it is well suited to be also used for the exchange of glycan - related data between distributed computers. taken together, it seems like the field has matured to the point where it is feasible to establish an online connection of distributed databases, at least between the larger of the established projects. the bacterial carbohydrate structure database (bcsdb) [(14), for urls see appendix ] is a database containing data on natural carbohydrates with known structure. in addition to the structure and bibliography, each record in the bcsdb contains the abstract of the publication, data on the carbohydrate source, methods of structure elucidation, information on the availability of spectral data and assignment of nmr spectra when available, data on conformation, biological activity, chemical and enzymatic synthesis, biosynthesis, genetics and other related data. the search criteria can be fragment(s) of the structure ; fragment(s) of the nmr spectrum ; and indexed tags, including microorganism, bibliography and keywords. currently, the bcsdb contains 8200 records on bacterial carbohydrates, including the corresponding part of carbbank (18) (3500 records on structures reported before 1995). data from both literature and carbbank have been carefully checked for consistency before the upload, and corrected when necessary. the bcsdb interface includes the web - based user part, web - based administrator part and programming gateways for the automated data interchange. the bcsdb is available on the internet for free usage and validated user data submission. the glycosciences.de portal (7) is an attempt to link glycan - related data originating from various resources through a unique structural description. the linucs (linear notation for unique description of carbohydrate sequences) (10) notation is used to uniquely encode fully characterized glycans. currently, the glycosciences portal provides access to 24 000 different entries with nearly 14 000 different carbohydrate moieties. these structures are sourced from a number of sources, including the former carbbank and sugabase - project (19), automatic extraction from the protein data base (pdb) (20), and the curation of new entries altogether. the structure - oriented approach to the database allows the data related to a single glycan, but originating from various sources (e.g. experimental nmr spectra, theoretically calculated fragment ions for mass spectra interpretation or experimental or simulated 3d structures) to be easily linked and accessed using a single database query. according to the varying needs of specific research questions, the glycosciences portal provides several structure - oriented options to recall glycan - related data. the retrieval of glycans matching an exact structure is the most traditional way to access a database. the motif search enables to retrieve all entries, which possess substructures having names such as lewis, blood group h antigen or gm3. all glycan - related scientific data of the glycosciences.de portal are freely accessible via the internet following the open access philosophy : free availability and unrestricted use. the soap - based web - services are available on the websites of the two projects and are documented in the form of wsdl (for urls see appendix) descriptions that provide the possibility of platform - independent formalization of server - side features. wsdl files can be easily integrated into the existing code by using features from various soap libraries which allow the transparent work with the soap interface under perl, php, java, etc. glycan data exchange (glyde) version 1.2 (17) was chosen as the structure exchange format. it supports almost all known peculiarities of carbohydrate structures, such as uncertainities in configuration and ring sizes, various combinations of repeating and non - repeating parts, non - carbohydrate linkers, cyclic structures, etc.. glyde uses a tree - based approach to structure description. within this approach the tree root is the reducing and or the rightmost residue in the repeating unit, while all the substituents are the children of the residue they are attached to. configurations, ring - size and other related information is stored as attributes of the residue. two approaches are used : the raw data (as array of strings corresponding to authors, title terms, journal name, etc.) or pubmed xml. a well - known identifier for an organism is a taxid provided by ncbi taxonomy database. however, the ranking of taxid is limited to species ; thus, no possibility to cross - search for particular strains / serogroups is provided. as this detailed ranking is significant mainly for bacteria, the capability to perform deep species searching taxids are stored in the glycosciences.de database together with structures, while bcsdb generates taxids based on genus and species name, making use of an ncbi web service. example 1, using the bibliographic search of glycosciences.de, shows all references found in both resources for author brade in year 2002. example 2 depicts a substructure search containing a specified disaccharide fragment [-d - neup5nac-(2 - 3)--d - galp ] in glycosciences.de. the data associated with two entries containing the disaccharide fragment are shown in example 2b. example 3 demonstrates a substructure search in bcsdb using glycosciences.de to input the trisaccharide fragment -d - galp-(1 - 3)--d - manp-(1 - 4)--l - rhap. substructure search for a - d - neup5nac-(2 - 3)-b - d - galp in glycosciences.de using the bcsdb input wizard. result querying glycosciences for all structures containing a specified disaccharide fragment -d - neup5ac-(23)-d - galp. querying bcsdb for all structures containing the specified trisaccharide fragment -d - galp-(1 - 3)--d - manp-(1 - 4)--l - rhap. the glycosciences.de substructure input spreadsheet is used. the data associated with bcsdb entry 10147 are additionally shown. the capability of web services to make distributed scientific data accessible is clearly demonstrated. to our knowledge, the implemented mutual online access between bcsdb and glycosciences.de is the first reported attempt of a structure - based interconnection of two glyco - related databases. for users the advantages are obvious : they can use and have to learn only one interface, always have access to the latest data from both services, and the results of both searches are presented in a consistent way. for the database design and its functionality the establishment of a connection helped to find shortcomings and inconsistencies in both underlying data concepts and structural representations. for the maintenance of the databases, duplication of work can be easily avoided. it can be expected that more frequent use of both services will improve the quality of data. this will hopefully lead to a better worldwide acceptance of both services within the community of glycoscientists. since the exchange of data is accomplished through standard, well - documented xml - based descriptions and soap protocols ; other interested providers of glyco - related databases may easily be linked so that a larger network could grow. it can be envisaged that online connection of thematically related scientific data collections will have a bright future, and not only in the area of glycosciences. one of the main bottlenecks is currently that broadly accepted standard xml exchange formats are often not yet available. it will definitively be a time - consuming task to come to agreements about such standard descriptions within the various communities. with glyde 1.2 an xml - based encoding scheme of glycan structures exists, which is sufficiently flexible to link the vast majority of structures contained in bcsdb and glycosciences.de. however, glyde 1.2 has some shortcomings regarding uncertainties in terminal residues and other fuzzy encodings, which will become more important for glycomics projects. the current focus of discussion is to base a more flexible encoding on the concept of a connection table approach, instead of a tree - like structure as used in glyde 1.2. recently (september 2006, nih meeting frontier in glycomics), the seven larger projects already mentioned above have agreed to support glyde - ct as the main database format for the exchange of glycan structures. a less favourable situation would be that several exchange format exit and parsers must be available for each database. | functional glycomics, the scientific attempt to identify and assign functions to all glycan molecules synthesized by an organism, is an emerging field of science. in recent years, several databases have been started, all aiming to support deciphering the biological function of carbohydrates. however, diverse encoding and storage schemes are in use amongst these databases, significantly hampering the interchange of data. the mutual online access between the bacterial carbohydrate structure database (bcsdb) and the glycosciences.de portal, as a first reported attempt of a structure - based direct interconnection of two glyco - related databases is described. in this approach, users have to learn only one interface, will always have access to the latest data of both services, and will have the results of both searches presented in a consistent way. the establishment of this connection helped to find shortcomings and inconsistencies in the database design and functionality related to underlying data concepts and structural representations. for the maintenance of the databases, duplication of work can be easily avoided, and will hopefully lead to a better worldwide acceptance of both services within the community of glycoscienists. bcsdb is available at and the glycosciences.de portal at |
corneal biomechanics is a branch of science that studies deformation and equilibrium of corneal tissue under the application of any force. the structure and hence the properties of a soft tissue, such as the cornea, are dependent on the biochemical and physical nature of the components present and their relative amounts. the mechanical properties of a tissue depend on how the fibres, cells, and ground substance are organized into a structure. collagen and elastin are responsible for the strength and elasticity of a tissue, while the ground substance is responsible for the viscoelastic properties. all these terms are important because the cornea is considered a viscoelastic material and some devices try to measure and even differentiate between the different components of the biomechanical behavior of the living corneal tissue. in the specific case of the human cornea, collagen in bowman 's layer and stroma accounting for over 80% of the dry weight of the cornea the ground substance, formed mostly by proteoglycans and keratocytes or fibroblasts, would provide the viscous behaviour. the corneal epithelium accounting for 10% of the central corneal thickness could also contribute to the viscous behaviour. it is important to bear in mind that the corneal epithelium is easily deformable and is the reference surface for most of the biomechanical corneal measurements. over the past two decades, researchers have developed a variety of techniques that can alter corneal surface for refractive purposes or even for halting disease progression in corneas with mechanical decompensation. beside geometric corneal parameters, the additional influence of the biomechanical corneal properties has received little attention, mostly because of the lack of appropriate in vivo measurement techniques. however, in recent years, increasing interest has arisen in corneal biomechanics to predict corneal response to surgical or therapeutic interventions and to assist in the detection of early keratoconus [46 ]. additionally, increasing interest has also arisen in corneal biomechanical properties and glaucoma once corneal biomechanics have been shown to influence intraocular pressure (iop) measurements and may be also indicative of ocular globe biomechanics that could also be predictive of glaucoma susceptibility. corneal biomechanics have been assessed in in vitro studies by measuring stress - strain and young 's modulus in isolated corneas. in the recent years, two devices have been marketed : the ocular response analyser (ora, reichert, depew, nj) since 2005 and the corneal visualization scheimpflug technology (corvis st, oculus, wetzlar, germany) since 2011. many studies covering a wide range of topics have been conducted and published using the ora. the aim of the present review is to provide an overview of published results on corneal biomechanics obtained with ora under different ocular and systemic conditions. knowledge accumulated to date on this field will potentially help the ophthalmic community to gain a better understanding of the changes that the corneal tissue undergoes during different ocular and systemic conditions as well as to predict the outcomes of therapeutic and refractive therapies. new technologies under development will also be discussed briefly since there is currently a wide range of instrumentation under development to provide a better understanding of the biomechanical nature of the cornea and its implications in visual care, with particular relevance to the detection and management of sight - threatening conditions. to better understand the results of corneal biomechanical measurements, it is important to remember the meaning of some corneal properties such as elastic, viscous, or viscoelastic response, hysteresis, and stiffness, among other concepts.the elastic response of a material is attributed to the instantaneous and reversible deformation under an external load. in elastic materials, the deformation is proportional to the force applied and it is recovered instantly upon unloading. the constant of proportionality between stress and strain is the elastic modulus, also called young 's modulus. young 's modulus is defined as the ratio of the stress (load per unit area) and the strain (deformation / displacement per unit length). a high modulus indicates a stiffer material (i.e., not easy to bend). this also leads us to the definition of resistance, which is the capacity of a material to hold stress without deformation.corneal young 's modulus, measured in vitro, varies from 0.1 to 57 mpa [8, 1120 ] that might be explained by variations in testing conditions and methods used. more recently, hamilton and pye, using the orssengo - pye algorithm, reported on 100 healthy eyes with mean young 's modulus being 0.29 0.06 mpa (range 0.13 to 0.43 mpa). modulus was positively correlated with the iop measured with gat, assuming that young 's modulus itself affects the iop measurement.a material shows a viscous behaviour when the deformation velocity is faster than the relaxation rate. the slow relaxation is due to configurational rearrangement of the material during deformation.viscoelastic materials exhibit elastic and viscous behaviour at the same time, so they present characteristics of elastic and viscous materials. hysteresis.hysteresis in viscoelastic materials under periodic loading and unloading, curves in the stress - strain diagram (figure 1(c)) are not coincident with each other ; the gap between them is called hysteresis.the energy stored over one full loading and unloading cycle in a material is zero since the material returns to its initial configuration (elastic behavior). the area within the hysteresis loop represents the energy per volume dissipated in the material per cycle. the elastic response of a material is attributed to the instantaneous and reversible deformation under an external load. in elastic materials, the deformation is proportional to the force applied and it is recovered instantly upon unloading. the constant of proportionality between stress and strain is the elastic modulus, also called young 's modulus. young 's modulus is defined as the ratio of the stress (load per unit area) and the strain (deformation / displacement per unit length). a high modulus indicates a stiffer material (i.e., not easy to bend). this also leads us to the definition of resistance, which is the capacity of a material to hold stress without deformation. corneal young 's modulus, measured in vitro, varies from 0.1 to 57 mpa [8, 1120 ] that might be explained by variations in testing conditions and methods used. more recently, hamilton and pye, using the orssengo - pye algorithm, reported on 100 healthy eyes with mean young 's modulus being 0.29 0.06 mpa (range 0.13 to 0.43 mpa). modulus was positively correlated with the iop measured with gat, assuming that young 's modulus itself affects the iop measurement. a material shows a viscous behaviour when the deformation velocity is faster than the relaxation rate. viscoelastic materials exhibit elastic and viscous behaviour at the same time, so they present characteristics of elastic and viscous materials. their particular characteristics make it possible to define characteristic properties including one known as hysteresis. hysteresis in viscoelastic materials under periodic loading and unloading, curves in the stress - strain diagram (figure 1(c)) are not coincident with each other ; the gap between them is called hysteresis.the energy stored over one full loading and unloading cycle in a material is zero since the material returns to its initial configuration (elastic behavior). the area within the hysteresis loop represents the energy per volume dissipated in the material per cycle. hysteresis in viscoelastic materials under periodic loading and unloading, curves in the stress - strain diagram (figure 1(c)) are not coincident with each other ; the gap between them is called hysteresis. the energy stored over one full loading and unloading cycle in a material is zero since the material returns to its initial configuration (elastic behavior). the area within the hysteresis loop represents the energy per volume dissipated in the material per cycle. it uses a rapid air pulse to indent the cornea and an electrooptical system to record corneal deformation. it records mainly two applanation measurements : one while the cornea moves inward, reaching a first applanation, when the first pressure (p1) is registered and the other as the cornea recovers from a slight concavity as the air pump decreases pressure at an inverse rate so that the cornea moves outward passing through a second applanation (p2). therefore, these two values, p1 and p2, indicate the pressure necessary to flatten the cornea during the loading and unloading cycle (figure 2). thus, below we define one by one the terms and parameters that are relevant to the understanding and interpretation of the outcomes obtained by the ora according to the literature.p1 and p2 : air pressures corresponding with the two applanation states of the cornea.corneal hysteresis (ch) is considered an indicator of corneal viscosity and is obtained by the difference between the 2 pressures : ch = p1 p2.the corneal resistance factor (crf) is considered an indicator of the overall resistance of the cornea and is expressed by the equation : crf = (p1 0.7p2). it is significantly correlated with central corneal thickness (cct) and goldmann applanation tonometry (gat). it has been also suggested that the crf could be mainly related to the elastic properties of the cornea. other authors suggested modifications on the original formula to crf = k1(p1 0.7p2) + k2, where k1 and k2 are constants [26, 27 ]. moreover, some authors evaluated the difference between ch and crf, but the meaning of this new parameter [28, 29 ] is not clear.iopg is an iop value equivalent to gat, which is an average of the two pressure values measured by ora, p1, and p2 and obtained by the following equation : iopg = (p1 + p2)/2.iopcc is a new iop value called corneal compensated iop and is obtained by the equation iopcc = p1 0.43p2. it is less affected by corneal properties than by the iop obtained with other tonometers and it is not correlated with the cct but it is correlated with ch [30, 31].corneal constant factor (ccf) is claimed to be an iop - independent corneal factor introduced by kotecha. and was derived from the changes of p1 and ch for every 1 mm hg of change in gat iop. it describes an iop - independent biomechanical property that increases with thicker cct and decreases with aging and yet explains more of the interindividual variation in gat iop than does cct. it is very similar to crf proposed by reichert and is expressed by the equation : ccf = p1 0.79p2. p1 and p2 : air pressures corresponding with the two applanation states of the cornea. corneal hysteresis (ch) is considered an indicator of corneal viscosity and is obtained by the difference between the 2 pressures : ch = p1 p2. the corneal resistance factor (crf) is considered an indicator of the overall resistance of the cornea and is expressed by the equation : crf = (p1 0.7p2). it is significantly correlated with central corneal thickness (cct) and goldmann applanation tonometry (gat). it has been also suggested that the crf could be mainly related to the elastic properties of the cornea. other authors suggested modifications on the original formula to crf = k1(p1 0.7p2) + k2, where k1 and k2 are constants [26, 27 ]. moreover, some authors evaluated the difference between ch and crf, but the meaning of this new parameter [28, 29 ] is not clear. iopg is an iop value equivalent to gat, which is an average of the two pressure values measured by ora, p1, and p2 and obtained by the following equation : iopg = (p1 + p2)/2. iopcc is a new iop value called corneal compensated iop and is obtained by the equation iopcc = p1 0.43p2. it is less affected by corneal properties than by the iop obtained with other tonometers and it is not correlated with the cct but it is correlated with ch [30, 31 ]. corneal constant factor (ccf) is claimed to be an iop - independent corneal factor introduced by kotecha. and was derived from the changes of p1 and ch for every 1 mm hg of change in gat iop. it describes an iop - independent biomechanical property that increases with thicker cct and decreases with aging and yet explains more of the interindividual variation in gat iop than does cct. it is very similar to crf proposed by reichert and is expressed by the equation : ccf = p1 0.79p2. the deformation signal waveform produced by the corneal deformation signal (characteristic shape illustrated in figure 2) can provide a unique description of each eye. further analysis of the waveform signal delivered by the electrooptical system of the instrument has provided more parameters with potential interest to allow a refined evaluation of the corneal properties. recently, 37 new parameters were derived from the new ora software allowing a detailed analysis of the deformation signal waveform. each one of these parameters describes a morphological feature of the waveform and 23 parameters are derived from the upper 75% of applanation peak height and 14 are derived from the upper 50% of the applanation peak height (figure 2). these new parameters are defined in appendix a. most of these parameters depend on p1 and p2 defined at the beginning so, in some way, these parameters could be intrinsically linked and their clinical significance and the manner in which these individual parameters represent biomechanical properties are currently unknown. several studies have investigated the clinical relevance of the new waveform parameters and reported that they could be more useful in diagnosis and prognosis after refractive surgery, and as stated in the following sections, some of these parameters seem to be promising as being more sensitive than others to detect corneal changes in specific corneal conditions [28, 3336 ]. the possibility to evaluate the biomechanical properties of the cornea provides a new diagnostic tool that will allow detecting differences in corneal biomechanics between normal eyes and pathological eyes and eventually detecting weaker corneas at a subclinical state before they evolve in some kind of ectasia or avoiding postsurgical ecstatic disease. since the introduction of ora in clinical practice, many research studies have been conducted looking for associations between both ch and crf and different parameters like age, corneal thickness, iop, progress of glaucoma, or presence and severity of a given condition such as keratoconus. according to luce, corneas with low ch are less capable of absorbing energy than normal eyes and they may be candidates for several ocular diseases. moreover, low crf indicates that the overall corneal rigidity is lower than normal. it is observed that both ch and crf vary in a rather wide range in the normal population and that a comparison between studies for both parameters is difficult. lim., in a study with 271 children, reported that ch and crf did not vary significantly with age but the range of ages was quite narrow. notwithstanding, as the authors observed, the values of ch and crf measured were slightly higher than those in other adult studies. the same was observed by kirwan. in children and adolescents who also found no correlation between age and ch. however, when compared with other studies, the values of ch were again slightly higher. on the other hand, some studies have shown that ch significantly decreases with age [4, 26, 4345 ]. kamiya. evaluated 204 eyes of healthy subjects and found a small but statistically significant negative correlation between ch and crf with age without significant differences in central corneal thickness (cct) or iop across the sample. ortiz. only found significant differences in ch and crf between subjects younger than 14 and older than 60, but a linear correlation between these two biomechanical parameters and ageing did not exist. kotecha. observed a reduction in ch of approximately 0.28 mm hg / decade, while foster. found that the crf declined significantly with age at a rate of 0.31 mm hg / decade, as did ch by 0.34 mm hg / decade. in any case, due to the potential limitations of these studies for instance, in one of these studies the sample was quite limited, with only fifteen subjects. in another study, the changes are possibly confounding because of the proportion of the participants affected by ocular hypertension, glaucoma, or pigment dispersion syndrome. due to age - related changes in corneal structure such as an increase in collagen fibril diameter or intermolecular bragg spacing in fact, ex vivo studies have shown an increase in corneal stiffness with ageing and that young 's modulus of the human cornea approximately doubles between the ages of 25 and 100. considering this, if the crf is a real indicator of corneal rigidity, it should change with ageing as well. nevertheless, due to the intersubject variability and the differences among the results published in the different studies, we can not conclude, based on present data, that ch and crf parameters are able to confirm in vivo and in the clinical routine the expected changes towards a stiffening of the cornea. several studies investigated the potential effect of cct on the biomechanical properties of the cornea measured with ora. in fact, many studies reported a positive correlation between cct and ch [3, 24, 30, 42, 50, 51 ] and also with crf [24, 30, 40, 44, 51 ]. these studies included healthy subjects from different races / ethnicities and with a wide range of age. recently, leite. found that black subjects had lower ch values compared to white subjects, but although they attributed those differences in ch to differences in corneal thickness between the two groups, they did observe a statistical trend towards lower ch among black subjects even when adjusting for cct. a similar result was observed in a study with a strong statistical power by haseltine.. these results are in agreement with the expected response because a thinner cornea will be easier to deform, while a thicker healthy cornea containing more collagen fibers and ground substance will present a higher resistance against deformation and a higher damping capacity. consequently, the stronger the corneal tension, the faster the cornea recovers its original position following deformation. there are a couple of articles where the 24-hour changes of cct and corneal biomechanical properties were analysed [44, 54 ]. despite a significant change between the nocturnal and diurnal cct values, these results could be explained considering that nocturnal cct increase is related to increase in corneal hydration instead of collagen fibril or ground substance changes that would potentially reflect more directly on the biomechanical behaviour of the cornea. furthermore, it has been claimed that longer eyes are associated with flat corneal curvature and thinner corneas. furthermore, longer eyes had thinner sclera walls and possible thinner choroidal structure. in this way, according to previous section, if the highly myopic eyes have thinner corneas and if corneal biomechanical response might be somewhat related to the whole - eye biomechanical response, it would be expected that that more myopic eyes have lower ch values. it has been the goal of some studies to test the hypothesis that the weaker scleral structure of highly myopic eyes might be reflected and quantified in some way through the biomechanical analysis of the cornea. studies performed in chinese subjects [41, 56 ] and caucasian subjects with a wide range of refractive errors observed a significant negative correlation between ch and myopia. shen. found lower ch in highly myopic eyes (9 d) and no statistically significant differences in ch between emmetropes and low myopes (+ 0.25 to 2.75 d) or moderate myopes (> 3.00 to 6.00 d). similar results were reported by jiang., but the reason of this decrease was not fully explained. however, although variation was not observed neither in cct nor in crf among subjects with different myopia degree, it is possible that the changes are related to the different characteristics of the cornea rather than weaker sclera structure which is characteristic of the highly myopic eyes., in a study of subjects with myopic anisometropia, reported a significant lower ch in high myopic eyes compared to contralateral normal eyes. in this study, the difference in al between the two eyes that resulted in anisometropia and ch was correlated with al and cct in high myopic eyes, whereas in the contralateral eyes, it was only correlated with cct. additionally, since differences in iopg and iopcc between the high myopic and contralateral eye were not observed, the authors suggest that the difference in al does not occur by virtue of higher iop, but it is possible that eyes with lower ch and thinner scleral structure may be easier to elongate [58, 59 ]. however, these studies do not permit elucidation if the lower ch and thinner scleral structure are the cause or the consequence of the increasing myopia of those eyes. yet, despite above studies indicate that the mechanical strength of the anterior segment of the eye is somehow compromised in high myopia, other previous studies did not show a correlation between refractive error and ora measurements [40, 59, 60 ]. the study conducted by radhakrishnan. evaluated 95 normal myopic adult subjects (19 to 48 years) and found that ch was not significantly correlated with refractive error, while crf showed a statistically significant but very weak correlation with spherical equivalent refractive error (r = 0.04). however, the mean spherical refractive error was 1.78 2.26 d and both parameters showed a considerable scatter across the sample under analysis. the goldmann applanation tonometer (gat) is the reference method to measure the iop but when the iop is measured with gat it is assumed that the cornea is uniformly thick and perfectly elastic and behaves like a thin and perfectly flexible membrane. actually, none of these assumptions applies to the anatomical structure and physical behaviour of the living cornea under applanation forces. the pressure required to applanate the cornea depend on the iop and the corneal rigidity, and it is well known that the iop measures are influenced by cct with thicker corneas requiring stronger force to applanate than thinner corneas, independent of iop. many published articles have proposed linear correction factors to convert measured iop into true however, reported correction factors are different and mostly dependent on the population under study and can lead to corrections that may be wrong in magnitude and in direction such as correcting down when the true pressure is actually higher. in fact, corneal biomechanical properties seem to be stronger predictors of iop measurement error than does cct alone ; this might explain the success of the ora over the last 8 years for the iop measurement in several corneal conditions. iopg provided by ora is analogous to standard noncontact tonometry iop measurements whereas iopcc takes into account the biomechanical properties and is independent of the cct as explained above. although some studies find no mean difference between gat and both ora iop measurements [24, 31, 63 ], other studies found poor agreement between gat and iopg and iopcc with a significant overestimation of iopg and iopcc compared to gat [27, 64 ]. medeiros and weinreb found that gat iop was significantly correlated with cct and significantly influenced by crf, while iopcc was not, and similar results have been confirmed by others [27, 64, 65 ]. therefore, the effect of cct on iop overestimation may be explained by crf and the resistance against deformation of the cornea which is also higher in eyes with higher iop values. in contrast, some studies reported the lack of association between ch and both gat and iopg [30, 64, 65 ], suggesting that ch is independent of iop, while other studies suggest a relationship between ch and iop. ch has been shown to decrease as the iopcc increases [30, 46, 66, 67 ]. found iop as a significant explanatory variable relevant to ch, while gonzlez - meijome. found a significant correlation between changes in iop and changes in ch during the day in healthy eyes. also, ch has been shown to increase when iop was lowered to normal range in patients with chronic primary angle - closure glaucoma. considering the previous results and despite some controversy, it is expected that in corneas with higher ch and higher crf and therefore higher resistance to deformation, the values of gat iop or iopg may be higher than the actual values and iopcc could be a more reliable measure in those cases. the opposite might hold true in cases of lower ch and lower crf where the actual iop might be higher than actually measured by conventional methods. again reduced oxygenation of the cornea during contact lens (cl) wear is known to produce corneal edema that is reflected in an increase in corneal thickness (swelling). in fact, in a recent study, it was observed that the myopic subjects wearing soft contact lenses have higher values of ch and crf than noncontact lens wearers. the corneal swelling response with contact lens wear and eye closure averaged from 3% to 10% [71, 72 ] and some studies have analysed these effects on ora measurements [70, 73, 74 ]. lau and pye induced corneal edema wearing soft contact lens for three hours and found no change in ch even with 13.1% corneal swelling, while crf was elevated by a maximum of 0.6 mm hg immediately after lens removal and was followed by a gradual recovery to normal values. additionally, there were significant increases in iopg but not in iopcc and there were significant but weak correlations between changes of cct and iopg and iopcc and crf. lau and pye found that ch and crf respond to corneal swelling in dissimilar ways : ch was reduced by 0.6 mm hg immediately after lens wear before returning to baseline, while crf was elevated by a maximum of 0.6 mm hg. in addition, the ability of cct to predict both ch and crf was significantly different between control and monocular closed - eye contact lens wear and the gat overestimation observed is associated with an overall increase in crf caused by small amounts of corneal swelling. differences in the study population as well as in the amount of corneal swelling induced are likely contributors to the differences in the results between the two studies. however, the results suggest that ora - generated parameters may be different in subjects with and without contact lens wear when significant amounts of edema are present. this kind of response, commonly observed in aphakic patients with overnight wear of thick cl, is not expected with regular use of silicone hydrogel contact lenses under daily wear conditions by patients within the normal range of refractive errors. orthokeratology (ok) is a technique that uses special gas permeable cl to temporarily reduce myopia by flattening the cornea. therefore, the epithelial corneal thickness profile is changed and the cornea is significantly flattened by the use of these cl [75, 76 ] and the corneal biomechanical properties could be affected by these changes. biomechanical properties of the cornea may help to understand the different responses to ok among different subjects. a study published in 2008 investigated the changes of ora measurements, cct, and topography in subjects three hours after wearing ok lenses and three hours after removing the cl in order to assess the effect of corneal biomechanical properties on response (corneal flattening) and recovery (corneal steepening) during ok lens wear and after removal, respectively. the authors found that corneas with high values of ch showed a slower response and slower recovery to the ok treatment in the short - term treatment (3 hours of treatment). in another study, during short - term ok treatment, crf was shown to decrease with increasing duration of lens wear, while there was no significant change in ch. on the other hand, a significant decrease in ch and crf however, crf and ch returned to original values and remained unchanged thereafter. according to the authors, the early reduction in ch and crf may be due to a temporal response of reshaping of the corneal surface, rather than changes in the corneal microstructure. this may explain why there is a trend for ch and crf to be reduced during the first month of treatment and after 1 year of treatment ; when this is interrupted, ch and crf show a trend to return to baseline values. the knowledge of these associations could help to have a better predictability of the ok effect [5, 80 ] and then to choose the suitable patients to undergo ok treatment or to predict the speed of onset and recovery of the effect. several studies showed invariably a significant reduction of ch and crf by about 1 to 3 mm hg approximately after different laser refractive treatments [4, 6, 25, 30, 8187 ]. studies comparing different laser refractive techniques showed a higher decrease in both ch and crf in lasik eyes when compared with photorefractive keratectomy (prk). similar decrease in ch has been documented for lasik and laser - assisted subepithelial keratectomy (lasek). this biomechanical effect was correlated with deeper ablation because more central collagen and matrix material would be removed [4, 81 ] or with the potential effect of flap preparation that itself causes a reduction in both ch and crf [82, 88, 89 ]. ortiz. found a moderate correlation between the refractive error correction and the change in ch (r = 0.5, p =.007) and crf (r = 0.6, p =.001) in myopic lasik, while a smaller decrease in ch and crf was found in hyperopic lasik eyes than in myopic lasik and lasek eyes, supporting the predominant effect of tissue ablation. found a reduction in both ch and crf with microkeratome - assisted flap creation alone. found that despite similar changes in ch and crf in the myopic lasik and myopic lasek groups, there were significantly greater postoperative changes in the ora waveforms in the lasik groups than in the lasek group with the amplitude of peak 1 being less reduced in the group of lasek, suggesting that the creation of a flap has a greater effect on these waveform parameters than the depth or location of the stromal ablation. similar results were reported by franco and lira who found that, as a result of induced changes in viscous and elastic properties by lasik, the time needed for the first applanation of the cornea (time in) was higher in normal than in post - lasik eyes and that the post - lasik eyes needed more time to recover their shape (time out parameter). studies reporting the time course of ora parameters after different surgical techniques showed that the largest changes occurred within the first few weeks after surgery and then became nearly stable or even showed a slight recovery in the medium and longer term [84, 86, 87 ]. surgically induced corneal ectasia is a rare complication of refractive surgery and is thought to be a result of biomechanical decompensation due to an insufficient residual stromal bed thickness after the surgery or when surgery is performed on unidentified subclinical keratoconic cornea. thus, the possibility of using ora parameters for assisting in the detection of corneas at risk has been very promising since the ora was marketed. although a low ch (< 8 mm hg) might be a predictive index of a preectatic conditions [3, 33 ], the overlap in the distribution of both ch and crf values within the normal population does not support a role for ch and crf measurement as single predictors to detect early ectasia or to predict its onset before surgery. instead, waveform analysis of ora signals [33, 82, 92 ] has shown that the morphology of the signal may provide additional information. for instance, in a case of iatrogenic ectasia after lasik, kerautret. found a lower peak 1 height in the ectatic eye than in the fellow nonectatic eye, despite the similar ch and crf values in the 2 eyes. these findings may suggest that a higher peak 1 is associated with a stiffer cornea. considering that recent studies seem to indicate that the new ora parameters represent a significant improvement over ch and crf alone, more research is needed to confirm and improve the sensitivity and specificity for preoperative detection of at - risk corneas. cross - linking (cxl) is a minimally invasive procedure which presumably induces the formation of new molecular bonds between the corneal collagen fibrils and lamellae using riboflavin and uv light. this procedure of reinforcing the collagen meshwork with cxl has shown to be effective in the treatment of surgically induced ectasia and in halting progression of keratoconus [9496 ]. in corneal cxl, the cornea is stiffened and a high increase is observed in young 's modulus by nearly 300%. it would be expected that the biomechanical properties of the cornea will change as a result of the treatment, particularly corneal rigidity parameters. differences in ch and crf were observed during the first weeks after cxl treatment that returned to baseline values later. the effect of matrix reorganization or cct changes immediately after the procedure may explain these differences in ch and crf [97, 98 ] ; however, sustainable changes in ch and crf parameters alone that can be correlated with the assumed increase in corneal stiffness induced by cxl [34, 36, 97, 99 ] were not found and the clinical results did not confirm the ex vivo results. from the analysis of the new ora parameters based on waveform signal analysis, a significant increase (35%) in area under peak 1 and peak 2 was observed after six months of treatment, suggesting that this can be the result of a modified corneal surface after cxl, which provides better reflectivity due to an improvement of corneal homogeneity [34, 97 ]. these recent studies seem to indicate that additional parameters derived from signal analysis provide supplemental information to evaluate the potential positive effect of cxl and to measure the long - term effects of this procedure. intrastromal corneal ring segments (icrs) are primarily used for the treatment of primary keratoconus and secondary keratectasia following refractive surgery. the insertion of the icrs induces a flattening of the central cornea by adding extra material within the corneal paracentral area, improving regularity of the corneal shape, and preventing additional degradation of vision. knowledge of the biomechanical properties of the corneal might help to decide the best treatment approach, predict the success of the treatment, and eventually monitor the postsurgical corneal behaviour. no significant differences were found in ch in the short - term (< 3rd month) postoperative period [104106 ] which may indicate that the icrs alter corneal curvature without changing the viscoelastic response of the corneal tissue. a study conducted on 20 patients with keratoconus showed a stable corneal flattening and a decrease of the astigmatism with no statistically significant changes in ora parameters, 18 months after icrs implantation. better visual outcomes could be expected for corneas with lower biomechanical corneal resistance due to easier deformation by the ring implantation. reported significant changes in ch, 6 months after icrs implantation, and the authors suggested that these changes may limit the prediction of the ring segment effect in the long term. however, this hypothesis could not be confirmed by a recent study, contradicting previous results obtained by the same authors. although the authors claim in the second publication that prediction of visual acuity (va) by ora parameters is feasible in the short term, they could not confirm that in the first study using the same follow - up time of 6 months. regarding crf value, significant transient decrease was found during the first 3-month period after the femtosecond laser - assisted icrs implantation with no significant changes thereafter. new waveform parameters such as the amplitude peak 2, aplhf, uslope11, w11, path11, time1, and deltatime showed significant differences with respect to the preoperative conditions but those changes were not attributed to a modification of the biomechanical properties induced by the treatment but rather to corneal stabilization. interestingly, from the waveform analysis provided by ambrosio., it has been recently reported that the corrected and uncorrected distance visual acuity improved more as the pre - icrs implant biomechanical properties were weaker or less resistant before treatment. this might provide useful information to predict the visual outcomes of icrs implantation in keratoconus. studies that evaluated corneal biomechanics by ora showed that corneas after penetrating keratoplasty (pk) or deep anterior lamellar keratoplasty (dalk) present weaker ch and crf than normal corneas [29, 112114 ]. additionally, yenerel. found that ch and crf were higher in pk eyes than in forme fruste (ff) or advanced keratoconus (kc) eyes and both ch and crf parameters approach the range of normal eyes after corneal transplantation. on the other hand, shin. analysed the results of 26 subjects that had undergone pk for different reasons (bullous keratopathy, herpes keratitis, trauma, etc.) in one eye and compared the results with the contralateral nonoperated eye. they reported lower ch and higher crf post - pk compared with the fellow healthy eye, although these differences were not statistically significant. the effect of different keratoplasty techniques showed that post - pk eyes had lower ch and crf when compared with post - dalk eyes and post - dalk eyes had ch and crf values similar to normal eyes. this may be due to the action of descemet 's membrane which is preserved in dalk, which acts as a strong foundation for the rest of the corneal stroma which rests above it. opposite findings were reported by jafarinasab. that found lower values of ch and crf in the dalk group compared to pk group, but those differences were not observed 30 months after surgery. differences between the indications for keratoplasty or graft - related differences may explain the difference in the results of different studies. differences in cct have been considered as a risk factor for glaucoma [117, 118 ] and given the correlation between low cct and glaucomatous changes in the optic disc, a biological association shared by the cornea, sclera, and lamina cribrosa is conceivable [119, 120 ]. a number of recent reports have suggested a relationship between ch, crf, and glaucoma with evidence that ch is lower in glaucomatous eyes compared with normal eyes and eyes with ocular hypertension [3, 7, 42, 45, 90, 121124 ]. furthermore, normal tension glaucomatous (ntg) eyes show the lowest value among glaucomatous eyes according to some studies [121, 125 ]. even after pharmacologic iop lowering, ch was shown to be lower in glaucomatous eyes than in normal eyes. this suggests that eyes with lower ch and/or thinner than normal cct might exhibit structural weakness and it is possible that cct and ch could be considered as risk factors for glaucoma, independent of iop [121, 122, 127 ]. conversely, crf was found to be significantly higher in patients with ocular hypertension and in patients with primary open - angle glaucoma and low in ntg patients [90, 123 ]. this implies that gat iop should be expected to be overestimated as a greater force required to applanate a cornea with higher crf. this could suggest that crf could be also useful to differentiate between subjects with ocular hypertension and glaucoma. as both the sclera and the cornea are formed from continuous extracellular matrix, this might have some effect on the biomechanical relationship between the two tissues. compared ch in glaucomatous eyes with and without acquired pit of the optic nerve and reported that ch was lower in glaucomatous eyes with an acquired pit and hypothesized the possibility that corneal biomechanical properties reflect the attributes of the lamina cribrosa [120, 121 ]. several studies found that eyes with low ch are associated with increased severity of glaucomatous visual field defects [45, 122, 129, 130 ]. in contrast, wells. found a relationship between ch and deformation of optic nerve head with higher ch being strongly correlated with higher deformability of the optic nerve head. in untreated newly diagnosed poag patients, ch was the only factor significantly associated with both mean cup depth (r = 0.34) and cup - to - disc ratio (r = 0.41). in conclusion, as the elastic properties of the cornea are believed to reflect the elasticity of collagen fibres in the eyeball as a whole, there might be an opportunity to consider corneal biomechanics as an indicator of overall globe biomechanical properties in glaucoma. if this is true, corneal biomechanical properties seem to be a promising addendum to the complex issues of glaucoma and may constitute a pressure - independent risk factor for glaucoma detection, prognosis, and treatment. in keratoconus (kc), the normal corneal collagen - fibril meshwork is disrupted leading to a localized reduction of corneal radius of curvature and tissue thinning. a significant weaker stress versus strain response in kc eyes compared to normal eyes and a more disorganised collagen fibber network as well been shown [16, 133 ]. thus, changes in corneal biomechanics in kc eyes might be expected and it has been suggested that kc progression is characterized by a reduction of material properties that lead to a progressive thinning, increasing strain and stress redistribution, and lower keratocyte densities [134, 135 ]. ch and crf measurements have been shown to be reduced in kc eyes [4, 28, 39, 85, 136138 ] with stronger decrease as kc severity increases [110, 139141 ] even after controlling for differences in age, sex, and cct [141, 142 ]. this suggests that other structural alterations different from cct lead to lower lamellar adhesion and lower shear modulus and may be responsible for these lowering effects in ora measurements. however, there is large overlap of ch and crf between normal and kc corneas and both ora parameters showed low sensitivity and specificity in differentiating kc or suspecting kc from healthy corneas [137, 138, 140, 142, 144, 145 ]. recent studies demonstrated that the new parameters derived from waveform analysis of ora signals represent a significant improvement in detection and differentiation of the keratoconic cornea [28, 92, 110, 145, 146 ]. in fact, characteristics of the air pressure corneal deformation profile are more affected by keratoconus than the traditionally extracted ch and crf factors ; keratoconic eyes have significantly lower elasticity coefficient compared to normal eyes and the area under the second peak of the signal curve has been shown to produce the best results and seems more promising in distinguishing between normal and kc eyes [110, 137 ]. when the disease progresses, the number of endothelial cells decreases and corneal oedema increases affecting visual acuity. both crf and ch parameters were found to be lower in fcd eyes compared to normal eyes [3, 51, 148 ]. reported that crf was positively correlated with cct in control eyes while this correlation was negative in fcd eyes. according to the authors, these results may be related not only to corneal hydration but also to other aspects of corneal biomechanics since patients with fcd have decreased endothelial cell density and thicker descemet 's membrane, and the corneal central region is usually involved which can lead to reductions of viscous damping within corneal tissues and, consequently, viscosity reduces. additionally, the authors found that the lower the ch, the higher the iopcc in fcd eyes, but these results may be due to an underestimation error in iop measurement caused by the observed diminished ch and elevated cct. similar results were reported by clemmensen and hjortdal who found a ch and crf reduction in fcd eyes and that iopcc appears to overestimate iop in those patients. altogether, corneas affected by fcd point to a paradoxical condition in which thicker corneas are not related as expected to higher crf as shown in normal eyes. this might also point to a mechanistic explanation to interpret crf values. according to this, crf increases with increase in cct as long as this increase is justified by an increase in collagen material. conversely, when the increase is due to a massive hydration of cornea as in fcd, the effect is the opposite as the ground substance becomes more relevant in the overall context of the mechanical behaviour of the cornea. several structural changes in the cornea of diabetes patients have been reported [149, 150 ] and an influence on the biomechanical properties of the cornea could also be hypothesized. several studies have investigated the impact of diabetes on corneal biomechanical parameters ; however, the results are rather controversial among different studies [151156 ].. found that ch, crf, and cct were significantly higher in diabetic eyes compared to healthy eyes. hager. reported a significantly higher ch in diabetic eyes than in nondiabetic eyes after correcting for age, iop, and cct. reported that ch was significantly lower in diabetic patients, whereas crf was not significantly different from that of control subjects. the authors hypothesized that lower ch in diabetic patients may be explained by a decrease in the dampening effects of the cornea as a result of an alteration in the collagenous components in diabetic eyes due to collagen cross - linking. the reasons for such contradictory results among different studies lie in the differences in age range and cct and diversity of diabetes types and severity enrolled. in some studies most patients presented type 2 diabetes, while in others there were a similar number of patients with type 1 and type 2 of diabetes. in fact, as recently shown by scheler., biomechanical properties of the cornea seem to be altered depending on the glucose control. in their study, scheler. found that in diabetes, ch and crf were significantly correlated to glycated haemoglobin (hba1c) ; diabetic patients with elevated hba1c showed an increased ch indicating an increase in the viscosity of the ground substance that is associated with higher corneal shearing strength and increased damping most likely due to a nonenzymatic glycosylation of proteoglycans and glycosaminoglycan that affects the corneal damping behaviour. given the promising nature of the possibility of measuring corneal biomechanics in vivo, there has been an increasing interest in the development of methods that allow minimally invasive mechanical test of the cornea which may permit a better understanding of the differences in corneal properties between a wide range of ocular conditions and healthy eyes as well as an improvement in the early detection of potential problematic corneas. until now, many studies covering measurement of corneal biomechanical properties in a wide range of topics have been performed and published using the ora device as previously described, but other new in vivo techniques of corneal biomechanical measurements are under development. however, with the exception of the corvis st, most of these new noninvasive or minimally invasive techniques are experimental prototypes that despite being promising still have many drawbacks such as not being commercially available, being of high costs, and lacking evidence of accuracy and availability for clinical purposes that need to be overcome. one technique is the corneal visualization scheimpflug technology (corvis st ; oculus, wetzlar, germany) which is commercially available since 2011. this device is based on a noncontact air puff tonometer combined with an ultrahigh speed scheimpflug camera. the scheimpflug camera records 4330 images per second along an 8 mm horizontal corneal coverage during corneal deformation under an air puff indentation. this camera allows a dynamic inspection of the deformation process of the cornea and provides further detailed information of biomechanical characterization of the cornea. the corvis st output parameters include time and length of the flattened cornea in the first applanation ; corneal velocity during the first applanation moment ; time from start until the second applanation ; length and corneal velocity during the second applanation moment ; time from start until the highest concavity of cornea is reached ; and maximum deformation amplitude (from start to the highest concavity) at the corneal apex, among others. however, the machine is still under development and new parameters are being continuously added to the output and only available for research purposes. a definition of the parameters currently available in the commercial version of the instrument is provided in appendix b. clinical outcomes are limited and preliminary results have found significant differences of corneal deformation response among normal and keratoconic corneas for many parameters such as corneal speed during deformation, corneal applanation length, and deformation amplitude. all of them seem to be relevant parameters to define the corneal stiffness and corneal viscoelastic properties and are promising in the evaluation of several corneal conditions and the outcomes of different surgical procedures [158161 ]. another prototype device is the dynamic corneal surface topography that involves surface topographic corneal imaging, with a dynamic rasterstereographic corneal topography (d.rct) with off - axis geometry, during an air puff indentation by an nct. this device includes an imaging arm, a calibrated grid arm, and a digital camera. when fluorescein is instilled into the cornea and the fluorescent emissions are excited by the projected grid, an image of which is then captured that contains the three - dimensional information from the corneal surface. after approximately 12 ms from the beginning of the air puff, when the air puff pressure is maximum, another image is taken, which corresponds with the largest corneal deformation. from the two images that are acquired (predeformation and middeformation), biomechanical properties can then be determined using a model of corneal viscoelasticity, based on the applied force and the stress - strain relationship of discrete surface segments across the cornea by measuring corneal shape and displacement between the predeformation state and the middeformation state. another novel method is based on high speed swept source ocular coherence tomography (ssoct) combined with an air puff nct. the cornea is deformed by the air puff, and during the 20 ms of applanation time, the ssoct acquires multiple a - scans at the center of the air puff, allowing observation of the dynamics of the anterior and posterior corneal surfaces. from the analysis of the scan, one can obtain information about the biomechanical behaviour of the cornea during the applanation process. however, the system needs improvements particularly in a faster acquisition system and a large clinical study is required to fully understand the potential of the system in the clinical setting. brillouin optical microscopy is another noncontact technique that uses the combination of a confocal microscope with an ultrahigh resolution spectrometer to perform brillouin imaging of the cornea. it has the ability to visualize corneal elasticity and measure the depth - dependent variation of elastic modulus within the cornea noninvasively with three - dimensional resolution. this device was firstly used in bovine corneas and is currently in development for use in human eyes. shear wave propagation velocity has been used to measure corneal biomechanical properties in vivo, through the use of linear elastic model approximation, in which the young 's modulus and poisson 's ratio can be estimated from the shear wave speed [18, 167, 168 ]. however, corneal strain and corneal hydration strongly affects the wave speed by attenuating high - frequency shear wave and do not reproduce the nonlinear properties of the cornea. recently, a new method has been developed : the quantitative ultrasonic spectroscopy (qusi). the qusi has improvements in the form of wave propagation that are not available in clinical ultrasound and derives more information of the reflected full - wave forms. once corneal acoustic and elastic properties have been shown to correlate, this method is currently being developed to map corneal elastic properties and so to determine an elastic constant of the cornea called the aggregate modulus, which provides a measure of its stiffness. corneal transient elastography (cte) is another technique that is under development for ophthalmologic use and was adapted from a technology in current use for the analysis of breast tissue imaging. it combines the generation of a remote palpation in the cornea and ultrafast (20 000 frames / s) ultrasonic images of the resulting corneal displacements that evolve into a shear wave propagation whose local speed was directly linked to local elasticity. the mainly improvements was at the level of the echographic probe that was specifically designed to couple a homogenous transverse compression wave to the tissue (supersonic mode) and an ultrafast echographic acquisition mode, allowing high resolution and quantitative maps of the whole corneal elasticity. optical interferometric techniques were also used to measure corneal biomechanical properties because they are noncontact, highly sensitive, and capable of simultaneously recording information from across the whole surface. electronic speckle pattern interferometry (espi) was used to quantify the effect of microkeratome flap creation on the displacement response of the sheep cornea ; however, these techniques are extremely sensitive to environmental disturbances such heat and vibration that may influence its accuracy. radial shearing speckle pattern interferometry (rsspi) is an interferometric technique where the two images contain information on the topography of the surface location which changes as applied pressure is altered and is much more resistant to physical disturbances. the differential magnification between the two images allows a mathematical analysis to detect changes in radial strain. it has been used to describe the progressive increase in corneal young 's modulus as a function of aging in human corneas and to quantify the magnitude of the stiffening effect of corneal cross - linking. another technique uses a physical probe to indent the central cornea with an electronically controlled microprecision motor coupled with simultaneous video - topography imaging of the cornea. it is called dynamic corneal imaging (dci) and measures the change in curvature of the cornea as it bends. in this technique, greater difference flexing curves have been demonstrated with lower iop, thinner corneas, and in keratoconic versus normal corneas as well, which is consistent with more easily deformable corneas. another technique uses optical coherence tomography elastography to generate in vivo 2d maps of corneal deformation as it is indented by a concave curved lens to preserve the curvature of the cornea as it deforms. it has the potential to measure local and depth variations in the mechanical properties of the cornea owing to its ability to measure strain throughout all the stroma, providing measures of local viscoelastic properties such as elastic modulus, shear modulus, and hysteresis. current efforts include the development of 3d analysis routines and stress sequences for in vivo use. the published literature sheds light on the potential utility of the biomechanical corneal properties to a better comprehension of the mechanical behaviour of this complex tissue. however, it also shows some to some controversial results in relevant areas such as their impact on intraocular pressure measurement, preoperative refractive surgery assessment, and surgical treatment of keratoconus. new parameters derived from a more detailed analysis of the outcomes as well as new technologies are promising in consolidating the utility of the biomechanical corneal properties as a clinical tool and a very relevant field for the future improvement of safety and efficacy of different eye health care strategies. | several refractive and therapeutic treatments as well as several ocular or systemic diseases might induce changes in the mechanical resistance of the cornea. furthermore, intraocular pressure measurement, one of the most used clinical tools, is also highly dependent on this characteristic. corneal biomechanical properties can be measured now in the clinical setting with different instruments. in the present work, we review the potential role of the biomechanical properties of the cornea in different fields of ophthalmology and visual science in light of the definitions of the fundamental properties of matter and the results obtained from the different instruments available. the body of literature published so far provides an insight into how the corneal mechanical properties change in different sight - threatening ocular conditions and after different surgical procedures. the future in this field is very promising with several new technologies being applied to the analysis of the corneal biomechanical properties. |
cardiomyopathies are heterogeneous diseases in which alterations of heart muscle structure and function are the main characteristics. dilated cardiomyopathy (dcm) is characterized by cardiac chamber dilation and systolic dysfunction in the absence of coronary artery disease or other conditions associated with pressure or volume overload. right ventricular dilation and insufficiency may be present, but it is not necessary for the diagnosis. beyond extrinsic factors that cause myocardial injury with evolution towards cardiac dilation (ischemia, infections, medications, hormonal disorders, nutritional deficiencies), they produce the so - called idiopathic dilated cardiomyopathy which is consistent with the familial form of dcm from the european society of cardiology classification of cardiomyopathies. there are approximately 30 genes related to the pathogenesis of dcm, encoding proteins with very different functions in myocardial cell physiology, involved in contraction and relaxation, calcium homeostasis, cytoskeleton proteins, proteins involved in transmitting mechanical forces, and nuclear membrane proteins with a role in nuclear stability and regulation of ene expression, rna splicing, transcription and energetic metabolism. however, only 2030% of the patients with primary dilated cardiomyopathy have a known genetic defect. this area is still open to research, with enormous progress in identifying new genes or new mutations in the known genes involved in the pathogenesis of this disease. the prevalence of dilated cardiomyopathy in the general population is unknown, but in adult population it is estimated to approximately 1/2500 individuals, representing 50% of all patients with the diagnosis of dcm. the inheritance of the mutation responsible for the disease is estimated to approximately 90%, influenced by incomplete, age - dependent penetrance (meaning that not all carriers of the genetic defect will develop the disease and that the phenotype becomes manifest usually after the fourth decade of life) and variable expression (only certain features of the disease may be present in some individuals). the disease transmission within the family is probably underestimated and sometimes favors erroneous classification as a de novo mutation. genetic defects involved in the pathogenesis of dcm can be transmitted in a autosomal dominant, autosomal recessive, x - linked or mitochondrial manner. the diagnosis of familial dilated cardiomyopathy is established if the condition (primary dilated cardiomyopathy) is affecting at least two first degree relatives. the presence of positive family history is a less sensitive criterion for the diagnosis of familial disease, a family member may be completely asymptomatic and without ecg changes but with left ventricular dysfunction and dilation identifiable with imaging investigations, hence the importance of screening family members. when the full definition of the disease was used in the cardiovascular screening the familial form was found in 2035% of cases, but when the left ventricular dilatation was used as the sole criterion for diagnosis the prevalence was 48%. the present review article intends to summarise the latest information on dilated cardiomyopathy associated with lmna gene mutations. the research on genetic causes and their clinical and therapeutical impact are continuously developing generating results with great influence on diagnostic and therapeutical conduct. until recently the genes reported as most frequently involved in the development of dcm have been tnnt2 (cardiac troponin t), lmna (a / c nuclear lamins) and myh7 (myosin heavy chain). a recent study sequenced the entire ttn gene which encodes titin, the largest human protein ; their study has showed that ttn truncating mutations are the most common known genetic cause of dilated cardiomyopathy. no clear relationship is established between the genotype (gene, mutation) and the clinical phenotype, except lmna gene, which proved to be associated with conduction defects, malignant ventricular arrhythmias and supraventricular arrhythmias preceding the development of left ventricular dilation and heart failure. lamins a and c are intermediate filaments located between the nuclear membrane and the chromatin, having an important role in maintaining the shape and nuclear structure, translation and transcription regulation, position and function of nuclear pores, and chromatin organization. lmna gene encodes two isoforms, lamin a and lamin c, resulting from alternative splicing. laminin a has 98 aminoacids in addition to laminin c. they are expressed in many types of differentiated tissues, forming nuclear lamina by polymerization of laminin molecules in the nucleus, resulting a network of intermediate filaments attached to the internal nuclear membrane which has a supporting role for chromatin organization, gene regulation, dna replication and rna splicing. mutations of this gene can cause several clinical entities, including dilated cardiomyopathy with minimal or no skeletal myopathy, emery - dreifuss muscular dystrophy, charcot - marie - tooth neuropathy, dunningan partial familial lipodystrophy, progeria and other overlapping syndromes, all known as laminopathies. they all have in common a certain degree of nuclear fragility, altered nuclear architecture, impaired nuclear signaling and transcriptional activation by altering abnormal adaptive and protective mechanisms. in vitro studies on fibroblasts have shown that the result of these changes is a nuclear susceptibility to mechanical stress rupture subsequently leading to cell death. in humans lmna mutations are heterozygous with minimal or no reduction of lamins a and c levels, but with various degrees of structure and function imparement, thus explaining the multitude of variations in the phenotype of the disease. multiple mutations in the same gene, known as allelic heterogeneity, may cause identical or completely different phenotypes. the most common clinical entities caused by lmna mutations are cardiolaminopathies, characterized by cardiac dilation with progressive heart failure, conduction abnormalities and arrhythmias ; skeletal myopathy may be present in varying degrees or absent. the disease has a poor prognosis with increased mortality, especially from sudden death and progressive heart failure. in familial lmna - dcm mutations are found with a frequency of 68%, but among patients with associated conduction defects their frequency rises to 30%. although there are no clearly established clinical criteria to differentiate the lmna - dcm from other dilated cardiomyopathies, the presence of conduction abnormalities or arrhythmias associated with ventricular dysfunction is suggetive of lmna - associated dilated cardiomyopathy (lmna - dcm), especially if skeletal muscle involvement is present, regardless of degree. the lmna mutations responsible for dilated cardiomyopathy are transmitted in a autosomal dominant manner meaning that the offsprings of a patient have 50% chances to inherit the genetic defect. genetic testing for dcm patients is now possible in many genetic laboratories by protocols for the most frequently involved genes in the ethiopathogenesis of the disease, but with high costs and with minimal influence on therapy, so it is not done routinely. but, for lmna mutations, the things are changing due to the high risk of sudden death of the mutation carriers, making the genetic analysis necessary. there are attempts in discovering a non - genetic, lower - cost, diagnostic test for lmna - dcm ; narula. used the quantitative expression of the mutated lmna (using the mrna levels in blood and the lamin a / c immunostain in endomyocardial biopsy) with a 100% sensitivity and a 87% specificity in predicting the lmna mutation. the expression of mran in blood was lower in lmna carriers comparing with wild - type lmna individuals or with dcm patients of another genetic cause, so it is a potential biomarker for the monitoring of the quantitative expression of lmna mutated gene. the onset of lmna - dcm is usually in adulthood, with a penetrance directly related to the age of mutation carriers, as pasotti. observed in their study. just like all types of dilated cardiomyopathy, the main characteristics are left ventricular dysfunction and dilation, associated in the majority of cases with arrhythmias and ecg abnormalities preceding the onset of ventricular dysfunction. the initial symptoms of the disease can be represented by conduction disorders, with or without symptoms of arrhythmia or heart failure, including thromboembolic events from ventricular mural thrombi. lmna - dcm has a worse prognosis compared to other forms of dcm with a higher mortality and faster progression towards end - stage heart failure. most patients suffer an adverse event by the fifth decade of life ; the most common events leading to death are malignant ventricular arrhythmias followed by en - stage heart failure. showed in their retrospective analysis that about 70% of patients with lmna - dcm develop a cardiac adverse event within 5 years from diagnosis due to malignant arrhythmias (66%) or terminal heart failure requiring heart transplant. conduction defects and rythm disturbancies that may occur in the natural course of the disease include atrioventricular block of any degree, bundle branch or fascicular blocks, minor intraventricular conduction delay, supraventricular arrhythmias, (atrial fibrillation, atrial flutter, paroxistical supraventricular tachycardias, supraventricular premature beats) and ventricular arrhythmias (ventricular premature beats, sustained or unsustained ventricular tachycardias, ventricular fibrillation). analyzed the temporal relationship between the onset of ecg changes and the onset of ventricular dysfunction in carriers of lmna mutations and calculated that the ecg changes precede the ventricular dysfunction with approximately 7 years. all the patients included in the study, except one, had ecg changes at the time of diagnosis of left ventricular structural or functional abnormalities. they considered that even the minor ecg abnormalities represent an early sign of the disease and a longstanding, severe dilated cardiomyopathy with no ecg abnormalities is unlikely to be produced by a lmna mutation. it is proven that the carriers of a lmna mutation are at increased risk for malignant ventricular arrhythmias and sudden cardiac death before the onset of heart failure and the risk rises afterwards. van rijsingen. analyzed in a retrospective study, the risk for malignant ventricular arrhythmias of lmna mutation carriers, disregarding their clinical state, and the eventual predicting factors of this risk. their study confirmed the increased risk of sudden cardiac death, ventricular arrhythmias and end - stage heart failure of these individuals and identified four independent risk factors of ventricular arrhythmias : unsustained ventricular tachycardia, ejection fraction below 45% at diagnosis, male sex and non - missense mutations (insertions - deletions, truncating or mutations affecting splicing). their multivariate analysis showed that the risk rises and the age of onset lowers with the number of these risk factors, hence the importance of periodic clinical evaluation. up to date, although the importance of cardiodefibrilator was unanimously accepted as life saving, the optimal moment of implantation was not established. consider the icd as being appropriate for the high risk patients with 2 or more risk factors, earlier than the guidelines recommend. the high penetrance and the aggressiveness of the disease together with the theory of the favorable effect of early pharmacologic treatment (betablockers, ace inhibitors) and non - pharmacologic (cardiodefibrilator, cardiac resynchronization) on prognosis added weight to the importance of familial screening. moretti. compared the clinical features of patients with dcm diagnosed through familial screening (non - proband patients) to those with sporadic forms of dcm (proband). their results showed that patients diagnosed through familial screening were younger, with less severe symptoms at diagnosis (nyha i and ii classes) and with later need for cardiac transplantation. there was no significant differences in clinical state, all caused mortality (including the moment of sudden cardiac death or malignant ventricular arrhythmias) and there was no diference in the rate of ventricular dysfunction progression, although through the entire study period the non - proband patients mantained a higher ejection fraction than those with sporadic form of disease. they have concluded that the familial screening helps in early diagnosis and treatment of dcm, improving the outcome but with no significant influence on the long - term natural history. the authors observed that in the non - proband group the pharmacologic treatment for heart failure was less intense comparing with the proband group, possibly with a negative effect on the outcome. the theoretical beneficial effect of the early treatment, before the onset of symptoms, on the long - term outcome is yet to be proven by future studies. lmna - dcm is a genetic, autosomal dominant disease with high, age dependent penetrance and variable expression. it has a severe prognosis, with a high risk of malignant ventricular arrhythmias and evolution towards end - stage heart failure and need for heart transplantation. early pharmacologic treatment and defibrilator implantation could favorably influence the long - term outcome of the mutation carriers. familial screening through clinical examination, ecg and echocardiography has a significant importance in early dcm diagnosis. | lamin a / c gene (lmna) associated cardiomyopathy is a form of dilated cardiomyopathy with poor prognosis and high mortality, and a rapid evolution toward end - stage heart failure and malignant ventricular arrhythmias associated with increased risk of sudden cardiac death. it is transmitted in a autosomal dominant manner and is characterized by age - dependent high penetrance and variable expression. screening of first degree relatives of proband patients by means of clinical evaluation, electrocardiogram, echocardiography and genetic analysis is useful for the early diagnosis of the disease. drug therapy and non - pharmacological measures in the early stages of the disease seem to improve the prognosis of these patients. |
in the united states (usa), cigarette consumption has steadily declined during the past decade. however, sales of cigars, particularly little cigars and cigarillos, have markedly increased. of the three types of cigar products available in the usa, trend data from 1993 to 2006 suggest that the sales of large cigars decreased from 37% to 47%, while the sales of cigarillos increased from 25% to 32% and little cigars increased from 37% to 47%. increased consumption of small cigars (i.e., little cigars and cigarillos) presents a new challenge for tobacco control researchers. small cigars are similar to cigarettes in their size, shape, and filtering, are often inhaled, and are flavored. small cigars deliver considerable amounts of nicotine to sustain dependence and increase the carbon monoxide levels of smokers [3, 4 ]. small cigar smoking is a public health threat and is not a safer alternative to cigarette smoking. 65% of young adults have heard about small cigars, and 26% report ever use of small cigars. young adults with a history of tobacco use have an elevated risk for cigar smoking, with recent studies indicating that 12.016.0% of young adult cigarette smokers have smoked some cigar variant (cigars, little cigar, or cigarillos) in the past 30 days [68 ]. the increased popularity of small cigars among young adult smokers may be due, in part, to their exposure to advertisements and promotions of small cigar products. although magazines and point - of - sale advertisements (which accounted for 24.1% and 13.0% of cigar advertising and promotional expenditures in 1999) are traditional avenues for cigar advertisements, tougher restrictions on tobacco advertising may entice the tobacco industry to use creative marketing strategies to promote small cigars. in 2012,, also known as snoop dogg, announced his launching of a new brand line of cigarillo products, called executive branch. several widely circulated music magazines and press releases reported that snoop dogg planned to unveil the new product during his upcoming performances at the 2012 coachella music and arts festival in indio, california. the music festival mainly attracts an international audience of adults aged 35 and younger [10, 11 ]. a recent study by richardson and colleagues recently documented snoop dogg 's promotion of executive branch small cigars on social media sites, such as instagram. the tobacco industry historically has relied upon hip - hop and other forms of music to promote smoking, as music can be used as a form of direct, targeted marketing. hip - hop music has a wide appeal and a varied fan base across different genders, racial / ethnic backgrounds, and nationalities (i.e., usa versus international countries). endorsements by its celebrity music artists can introduce their youthful fan base (primarily under 30 years old and including many fans under the age of 18) to new tobacco products and smoking behavior. as such, exposure to a hip - hop music artist 's endorsement and promotion of a brand - specific small cigar product may be associated with young adults ' small cigar smoking behaviors. in 2012, the surgeon general 's report concluded that there is a causal association between exposure to smoking in the movies and youth smoking. the report also concludes that traditional advertising causes youth smoking [14, 15 ]. to our knowledge no study has examined the association between small cigar smoking behavior and exposure to a celebrity music artist 's advertisement and promotion of small cigars. we surveyed a random sample of young adult cigarette smokers who attended the coachella festival in indio, california, to assess their exposure to snoop dogg 's endorsement and advertisement for executive branch small cigars. we focused on young adult cigarette smokers because extant evidence suggests that they may be considered at high risk for small cigar use [6, 7, 16 ]. we hypothesized that young adults who reported exposure to the artist 's small cigar advertisements, compared to those who did not report exposure, would have greater susceptibility or intention to smoke executive branch small cigars in the future. our hypothesis is supported by ample evidence that has reported that exposure to cigarette smoking advertisements is associated with a susceptibility to smoke cigarettes [1720 ]. our rationale for use of intention to smoke small cigars is that it is a proximal predictor of smoking behavior and is the most appropriate outcome to assess in a cross - sectional design. eligible participants were young adults, aged 1835, who attended the coachella festival and reported smoking at least one cigarette in the past 30 days. approximately 85,000 concertgoers attended the festival per day over a three - day period. according to a small survey of concertgoers (n = 238) posted on the festival 's webpage (http://www.coachella.com), 31.5% were aged 2529 years, 29.8% were aged 2024 years, and 12.2% were aged 3034 years old. venue - based sampling procedures, in which the venue is the primary sampling unit, were used to select and survey eligible young adults at the festival. to construct our sampling frame, the study team (k.s. and n.p.) utilized ethnographic sampling techniques to survey the area and identify locations within the festival where young adult smokers would gather or pass through. we identified three locations within the venue that fit this criterion : two beer gardens where festival attendees purchased and consumed alcoholic beverages and a smoking lounge., we implemented area - based sampling and approached each individual for inclusion into the survey. for the two beer gardens, we identified young adults who were smoking cigarettes and randomly selected them by systematically intercepting every third cigarette smoking attendee that crossed a predetermined point. the two study team members collected data from attendees over a two - day period from festival open to close. upon interception, each team member approached festival attendees, gave them a short description about the study, and obtained verbal consent. if the attendee refused, the study team member noted their refusal and continued the process with the next random participant. festival attendees who appeared to be visibly impaired by alcohol or other substances were not approached. the study team determined the eligibility status of festival attendees that were intercepted and who were willing to participate using a brief screener that verified the attendant 's age and cigarette smoking status. attendees who did not meet the eligibility criteria were not surveyed. over the two - day data collection period, data were lost for 22 respondents due to a synchronization error between the server and the device and could not be retrieved. the majority of the 121 respondents were male (55.4%) and between the ages of 18 and 30 years old (88.0%). regarding race / ethnicity, most respondents were white (75.2%) and lived in a country outside of the usa (52.4%). overall, 70% reported past 30-day nonmentholated cigarette smoking, while 36.4% smoked menthol cigarettes. survey items were entered in a survey software app, isurveysoft (http://www.isurveysoft.com), and were uploaded to the handheld device (ipod touch 5th generation). the brief survey consisted of 21 items including demographic information, smoking history and current tobacco use, and exposure to small cigar advertisements. respondents were asked about their age, gender, race / ethnicity, and country and state of residence. age categories included < 18 years old, 1824 years old, 2530 years old, and 31 years or older. racial / ethnic categories included asian / pacific islander, american indian / alaskan native, black / african - american, hispanic, white, and other. respondents were allowed to select one or more of the racial / ethnic categories. respondents were asked if they had used any of the following tobacco products in the past 30 days : cigarettes, nonmentholated (i.e., marlboro), mentholated cigarettes (i.e., newports), cigars, and small cigars (e.g., black & mild, swisher sweets). brand names were provided for each tobacco product to help respondents better recognize the tobacco product. respondents were allowed to select one or more of the tobacco products if they used them in the past 30 days. those who selected yes for the past 30-day use of any of the tobacco products were classified as current users. respondents were asked two questions : (1) if they intended to smoke executive branch small cigars and (2) if they intended to smoke any other small cigar in the future. response categories for both questions were yes or no. we assessed festival attendees ' exposure to (1) the snoop dogg 's advertisements of the executive branch small cigars and (2) other advertisements for other small cigar products (e.g., swisher sweets). if a respondent reported exposure to the product, they were asked to recall where they had seen the advertisement. response categories included in a newspaper, in a magazine, on the internet, on social media sites (e.g., facebook, twitter, etc.), and other. urge to smoke executive branch small cigars was assessed by asking respondents how much they wanted to smoke the product after exposure to snoop dogg 's advertisement. the urge to smoke variable is similar to the one used by sargent and colleagues. a four - point response category was used to assess urge to smoke and included 1 = strongly agree to 4 = strongly disagree. we assessed exposure to and urge to smoke other small cigar products with similar questionnaire items, by replacing the phrasing respondent demographics, smoking - related behaviors, advertising exposure, and urge to smoke variables were explored using descriptive statistics. we assessed the prevalence of the past 30-day small cigar smoking among our sample of young adult cigarette smokers. to understand the demographic and tobacco use characteristics of small cigar users in our sample, we conducted descriptive analyses comparing survey responses from those young adult cigarette smokers who reported past 30-day use of small cigars to those who did not report use. descriptive statistics were also used to assess exposure to small cigar advertisements (celebrity - endorsed and other) and the urge to smoke small cigars after viewing the ads. bivariate and multivariate logistic regression analyses were conducted to assess the associations between small cigar smokers and nonusers and the demographic, smoking - related, exposure, and urge to smoke variables. of the 121 young adult cigarette smokers in our sample, 25.6% reported smoking small cigars at least once in the past 30 days. table 1 shows the distribution of demographic characteristics by small cigar smoking status. the majority of small cigar smokers in our sample were male and aged 1824 years old, although most small cigar smokers in our sample were white, significantly more african - americans reported smoking a small cigar in the past 30 days than not smoking a small cigar (p = 0.001). notably, over half of the small cigar smokers in our sample lived in a country outside of the united states. small cigar smokers (n = 31) in our sample reported concomitant use of other tobacco products. over two - thirds (71.0%) of small cigar smokers reported smoking nonmentholated cigarettes in the past 30 days, while one - third (32.3%) reported using mentholated cigarettes. small cigar smokers also reported current cigar smoking, with 19.4% reporting smoking cigars at least once in the past 30 days. approximately 14.0% of respondents reported seeing snoop dogg 's advertisement of executive branch small cigars. the majority of respondents (33.3%) reportedly saw these advertisements on the internet, while 27.8% reported seeing them on social media sites, such as facebook or instagram. of the respondents who were exposed to the executive branch advertisements, 82.4% said seeing the advertisement made them want to try the product. respondents reported seeing advertisements for the other small cigars in magazines (35.1%), on the internet (23.0%), in convenience stores or bodegas (12.4%), in smoke shops (6.6%), and on social media sites (5.4%). over half of respondents (55.1%) wanted to try the small cigars after being exposed to the advertisements. forty - five percent (45.4%) of 121 young adult cigarette smokers in our sample reported an intention to smoke executive branch small cigars in the future ; of these, 40.9% were current small cigar smokers. bivariate and multivariate logistic regression analyses were conducted to assess whether the demographic and smoking - related variables and the exposure to snoop dogg 's executive branch small cigar advertisements were associated with susceptibility to smoke the product in the future. though demographic variables were not associated with susceptibility, bivariate analyses found that past 30-day cigar (or = 4.90, 95% ci 1.26, 19.13, p = 0.02) and small cigar (or = 2.98, 95% ci 1.19, 7.42, p = 0.02) smoking were significantly associated with an intention to smoke executive branch small cigars. exposure to snoop dogg 's advertisements was marginally associated with intention to smoke executive branch small cigars (p = 0.08). given the exploratory nature of our study, variables from the bivariate analysis that were significant at p < 0.10 were included in the multivariate analysis. respondents who reported current cigar smoking were five times more likely and those who smoked small cigars were three times more likely to be susceptible to smoking executive branch small cigars. respondents who reported seeing the artist 's advertisement for executive branch small cigars were three times more likely to be susceptible to smoking those small cigars than those who were not exposed to the advertisement. over half (53.7%) of our respondents intended to smoke other small cigars in the future bivariate analyses found that past 30-day cigarette (p = 0.06), cigar (or = 3.75, 95% ci 1.00, 14.00, p < 0.05), and small cigar smoking (or = 16.34, 95% ci 3.65, 73.18, p < 0.001) were significantly associated and exposure to other small cigar advertisements (p = 0.06) was marginally associated with the susceptibility to smoke other small cigars. table 2 shows the results of our multivariate model for susceptibility to other small cigars. past 30-day cigar and small cigar smoking were significantly associated with susceptibility to smoke small cigars in the future. exposure to other small cigar advertisements was not significantly associated with susceptibility to smoke other small cigar products, however. small cigar smoking is a growing public health concern among young adults, particularly for those with a history of tobacco use. among our sample of young adult cigarette smokers, over 25% concurrent use of both products is a public health concern, as it may increase young adults ' risk for developing nicotine dependence [2, 26 ], may lead to an escalation of tobacco use, and may make it difficult to quit smoking and achieve long - term abstinence. our preliminary findings suggest that exposure to small cigar advertisements may explain, in part, young adults ' awareness of small cigars. over 60% of our respondents reported exposure to other small cigar advertisements, and 14% reported exposure to snoop dogg 's executive branch small cigar advertisement. although traditional outlets such as magazines and convenience stores were also sources of advertisement exposure, respondents reported exposure to small cigar advertisements on the internet, particularly on social media sites. this is consistent with growing body of evidence that has found protobacco images and references [2729 ], in particular small cigar smoking [12, 30 ] on internet social media sites. we have little evidence to support the claim that the tobacco industry paid for or supported the hip - hop artist 's endorsement of this small cigar product. however, the federal trade commission report of 1999 documented the cigar industry 's payments to celebrities for their endorsements, appearances, and cigar product placements. it is well documented that cigarette smoking has been promoted through rap and hip - hop music [15, 31 ] and through music - themed campaigns (i.e., the kool mixx campaign by brown & williamson, and concerts and festivals such as camel 's smooth moves and speakeasy tours). rap and hip - hop music genres have a wide reach. hip - hop artists appeal to individuals across gender, nationalities (usa versus international), racial / ethnic backgrounds and ages (although as noted previously, to youth in particular). taken together, the artists ' endorsement of a cigar product may establish brand loyalty and influence the smoking behaviors of large segments of their fans. perhaps the use of celebrity rap and hip - hop artists to promote small cigar products and smoking is a strategy that is being used by the cigar industry to circumvent tobacco advertising restrictions. exposure to the hip - hop artist 's small cigar advertisements appeared to influence small cigar smoking susceptibility among the young adult cigarette smokers in our sample. those who reported exposure to the artist 's executive branch small cigar advertisements were three times more likely to intend to smoke these small cigars in the future. notably, over 40% of those who reported an intention to use the executive branch small cigars were already past 30-day small cigar smokers. although not previously documented for music entertainment, our findings are consistent with studies that found an association between exposure to prosmoking depictions in films and smoking behavior among adolescents. future studies that examine the association between exposure to small cigar advertisements and intention to smoke among other young adult smoking samples are needed to substantiate our findings. an important limitation of this study is its generalizability, as the survey sample largely consisted of a sample of young adult festival attendees in the united states. while we broadly assessed other small cigars, this study specifically examined exposure to snoop dogg 's advertisements and promotions of executive branch small cigars. as such though these items may estimate small cigar use more accurately, their use may also limit comparability to studies that used other measures to capture small cigar use (i.e., single finally, the items that assessed small cigar use in our study did not specifically ask if participants were smoking tobacco in the small cigar. despite the use of this item to measure small cigar smoking in other studies, its use may bias findings because it may also capture use of marijuana. thus our study documented young adult smokers ' exposure to a celebrity hip - hop artist 's advertising of executive branch small cigars. we also provided preliminary evidence of an association between exposure to a celebrity artist 's small cigar promotion and susceptibility to smoke the brand - specific small cigar. our findings indicate that celebrity endorsement is a potentially important source of marketing small cigar products to young adults and should be monitored. | small cigar smoking among young adult cigarette smokers may be attributed to their exposure to its advertisements and promotions. we examined the association between exposure to a celebrity music artist 's endorsement of a specific brand of small cigars and young adult cigarette smokers ' susceptibility to smoking that brand. venue - based sampling procedures were used to select and survey a random sample of 121 young adult cigarette smokers, aged 1835. fourteen percent reported exposure to the artist 's endorsement of the small cigar and 45.4% reported an intention to smoke the product in the future. the odds of small cigar smoking susceptibility increased threefold for those who reported exposure to the endorsement compared to those not exposed (or = 3.64, 95% ci 1.06 to 12.54). past 30-day small cigar use (or = 3.30, 95% ci 1.24 to 8.74) and past 30-day cigar use (or = 5.08, 95% ci 1.23, 21.08) were also associated with susceptibility to smoke a small cigar. an association between young adult cigarette smokers ' exposure to the music artist 's small cigar endorsement and their susceptibility to smoke small cigars was found. this association underscores the importance of monitoring small cigar promotions geared toward young people and their impact on small cigar product smoking. |
infections caused by opportunistic pathogens, such as yeasts, are becoming important reasons of morbidity and mortality because of alterations in the immune system and invasive hospital procedures 1. aids, organ transplantation, chemotherapy, invasive procedures and radiotherapy increased the prevalence of immunocompromised individuals and also diabetes mellitus, and the over use of extended spectrum antibiotics made an increment in these infections 2 - 4. infections due to yeast like fungi increase at intensive care units 5. in the last two decades yeast like fungi are the fourth agent in the blood stream infections. c. albicans, c.tropicalis and c. parapsilosis are the most common yeast like fungi causing blood stream infection 6. the aim of our study was to evaluate the distribution of yeast like fungi isolated from clinical specimens at tepecik education and research hospital infectious diseases and clinical microbiology department mycology laboratory which was recently opened. yeast like fungi were isolated from the various clinical specimens (wound, urine, blood, respiratory specimen) between 13.01.2010 and 19.08.2011 at tepecik education and research hospital infectious diseases and clinical microbiology department mycology laboratory. all the isolates were identified to species level by the germ tube test, chrom agar candida (salubris, istanbul, turkey), cornmeal tween 80 medium (salubris, istanbul, turkey) and carbohydrate assimilation profile using the id32c yeast identification system (biomerieux, france). these samples were received from intensive care units (icus), pediatrics, internal medicine, infectious diseases and clinical microbiology, urology and ear nose throat departments. the specimens consisted of 144 urine (42.7%), 155 blood culture (45.9%), 13(3.8%) respiratory specimen and 25 (7.4%)'wound. the isolated yeast strains were 130 (38.6%) c.albicans, 47 (13.9%) c.tropicalis, 96 (28.4%)c. parapsilosis, 25 (7.4%) c.glabrata, 13 (3.8%) c.krusei, 10 (2.9%) c.kefyr, four (1.2%) c. guillermondii, five (1.5%) c. pelliculosa, four (1.2%) c.dubluniensis, two (0.6%) c. famata, and one (% 0.3) c. lusitaniae. the distribution of yeast like fungi acoording to specimen type and various departments were shown in table 1 and table 2. candida are prevalent all around the world and cause infectios within a spectrum of noninvasive infections to invasive opportunistic infections. exogeneous infections are due to hands of hospital staff, contaminated biomaterials and catheters 7, 8. hematologic disorders, immunosuppresive therapy, bone marrow transfer, organ transplantation, use of extended spectrum antibiotics, radiotherapy, burns, and longer duration in intensive care units are the main risk factors 9. investigated the yeast like fungi isolated from clinical specimens between august 2003 - 2005 at mersin university hospital microbiology laboratory. 872 yeast like fungal strains from 471 patients and 811 clinical specimens were taken into the study. c. albicans was the most common yeast like fungi isolated in all clinical specimens and c. parapsilosis (51.8%) was the one in blood culture strains. there was a decrease in c. tropicalis. c. albicans was the most common isolated strain overall ; but there is a increase in non c. albicans especially in intensive care units 10. investigated 390 fungi like yeast isolated from intensive care units over a four year period. blood culture, tracheal aspirate and urine were the most common specimens and c.albicans was 53.3%, c. tropicalis 14.5%, c. glabrata 12.2% and c. parapsilosis 6.5%.detected. according to the authors c. albicans was the most common isolated strain and an increase in non albicans isolates such as c. glabrata and c. tropicalis was detected 11. evaluated the blood culture of twenty patients in one year period and the most common isolated strain was c. albicans 12. in our study the most frequently isolated strain from blood cultures was c.parapsilosis and c.albicans was in second place. these consisted of c.albicans (49%), c. parapsilosis (23%), c. tropicalis (14%), c. glabrata (12%), one c.guillermondii and one c. krusei. according to the authors the most common isolated strain was c. albicans and the most prevalent non c. albicans isolates were c. parapsilosis and c. tropicalis. they also think that when considering candidemias, studies evaluating predisposing factors should be held in order to decrease morbidity and mortality and also to take the preventive measures 13. kocoglu. investigated the distribution and the antifungal susceptibility of yeast like fungi at gaziantep university hospital mycology laboratory from various clincal specimen in an one year period. the most common one was c.albicans (56.8%), secondly c.tropicalis (7.7%) and thirdly c. sake (6.8%). evaluated the newborn candidosis cases at the newborn intensive care unit between august 2005-january 2006 in italy. twenty two cases were investigated and the clonal spread of c. albicans isolates were shown 15. motta. investigated the distribution and the antifungal susceptibility of yeast like fungi isolated from blood cultures at a tertiary education hospital in brazil in 2006. the isolated yeast like fungi were c.albicans (52.2%), c. parapsilosis (22.1%), c. tropicalis (14.8%) and c. glabrata (6.6%), respectively. according to the authors candidemia incidence is high and the distribution of candida species and their antifungal susceptibility should be known 16. dimopoulos. evaluated the candidemia cases that took place after hospitalization at icu. 64.3% of the cases were c. albicans and 35.7% were non c. albicans 17. investigated the mucosal candida colonisation in 273 hiv seropositive patients in a two year period in iran. 273 oral and 86 vaginal specimens were examined. among these 50% c. albicans was isolated and this was followed by c. glabrata (21.4%), c. dubliniensis (13.3%), c. krusei (9.8%), c. kefyr (3.1%), c. parapsilosis (1.6%) and c. tropicalis (0.8%) 18. investigated the ditribution and antifungal susceptibility of yeast like fungi isolated from blood culures during a ten year period in mycology reference laboratory in kuwait. c. albicans (39.5%) was the predominant strain and followed by c. parapsilosis, c. albicans and c. krusei. c. albicans, c. albicans, c.tropicalis and c. glabrata were all susceptible to amphotericin b. c. parapsilosis isolates were 2% resistant to amphotericin b. nine c. albicans strains were resistant to fluconazole. the authors think that the data in this study is similar to various other studies. although there is over use of fluconazole ve amphotericin b in clinical practice, there is not an increase in resistance rate 19. recently there is an increase in invasive infections caused by candida due to immunsuppresive individuals, patients with organ transplantation and hospitalization at icus. in this study, isolated yeast strains were 130 (38.6%) c.albicans, 47 (13.9%) c.tropicalis, 96 (28.4%) c.parapsilosis, 25 (7.4%) c.glabrata, 13 (3.8%) c.krusei, 10 (2.9%) c.kefyr, four (1.2%) c. guillermondii, five (1.5%) c. pelliculosa, two (0.6%) c. famata, one (% 0.3) c. lusitaniae and four (1.2%) c.dubluniensis. in our study the most common isolated strain was c. albicans and this is similar to most of the studies stated above. c. parapsilosis was the most isolated strain in blood cultures and this may be due to invasive procedures and the use of catheters. five c.pelliculosa isolates were identified from blood culture at the same department and this may indicate nosocomial infection due to application of invasive procedures in that unit. the prevalence of non albicans isolates c. glabrata and c. krusei were low. because of this situation azolles could be a choice in antifungal treatment. since our mycology laboratory has been recently developed, the greater number of received samples and identified candida species will help us to monitor and choose appropriate antifungal treatment at our hospital in the future. | objective : candida infections have increased due to transplant patients, prolonged icu stay and invasive procedures. the most common isolated strain is c. albicans. the aim of this study was to evaluate the distribution of candida isolates at tepecik education and research hospital.materials and methods : yeast like fungi were isolated between 13.01.2010 and 19.08.2011 at mycology laboratory. the identification was done by conventional methods and carbohydrate assimilation profile using the id32c identification system (biomerieux, france).results : yeast like fungi were isolated from 337 clinical specimens. they consisted of urine, blood culture, respiratory specimen and wound. the most isolated yeast strains were c.albicans (38.6%), c.tropicalis (13.9%), c. parapsilosis (28.4%), c.glabrata (7.4%), c.krusei (3.8%).conclusion : recently there is an increment in candida infections. in this study the most common strain was c.albicans and the rate c. glabrata and c. krusei isolates were lower than expected. c. parapsilosis was the most isolated strain in blood cultures and this may be due to invasive procedures and the use of indwelling catheters. |
asked how they make a decision, health professionals, either individually or as part of a multidisciplinary medical team, will often say something liketogether with the patient we look at the available options to see how well each performs on the main effect benefit, then take into account the side effect and adverse event harms, the burdens of the treatment and so on, finally weighing the benefits and harms and any other considerations to arrive at a conclusion as to the best option. we naturally bear in mind what the most recent relevant high quality guidelines have to say. together with the patient we look at the available options to see how well each performs on the main effect benefit, then take into account the side effect and adverse event harms, the burdens of the treatment and so on, finally weighing the benefits and harms and any other considerations to arrive at a conclusion as to the best option. we naturally bear in mind what the most recent relevant high quality guidelines have to say. a patient responding to the same question will probably come up with something similar, albeit expressed in different words such as taking the pros and cons into account ' and giving all the considerations due weight. clearly, these are not accurate characterizations of all clinical decisionmaking processes, but would seem to be reasonably descriptive of many. more importantly, they would certainly be common responses when the prescriptive question is asked : how should a clinical decision be made? these sorts of statements indicate that we operate in a healthcare system where some form of shared decision making is accepted as the aim. the majority of health professionals routinely talk the talk of informed choice and patientcentred care, increasingly emphasizing patientimportant outcomes as promoted by the grading of recommendations assessment, development, and evaluation (grade) collaboration1 and the newly established patientcentered outcomes research institute (pcori) (http://www.pcori.org) among many other individuals and groups. they do so with genuine conviction and intent, but find it more difficult to walk the walk2, 3 and even to agree on what the key steps should be in terms of pace, direction and support. the presence of cultural and socioeconomic variations, together with great individual heterogeneity within cultures and classes, is at the heart of the challenge posed in pursuing shared decision making (and informed choice) within an overall philosophy of person and patientcentred care. the challenge to the professionals is mirrored by that of the individuals with whom they engage. all parties lack a simple and generic way to picture and communicate about the decisions that need to be made in health care. we seek to address this major handicap to progress towards all three goals. for convenience, the discussion is focused on the encounter between individual clinician and patient, but we regard our proposal as applying beyond the microclinical setting, to the meso and macrolevels of healthcare decision and policymaking. as it takes some form of argumentation conducted in words, even if it refers to numbers as inputs, we feel an appropriate shorthand term for it is verbal multicriteria decision deliberation (mcdd). it embraces all forms of decision making that occur through deliberative processes, including those which are based on decision aids and support grounded in descriptive theories of human decision behaviour, usually involving descriptive theories of expert decision making.4 it dominates recent work in relation to shared decision making and patientcentred decision support.5, 6, 7 mcdd is a useful term because it highlights the key similarities and differences with the alternative decision (and decision support) technology that we argue should be included in the portfolio of clinical decisionmaking competencies of both health professionals and patients. this alternative is based on the wellestablished, theoretically grounded, prescriptive technique of multicriteria decision analysis (mcda).8 to make the comparison with verbal mcdd even clearer, we can imagine the adjective numerical preceding it. in short, we are suggesting, along with dolan,9 van hummel and ijzerman10 and liberatore and nydick11 that numerical mcda (hereafter simply mcda) be added to the competency portfolio of all those involved in clinical decision making. we regard their studies as establishing that mcda based clinical decision support systems can be successfully developed and deployed. however, despite the high quality of the efforts of these researchers, the implementation of mcdabased decision support in health care has been fairly limited not that the success of mcddbased aids in routine practice has been spectacular to date.12, 13 the reasons for this undoubtedly include the usability and communicability of the current software implementations of mcda technique, computerization being a necessary condition for its application, in contrast to mcdd. in this respect, we believe it reasonable to infer that an implementation which is superior in these respects will be more successful and can be regarded a priori as a workable clinical decision support system. but the reasons also trace back to the fundamentally different theoretical paradigm from which mcda itself emanates, compared with that underlying current clinical practice and the majority of decision aids built for use within it (a comprehensive inventory of patient decision aids is available at http://decisionaid.ohri.ca/index.html). there are two key similarities between the two broad modes of multicriteria decision making, the umbrella term. first, both imply that in every clinical decision, two sorts of judgement are needed : (i) on the performance of each of the available options on each of the multiple relevant considerations and (ii) on the relative importance of those multiple considerations. second, that these conceptually different types of input must be integrated / synthesized / combined in some way to arrive at a decision. the key differences are reflected in the final words of the labels deliberation and analysis and in the preceding, implied adjectives verbal and numerical. (in this paper, we include a graphical representation of data within the scope of the latter term.) it might be asked why we characterize the distinction as a verbal / numerical contrast, rather than a qualitative / quantitative one. we do so because it is crucial to accept that mcdd is replete with the quantification of magnitudes. this quantification is simply done in predominantly verbal ways during the decisionmaking parts of the discourse. this applies in relation to the performance magnitude judgements, for example, of different medications reducing the chance of pain, where terms such as low probability, good chance and very likely are used to characterize the chances of the criterion being met for this patient. it also applies to the relative importance judgements, for example, of the importance of pain reduction relative to medication side effects, where again a variety of terms such as paramount, trivial and major or simply very important and not very important are deployed. the word analysis is used because the explicit aim in mcda (and in fact of any version of decision analysis, including its costeffectiveness and costutility forms) is to arrive at a result an opinion is our preferred term by a process of analytical calculation on the basis of numerical judgments. of course, the process of arriving at those numerical judgements almost certainly involves extensive verbal, nonnumerical elements and hence deliberation, in the same way that the deliberative discourse of mcdd may contain many judgments of magnitudes, including some expressed numerically. deliberation on the other hand is an interpersonal process where the provenance of the emerging conclusion inheres in the social process adopted and the participants involved in it. unless the deliberation is structured as an mcda,14 the conclusion can not be detached from them in the form of a graphic summary, or equation, or set of numerical option scores. a process that would benefit from the perceived strengths of each approach is an attractive prospect, and such a hybrid form has been implemented by proctor and drechsler in the context of environmental policy formation.14 a stakeholder jury was used to structure an mcda through a deliberative process and populate it with the help of experts. mcdd then followed as the final stage. the extensive time and resources involved, as well as the environmental policy context, make the empirical conclusions of limited relevance, but the hybrid case is well made in general. however, such a hybrid involves compromise from both sides, and this is not easy once it becomes clear that paradigmatic principles are involved, not merely syntactic or semantic differences that can be addressed by this means establishing whether improvements can be made, identifying where improvements could be made and providing support that will lead to improvements. purely descriptive approaches, which focus on describing how decisions are made by individuals or by organizations, communities and other groups provide no basis for change as they have no basis for identifying what would be an improvement.15 purely normative approaches, which focus on establishing, without reference to how decisions are made, the fundamental principles and processes that an ideal decision maker would implement, are simply impractical. on what basis can such desirable, potentially decisionimproving prescriptions for decisionmaking processes be identified ? decision analysis is essentially the ideal processes of decision theory converted into processes that are practical, given the time, resource and cognitive constraints of the real world. lipshitz and cohen4 call prescription arrived at on this basis analysisbased prescription, and this is exactly what we mean when we say mcdabased decision aids are ' prescriptive '. they produce an opinion which reflects, as closely as practicable for many reasons this may not be very close at all the logical processes of an idealized decision maker. interestingly these principles and processes have been endorsed by many people if they are asked how a decision should be made, even when they do not follow the principles and processes themselves.4 the other possible basis of prescriptions for improvement in decision making is description of the decision processes of expert decision makers, on the one hand, in contrast to nonexpert decision makers on the other. identifying the differences between them what makes the experts expert can lead to what lipshitz and cohen call expertisebased prescription. despite the accuracy of this term, in the world of decision support the term those who favour the expertisebased approach prefer to characterize themselves as operating within an descriptive approach, which in many ways is true, even though, by definition, prescription is necessary to distinguish experts from nonexperts and good from notgood results.16 expertisebased description / prescription has been virtually the only route to improvement in decisions considered professionally acceptable in clinical medicine, and this is reflected in the curricula of medical schools and in clinical practice. decision analysis is rare to nonexistent in both the curricula of medical schools and in clinical practice. it is also the basis for the regular attacks on the expected utility / value principle which underlies analysisbased prescription. these critiques, most recently that of russell and schwartz,17 are always derived from the descriptive inadequacies of the expected value principle. but such inadequacies are ultimately irrelevant within a prescriptive paradigm because it is not derived from actual behaviour. as it is hard to conceive of unbiased crossparadigm evaluation, it is not surprising that this is never proposed in such critiques, which ultimately reflect the intuitive appeal of descriptive approaches that seek to take into account the complex characteristics, history and contexts of the individual. the issue is not whether these inadequacies and complexities exist are descriptively true but whether a user would prefer to be supported by analysisbased prescription or expertisebased description. the ethical responsibility is to make clear the paradigmatic origins of the type of support offered. the analysisbased prescriptive approach has one compelling advantage in the provision of patient / personcentred care and genuinely shared decision making. in its multicriteria form, decision analysis provides a generic approach to all decisions, that is, it is not condition specific and does not mandate the reasoning expertise and knowledge acquisition in the particular area (e.g. a disease) required to follow and share expertisebased prescriptions. as long as expertisebased prescription is the sole basis of the clinical encounter, patient empowerment will be a very difficult and demanding task. an mcdabased prescriptive approach allows the person / patient to input their preferences as importance weights for criteria in a straightforward manner and to have them transparently combined with the published evidence and the clinician 's expertise. this paper focuses on mcda as an appropriate technique for facilitating personcentred health care in relation to the adoption decision deciding what to do given the available options. it is important to distinguish this decision from two other decisions where we also regard mcda as an appropriate support technique. one is the quality decision deciding how good the decision just taken was, given the decision technology used. an mcdabased instrument for measuring decision quality has been developed and is presented in j. dowie, m. kjer kaltoft, g. salkeld and m. cunich (submitted). the other is the decision decision deciding how to decide, given the available decision technologies. in our case, the decision decision is whether the adoption decision is to be made by the exercise of the health professional 's clinical judgment, by some form of mcddbased decision making, or in conjunction with some type of mcdabased decision support ? meta question later. a great number of software implementations of mcda exist, reflecting both widely varying versions of the technique and particular judgements about the extent and type of complexity to be catered for and the time and cognitive resources required.18, 19 these range from implementations of a smart (simple multi attribute rating system) in a simple spreadsheet, implementations using the analytic hierarchy process (ahp) as executed either in a spreadsheet template or a dedicated software package, notably expert choice, http://expertchoice.com/products-services/expert-choice-desktop/, to specific mcda implementations such as v.i.s.a http://www.visadecisions.com, hiview http://www.catalyze.co.uk/index.php/software/hiview3/, webhipre http://hipre.aalto.fi/ and logical decisions http://www.logicaldecisions.com/. the latter two packages also contain an ahp option. the prime motivation for developing an mcda decision support system in the form of annalisa was that none of the existing implementations of mcda had, despite proving themselves as clinical decision support systems, made significant progress in health care. that was the situation when annalisa was first conceived and we feel it remains true now, despite the growing research in this area (see dolan,9 maarten ijzerman and colleagues,20 and other examples cited in the liberatore review).11 most of the increasing use of mcda in health care is at the policy and health technology assessment level, with the recent developments within the evidem framework and software in the forefront21, 22 confined to this setting. in deliberately implementing the simplest, compensatory however, annalisa seeks to provide enhanced interactive online usability by way of the numerous customizing and personalizing functionalities provided in the survey program elicia, into which the annalisa file is normally embedded. for example, annalisa enables personalization of the performance ratings of options on criteria on the basis of patient characteristics and personalization of the weightings of the criteria by the patient at the point of decision. thus, the focus of this paper is not to reintroduce mcda or confirm its value as the basis for clinical decision support systems, but to introduce a particular software template, annalisa, as a practical and personcentred implementation for use at the individual level, not only in shared decision making, but also in the community, especially in relation to cancer and other disease screening decisions and policies. the ahp has been the mcda implementation used most widely in the clinical healthcare context and warrants special mention. in an extensive series of papers, james dolan has expounded and investigated the ways it can contribute to both shared decision making and the wider issues involved in clinical decision support.8, 9, 23 however, in its standard form, ahp involves a level of complexity that imposes high demands on both the developers and implementers / users of ahpbased support systems. primarily responsible for this increased complexity is the hierarchical attribute structure which ahp permits and indeed encourages (hence its name) and the unique pairwise comparison method used to establish criteria importance weights and performance ratings, devised by its founder, thomas saaty.24 while this increased complexity can be seen as leading to a high level of performance by some standards of normative rigour and comprehensiveness,25 it creates the difficulties in the development and delivery of ahpbased decision support26 that have hindered its wider dissemination. we use them, as summarized by riabacke.,28 to highlight the basis of the claims of annalisa in each respect. all the implementations of mcda mentioned in this paper embody some version of the weightedsum principle which is at the centre of decision theory. annalisa implements this basic principle in a very simple way while still retaining its key principles. feasibility. the template was explicitly designed to reduce the complexity and resulting cognitive demands that is possible and tends to be facilitated by increasingly sophisticated implementations within the other available packages. no provision for sensitivity analysis is built into annalisa, but both the weightings and ratings can be directly varied and the effect on scores instantly observed. the weightedsum principle is not only explained and illustrated prior to the user 's interaction with annalisa, but also during interaction where appropriate, for example when and whether the individual panels (weightings, ratings, scores) are opened or closed at different stages during engagement. this criterion relates to the model developed in the software rather than to its basic functionalities. it is up to the user of annalisa to develop a model relevant to their context and setting. the embedding of annalisa in the online survey program elicia makes interactivity and cyclical iteration simple and efficient and leaves the amount of each entirely in the user 's control. all the implementations of mcda mentioned in this paper embody some version of the weightedsum principle which is at the centre of decision theory. annalisa implements this basic principle in a very simple way while still retaining its key principles. feasibility. the template was explicitly designed to reduce the complexity and resulting cognitive demands that is possible and tends to be facilitated by increasingly sophisticated implementations within the other available packages. no provision for sensitivity analysis is built into annalisa, but both the weightings and ratings can be directly varied and the effect on scores instantly observed. the weightedsum principle is not only explained and illustrated prior to the user 's interaction with annalisa, but also during interaction where appropriate, for example when and whether the individual panels (weightings, ratings, scores) are opened or closed at different stages during engagement. this criterion relates to the model developed in the software rather than to its basic functionalities. it is up to the user of annalisa to develop a model relevant to their context and setting. the embedding of annalisa in the online survey program elicia makes interactivity and cyclical iteration simple and efficient and leaves the amount of each entirely in the user 's control. the final point requires further development in the light of the increasing interest in the comparison and evaluation of decision aids on the basis of multiple criteria relating to development, performance, accessibility and impact. within its essential prioritization of simplicity over complexity, the functionalities provided by the annalisainelicia software create the possibility of building decision aids that should perform well on most criteria of adaptability and personalization. according to the eiring. coding scheme for the personalization of decision aids,29 the basic components of personalization are media content, user features, user model construction and representation and adaptive system behaviour. user features can broadly be classified into the users knowledge level, interests, preferences, goals / tasks, background, individual traits and context. adaptive system behaviours include adaptive navigation support, adaptive selection, organisation and presentation of content, adaptive search, adaptive collaboration and personalized recommendations. used in conjunction with elicia, any implementation of annalisa should provide a medium to high degree of personalization in all these respects and hence compare favourably with the 10 of the initial 259 decision aids that were subject to detailed classification in the eiringled study. it is up to the developer of a decision support tool within this software to determine what degree and type of adaptational flexibility and personalization to individuals or groups is to be offered in terms of attributes (such as content, language, connectivity and presentation). it is also up to the developer to determine whether these are to be provided on an optin or optout basis. while compared with some ideal decision aid there are limitations in all these respects, a tool built in the annalisa software is capable of technically matching, or possibly surpassing, any of the actual decision aids subjected to intensive analysis in the eiringled project. most differences that arise will not be for reasons of functionality, but be traceable to their mcda basis, because this is what influences what is offered and required and how it is offered and required by way of adaptation and personalization. this point is worth emphasizing because it is important that a particular decision aid is assessed as an implementation of the underlying technique and philosophy within which it is built. a tool using annalisa is based on the paradigm of analysisbased prescription, as opposed to that of expertisebased description within which all the other aids examined by eiring. what is paramount in the development and use of a particular decision support tool or system is to make the potential user very clear about its underlying paradigm, so that questions concerning lower levels of functionality are relevant to it. the appropriate evaluation of annalisabased aids is therefore in comparison with other mcdabased ones and such a comparison, involving annalisa and ahp (using expert choice), carried out within hiview, is that has been undertaken by pozomartin (personal communication). this makes the 2005 french and xu30 survey of the five mcda packages cited earlier hiview, v.i.s.a, webhipre, expert choice and logical decisions a survey of current decision analysis software, including full technical and operational details, is provided biennially by informs. fifteen packages offer some form of multicriteria da, but this is a purely descriptive listing of information provided by 24 vendors, with no comments or assessments added. french and xu compared the five programs in terms of the aspects in which they differed, notably (using annalisa terminology) decision structuring, weighting elicitation, rating elicitation, data presentation and sensitivity analysis. when annalisa, in conjunction with elicia, is added to the comparison, two things stand out. first, the package fails to provide the vast majority of the functionalities and features that these five offer, considerably augmented in the 8 years since the survey. these functionalities and features are entirely appropriate where complex analysis is of benefit, such as in major projects with numerous stakeholders involved and large amounts of resources used, but even in such contexts the limited use of these packages either in practice or as the basis of decision support templates is noteworthy. and even where used, the complexity of the analysis is rarely matched in, or warranted by, the extensive deliberation that follows, as exemplified in a swedish exercise in participatory democracy.31 somewhat paradoxically it is the failure of annalisa to provide alternative and/or more sophisticated and complex methods for key tasks (including determining the criteria and eliciting weights) that we regard as its positive virtue, because it will provide the potential for much wider use. the growth of product comparison websites and recommender systems within ecommerce32, 33, 34, 35 is a clear sign that multicriteria analysis is eminently accessible to large sections of the population, but only at an appropriate level of complexity. william buxton has pointed out that the speed of technological progress captured in moore 's law (a technology generation is 18 months and decreasing) is in complete contrast to his god 's law, which states that the capacity of human beings is limited and does not increase over time our neurons do not fire any faster, our memory does n't increase in capacity, and we do not learn or think faster as time progresses.36 the problem this creates for the evaluation of innovative and disruptive systems of all kinds is succinctly captured in martin buxton 's law it is always too early [for rigorous evaluation ], until suddenly it 's too late.37 the second difference is closely associated with the pace of change in both the hardware and connectivity within which any mcda software will operate. both french and xu and an earlier study by belton and hodgkin in 199938 saw three main settings for use of an mcda package : doityourself use by a single individual, an analystfacilitated group meeting, and offline analysis by a consultant sandwiched between facetoface meetings with decision makers. subsequent developments in communication technology and connectivity means that there are now many more possibilities, including one in which a prestructured (options, criteria) and evidencepopulated mcdabased decision aid is made available online. decision makers then need only to have their preferences (utilities, importance weights) elicited to obtain an opinion on the merits of the alternative options. in terms of data presentation and display, as well as interactivity, annalisa benefits from being developed specifically for online use within the latest technology. now written in html5, with tablet use prominently in mind, it has mobile presence on touchoperated devices using ios, android and other operating systems. such interactive mobile accessibility is likely to trump most other functional considerations in the coming years, making a level of complexity compatible with such operation a paramount consideration. annalisa was designed to embody the following practical principles : it should be possible to undertake an analysis within a very short time, such as the 510 min often available in time / resource pressured situations, to ensure that the possible benefits of even a modicum of slow thinking should not be lost.39 this was in no way, of course, intended to prevent weeks or months being devoted to generating the detailed structure and inputs if the time and other resources are available.irrespective of the time available at the point of decision (and therefore including 510 min), the decision owner should not be asked to make the necessary tradeoffs among more than 7 2 criteria.40, 41 (annalisa actually has a maximum of 10.)all the elements of the decision (preferences and evidence) and the outcome (best option) should be simultaneously visible on the screen, providing a complete picture of all elements of the decision and with the effects of changing any weighting or rating dynamically visible in real time.popups on the screen should provide access to additional information, especially the provenance of the option performance ratings (including external links where appropriate). it should be possible to undertake an analysis within a very short time, such as the 510 min often available in time / resource pressured situations, to ensure that the possible benefits of even a modicum of slow thinking should not be lost.39 this was in no way, of course, intended to prevent weeks or months being devoted to generating the detailed structure and inputs if the time and other resources are available. irrespective of the time available at the point of decision (and therefore including 510 min), the decision owner should not be asked to make the necessary tradeoffs among more than 7 2 criteria.40, 41 (annalisa actually has a maximum of 10.) all the elements of the decision (preferences and evidence) and the outcome (best option) should be simultaneously visible on the screen, providing a complete picture of all elements of the decision and with the effects of changing any weighting or rating dynamically visible in real time. popups on the screen should provide access to additional information, especially the provenance of the option performance ratings (including external links where appropriate). giving higher weight to practical considerations, annalisa adopts the simplest and most colloquially familiar form of mcda. in the decision matrix weightedsum approach, all attributes exist at the same level (there is no hierarchy of criteria and subcriteria) ; the performance of each option is directly rated on each attribute ; the importance of each attribute is directly weighted in relation to that of all the other attributes ; and the option scores are calculated by summing an option 's ratings on the attributes multiplied by the attribute weightings. an illustrative example of a completed annalisa screen is provided in figures 1 and 2. these might be seen as either those for two different patients or those of the same patient at two points of time (where fig. 2 is produced at the next encounter i.e. time 1). in the ratings panel of both instances, we can see that new treatment is better at maximizing the main effect benefit than current treatment (0.70 vs. 0.50), is better at minimizing the treatment burden than the current treatment (0.80 vs. 0.70), but is worse at minimizing side effects (0.20 vs. 0.50). the two are equally good in relation to minimizing adverse events (both 0.90). given the relative weightings of the four attributes in fig. 1, new treatment emerges with the highest score in a simple expected value calculation.score for current treatment=(0.500.50)+(0.500.30)+(0.900.10)+(0.700.10)=0.56 score for new treatment=(0.700.50)+(0.200.30)+(0.900.10)+(0.800.10)=0.58 figure 2 presents the scores when the weight assigned to minimizing side effects harm is increased, with correspondingly reduced weight to maximizing main effect benefit. current treatment now has the highest score, which means we interpret this option as the opinion emerging from the annalisa. from wherever and however they are derived, both the ratings and the weightings entered into annalisa are treated as measures on a ratio scale running from a (true) zero to 1 or 100%. zero on the ratings scale means either zero probability (literally, and in many case logically, no chance) or zero fulfilment of the attribute concerned ; 1 means 100% probability or complete fulfilment. similarly, zero on the weightings scale means of no importance whatsoever, and 1 means all important to the exclusion of all other attributes. the choice of the simple weightedsum approach, among many other decisions in the design and development of the annalisa template, was made in the light of our value judgments as to the weight to be assigned to particular considerations. preeminently, we have assigned high weights to relevant practical considerations in both development and delivery, in full recognition and awareness that these may lead to poorer ratings on other criteria, preeminently ones concerned with normative rigour. we do not see rigour / relevance and practicality / normativity as dichotomies, where one must make binary choices, but as matters of weighting and hence preference sensitive. annalisa, as with any implementation of mcdm including mcdd, embodies a particular view as to the criteria and weights to be used in deciding how to decide. this point continues to apply even when choosing among the candidates within the field of mcda. given that a patient faces multiple options and regards multiple criteria as relevant to choosing among them, should they stick with mcdd, the currently dominant decision technology, or move to mcda, at least as a decision support technology ? as discussed earlier, the two basic forms of mcdm and their many internal variations differ in important ways, as well as having key similarities. but from the clinical decisionmaking standpoint, we should not be thinking of making a choice between them at some general and abstract level. it hardly makes any sense to ask whether mcdd or mcda is better in general as a technique. neither is it particularly useful to ask whether ahp / expert choice or weightedsum / annalisa is a we need rather to focus on particular instantiations of each technique template in a tool for a particular clinical decision setting, where setting embraces such things as the organization, the professional, the patient and the condition involved. these criteria will probably include such higherlevel considerations as evidential strength and coverage, theoretical grounding, explicitness, precision, transparency, communicability and potential for social or institutional biasing. but given that this is clinical decision support, they should also include the basic resource requirements, such as the time and cognitive effort and commitment required from all parties, as well as any financial implications for them. it can be taken for granted that the performance of particular implementations of mcdd and mcda will vary on these criteria, not least because of conscious value judgementbased tradeoffs regarding the selection and weighting of the criteria made by individual parties in the case of mcdd and by the developers and implementers in the case of mcdabased decision support. for example, an mcdabased aid or mcddbased appointment designed to take no more than 20 min will (should) perform less well on a criterion such as coverage of the evidence than one assumed to have 40 min at its disposal. the various interactive decision support systems we are developing all allow customization of the support process to the time and other resources available, as well as personalization of the weightings by, and ratings for, the specific patient on the selected criteria. they explicitly assume, indeed emphasize, that such customization choices will impact on which aspects of the decision support will be accessed and that the personalization of weightings will affect the outcome (opinion) emerging from the analysis. thus, given that the decision on what decision procedure or decision support system to adopt involves multiple criteria and is therefore preference sensitive, it does not make sense to ask whether annalisa has, or ever can be, shown to work in some overall or average sense as the basis of a clinical decision support system. the answer will vary as a function of the particular decision maker 's preferences in the particular context as well as the quality of the instantiation. empirically, we can note that in a study with a small number of australian gps, 80% agreed that the demonstrated annalisabased tool for prostate cancer screening would be useful in discussions with their patients and half thought it would be useful and could be recommended for use in decisions on any health matter.42 pozomartin has recently established the preference sensitivity of decision support evaluation in a comparison of annalisa and the analytic hierarchy process for developing and delivering decision support for patients with advanced lung cancer in some spanish hospitals.43 finally, while it is not appropriate to report the full set of results of a rct involving annalisabased decision aids for psa testing here, table 1 provides information on the age of participants and their ratings on various criteria, such as difficulty in responding to the key items on criteria weighting, that confirm its accessibility. age of participants and individual ratings on criteria relating to usability of annalisa decision aids for psa testing given the resource requirements of decision making and decision supporting and hence their opportunity costs we suggest there is a strong case for deciding how to decide being approached analytically as an exercise in decision resource decision effectiveness analysis. this simply parallels in relation to the decision decision (should we adopt this or that way of deciding whether, for example, to adopt this new drug or device technology ?) the use of conventional costeffectiveness analysis in relation to the adoption decision (should we, for example, adopt this new drug or device technology or not ?). as implied above, both numerator and denominator in decision resourcedecision effectiveness analysis are appropriately conceptualized as multicriterial indexes. a template designed to facilitate generic online multicriteria decision support in personcentred health care is presented in this paper as a valid addition to the portfolio of decision support systems available to clinicians and their patients. it is essential that any comparative evaluation of decision support systems makes the theoretical basis of each aid and process very clear to all respondents and decision stakeholders. in the context of personcentred care, this comparison will involve multiple criteria, of which the paradigmatic basis of the aid or process is a crucial one. the choice will be preference sensitive, with the weighting sometimes leading to an instantiation of multicriteria decision deliberation emerging as the best way of deciding and at other times to an implementation of multicriteria decision analysis. ultimately whether annalisa and similar templates have a role to play in personcentred care the empirical question, which will need to be iteratively asked and reasked as technology and attitudes change, concerns the precise roles it can play in the increasingly complex world of translational health. the contribution of professor salkeld and dr cunich was supported by the screening and diagnostic test evaluation program (step) funded by the national health and medical research council of australia under program grant number 633003. jack dowie has a financial interest in the annalisa software, but annalisa and elicia are maldaba ltd. | abstractobjectiveto introduce a new online generic decision support system based on multicriteria decision analysis (mcda), implemented in practical and userfriendly software (annalisa).backgroundall parties in health care lack a simple and generic way to picture and process the decisions to be made in pursuit of improved decision making and more informed choice within an overall philosophy of person and patientcentred care.methodsthe mcdabased system generates patientspecific clinical guidance in the form of an opinion as to the merits of the alternative options in a decision, which are all scored and ranked. the scores for each option combine, in a simple expected value calculation, the best estimates available now for the performance of those options on patientdetermined criteria, with the individual patient 's preferences, expressed as importance weightings for those criteria. the survey software within which the annalisa file is embedded (elicia) customizes and personalizes the presentation and inputs. principles relevant to the development of such decisionspecific mcdabased aids are noted and comparisons with alternative implementations presented. the necessity to tradeoff practicality (including resource constraints) with normative rigour and empirical complexity, in both their development and delivery, is emphasized.conclusionthe mcda/annalisabased decision support system represents a prescriptive addition to the portfolio of decisionaiding tools available online to individuals and clinicians interested in pursuing shared decision making and informed choice within a commitment to transparency in relation to both the evidence and preference bases of decisions. some empirical data establishing its usability are provided. |
schizophrenia is a severe psychiatric disorder that affects 1% of the population worldwide ; it is characterized by a complex phenotype, including positive, negative, affective symptoms and cognitive impairments [1, 2 ]. schizophrenia psychosis usually occurs in late adolescence and early adulthood between ages of 18 and 25, a key period for the maturation of the prefrontal cortex. a recent neurodevelopmental model of schizophrenia has proposed that an imbalance of excitation and inhibition in the prefrontal cortex is involved in the pathophysiology of schizophrenia. data from human and nonhuman primate brains have provided evidence of n - methyl - d - aspartate (nmda) receptor hypofunction in schizophrenia, contributing to the excitation of pyramidal neurons indirectly by reducing the activity of inhibitory -aminobutyric acid (gaba)ergic interneurons. the activity of gaba interneurons is mediated by nmda receptors, while glutamate (glu) serves as a precursor for the synthesis of gaba and glutathione [58 ]. gaba interneurons, in turn, project to pyramidal neurons and influence their excitation [911 ]. postmodern studies have further shown reduced synapse density in glutamatergic pyramid cells in schizophrenia, linking changes of synaptic plasticity to excitation - inhibition imbalance. excessive excitatory synaptic pruning in the prefrontal cortex may cause progressive brain tissue loss and lead to the development of psychosis. gaba activity may be decreased in certain brain regions in schizophrenia, which may lead to reduce cortical plasticity and abnormal pruning. using advanced proton magnetic resonance spectroscopy (h mrs), neurochemical concentrations such as glutamate and -aminobutyric acid can be measured in vivo [12, 1416 ]. there is increasing evidence from mrs studies linking glutamatergic or gabaergic disturbances to cognitive deficits and the pathophysiology of schizophrenia [15, 1721 ]. thus, studies on the imbalance between gabaergic and glutamatergic systems should shed light into uncovering the mechanisms of schizophrenia [11, 22 ]. aberrant gabaergic or glutamatergic levels in schizophrenia have been separately reported in previous studies [18, 21, 2331 ]. meta - analyses focusing on h mrs studies have shown glutamate alterations across several brain regions in schizophrenia [12, 16 ]. marsman. reported lower glu and higher glutamine (gln) levels in the medial frontal cortex in schizophrenia. merritt. further suggested higher glutmate+glutamine (glx) levels of the medial frontal cortex in subjects at high risk for schizophrenia rather than first - episode or chronic schizophrenia. higher glu and glx levels in the basal ganglia, gln in the thalamus, and glx levels in the medial temporal lobe were also found in schizophrenia. reduced glx or glu levels in the medial prefrontal cortex (mpfc) and anterior cingulate cortex (acc) were observed in several studies on chronic schizophrenia but could not be repeated by other studies [30, 32, 33 ]. the mixed findings on glutamatergic alterations could be due to the illness progress, severity, duration, or pharmacological treatment. further studies on the ultrahigh risk for psychosis (uhr) and first - episode schizophrenia (fes), in particular, including drug - nave subjects, are necessary to exclude the effects of medications. in vivo gaba measurements have recently become available, aided by newly developed mrs sequences, meshcher - garwood point resolved spectroscopy (mega - press), and j - point resolved spectroscopy (jpress) [34, 35 ]. gaba alterations in schizophrenia are also controversial [15, 21, 27, 28, 3639 ]. several studies have reported lower gaba levels in the acc, bilateral calcarine sulci, and mpfc in chronic schizophrenia [15, 38, 39 ]. however, unchanged or elevated gaba levels in the acc and mpfc have also been observed [15, 28, 37, 39 ]. three studies on drug - nave or medicine - free fes patients showed reduced gaba levels in bilateral calcarine sulci and the left basal ganglia [27, 36 ]. in addition, a recent uhr study showed higher gaba in the dorsal caudate and mpfc. therefore, more studies on gaba alterations in both drug - nave uhr and fes are needed. one possible reason for the controversial findings of either gaba or glu alterations in schizophrenia could be that these two neurotransmitter systems interact with each other [22, 4043 ]. recently, several researchers have begun to examine the correlations between gaba and glutamatergic metabolites [17, 19 ]. kegeles. found a strong positive correlation between medial prefrontal gaba and glx levels across chronic schizophrenia and healthy controls (hc). similarly, de la fuente - sandoval. also suggested significant correlations between these two neurotransmitters in both the mpfc and dorsal caudate across uhr and hc. in addition, these correlations could differ among various brain regions in the two groups. for example, uhr subjects had a positive correlation between gaba and glx levels in the mpfc rather than the caudate, while hc had a positive correlation between gaba and glx in the caudate rather than the mpfc. these results suggested that correlations between gaba and glx could be an important feature for dysfunctional gabaergic and glutamatergic neurotransmitters. thus, more attention should be paid to studies combining the gaba and glu alterations in both drug - nave fes patients and uhr subjects. this holds great promise for uncovering the impact of gaba and glu alterations on the onset and course of psychosis. in the present study, we recruited drug - nave uhr subjects, drug - nave fes patients, and hc subjects and measured both gaba and glx levels in the mpfc. the mpfc was selected as the region of interest because it has been widely implicated in schizophrenia, including by the postmortem and mrs studies mentioned [17, 19, 46 ]. we hypothesized that (1) medial prefrontal gaba or glx levels should change in gradient with illness progression, and these changes would be more prominent in fes patients than in uhr subjects ; (2) gaba levels should be correlated with glx levels in fes and uhr subjects, and the correlations may differ across the three groups. twenty - one uhr individuals were recruited from the outpatients in shanghai mental health center (smhc). all uhr subjects met the criteria for at - risk mental state, as defined by the structured interview for prodromal syndromes (sips) and scale of prodromal syndromes (sops) [47, 48 ]. the method for identifying uhr subjects with prodromal symptoms was introduced in detail in our previous study. all uhr subjects were drug - nave and completed their assessments on the day of their first visit to smhc. other inclusion criteria were being between the ages of 16 and 40 years and having at least nine years of education. exclusion criteria included current pregnancy, major medical or neurological illness, or a history of suicide risk or alcohol or drug abuse. the mean duration of prodromal symptoms in the uhr group was 10.93 16.40 months. was also obtained from his / her legal guardian if the uhr subject was younger than 18 years old. sixteen fes patients were recruited from smhc and met the diagnostic criteria for schizophrenia or schizophreniform psychosis based on the structured clinical interview for dsm - iv (patient edition). these fes patients visited our hospital seeking help for the first time upon being diagnosed with schizophrenia and recruited, and they completed their assessments on the day of their first visit to smhc. inclusion criteria for fes patients were having a first - episode illness, no history of exposure to antipsychotics, being between the ages of 16 and 40 years, and having at least nine years of education. exclusion criteria included a current pregnancy, major medical or neurological illness, or a history of suicide risk or alcohol or drug abuse. clinical symptoms of each fes patient were assessed using the 24-item brief psychiatric rating scale (bprs, expanded version) [50, 51 ]. written informed consent was also obtained from his / her legal guardian if the fes subject was younger than 18 years old. other inclusion criteria were being between the ages of 16 and 40 years and having at least nine years of education. exclusion criteria included a current pregnancy, major medical or neurological illness, and alcohol or drug abuse. written informed consent was also obtained from his / her legal guardian, if the hc subject was younger than 18 years old. the study was approved by the research ethics committee of smhc and in accordance with the declaration of helsinki. handedness was determined by self - report from each participant, and all subjects were right - handed (see table 1). mri data were obtained using a 3-tesla siemens verio mr scanner with a 32-channel head coil (siemens ag, erlangen, germany). anatomical t1-weighted images were acquired using a three - dimensional magnetic preparation fast gradient echo (3d - mrpage) sequence with echo time (te) = 2.96 ms, repetition time (tr) = 2300 ms, field of view (fov) = 240 240 mm, 256 256 matrix, a slice thickness of 1.0 mm, and 192 continuous sagittal slices. the t1-weighted images were used to localize the volume - of - interest (voi) for the following mrs acquisition. all scans were reviewed by a senior radiologist who evaluated whether there were obvious artifacts, signal losses, or gross pathology. the mrs data were acquired using a mega - press sequence with tr = 1500 ms, te = 68 ms, and 128 averages with water suppression. the voi (30 30 30 mm) of the mpfc was localized in the midsagittal and coronal slices, as shown in figure 1. the voi included brodmann areas 24 and 32 (containing part of the anterior cingulate cortex). automated shimming followed by manual shimming was conducted to reduce the water signal full - width at half maximum (fwhm) below 25 hz. mrs raw data were processed using the lcmodel software (version 6.3 - 0i). absolute gaba and glx concentrations were quantified using scaling correction, the correction for relaxation, and partial volume effects based on the lcmodel package and lcmgui, respectively. the edited spectra were fit using lcm - basis functions that were generated from phantom measurements using the mega - press sequence with the appropriate acquisition parameters. the gaba peak arose at 3.01 ppm and the glx peak at 3.74 ppm. the criteria for selecting reliable metabolite concentrations were based on the % sd of the fit for each metabolite, reflecting the cramer rao lower bounds (crlb) for the lcmodel analysis [28, 55, 56 ]. only the results we obtained high spectral quality, as the % sd of all spectra from the three groups was lower than 15%. to calculate the proportion of grey matter (gm), white matter (wm), and cerebrospinal (csf) contained in the voi, the volumetric 3d mprage mri data were segmented using the spm8 software. in - house software developed in matlab (mathworks natick, ma) was then implemented to create a segmentation mask for each voxel, from which the proportions of gm, wm, and csf were calculated. to obtain the tissue - composition - corrected metabolite intensities, each metabolite value was corrected for the csf content of the voi using the following formula : corrected metabolite level = uncorrected metabolite level/(1 c), where c is the fractional csf content of the voi. the normality for the distribution of all independent variables, including age, years of education, and metabolite concentrations, was examined using kolmogorov - smirnov tests. demographic variables, gm, wm, and csf volumes within the voi were compared among the three groups using analysis of variance (anova). chi - squared tests were performed to assess the group effect on gender and handedness. one - way anova with the factor of group (uhr, fes, and hc) was conducted to compare group differences on gaba and glx levels. post hoc tests were performed between each two groups with bonferroni corrections for multiple comparisons (p = 0.017 was the significant threshold level). age, education, gender, and handedness were all matched among the three groups. these three groups did not differ in their signal - to - noise ratio (snr) (p = 0.65) and fwhm (p = 0.97) from the lcmodel. no between - group differences were found in the proportion of gm (p = 0.83), wm (p = 0.85), or csf (p = 0.81) in the voi of mpfc (shown in table 2). there were significant main group effects on both gaba (p = 0.002) and glx (p < 0.001) levels. post hoc tests demonstrated significantly lower gaba levels in fes patients (2.27 0.56 mmol / l) than hc (3.04 0.69 mmol / l, p = 0.003) and uhr participants (2.96 0.75 mmol / l, p = 0.01). post hoc tests also revealed significantly lower glx levels in fes patients (12.75 2.66 mmol / l) compared with hc (16.57 3.17 mmol / l, p < 0.001) and uhr subjects (16.37 2.56 mmol / l, p = 0.001). however, both gaba and glx levels in uhr subjects were comparable with hc subjects (p = 1.00 for gaba, p = 1.00 for glx) (shown in figure 2). removing the four smoking hc subjects there were positive correlations between gaba and glx levels in each group (r = 0.53, p = 0.009 for hc ; r = 0.56, p = 0.008 for uhr ; r = 0.79, p < 0.001 for fes), as shown in figure 3. further direct comparison of the correlation coefficients with z - transformation between the three groups did not indicate any significant differences (z = 1.35, p = 0.18 for hc versus fes ; z = 0.13, p = 0.89 for hc versus uhr ; z = 1.21, p = 0.23 for uhr versus fes). in addition, the glx / gaba ratio did not indicate a significant main group effect (f = 0.11, p = 0.90 ; 5.63 1.21 for hc, 5.79 1.24 for uhr ; 5.73 0.88 for fes). the present study measured both gaba and glx concentrations among drug - nave uhr subjects, drug - nave fes patients, and hc subjects. significantly lower gaba and glx levels in the mpfc were observed in drug - nave fes patients than uhr or hc subjects, while gaba and glx levels in uhr subjects were comparable with hc subjects. our findings, which are not confounded by medications, suggested that reduced gaba and glx levels should play an important role in the early stages of psychosis. reduced medial prefrontal gaba concentration in fes patients indicates the existence of a dysfunctional gaba neurotransmitter system in the early stages of schizophrenia. in vivo measurements of gaba concentrations however, findings showing gaba alterations in fes patients are relatively limited and less consistent [27, 36 ]. kelemen. found lower gaba / cr levels in the bilateral calcarine sulci in drug - nave fes patients. lower gaba / cr ratios were also found in the left basal ganglia in early - onset fes and remained after six months of treatment with antipsychotic medications in another study. however, unchanged gaba / cr ratios have been revealed in both the frontal lobe and parietooccipital lobe. all these results suggest that alterations in gaba levels are related to the onset of schizophrenia but depend on the specific brain region and medication [27, 36 ]. our findings demonstrating reduced medial prefrontal gaba levels in drug - nave fes patients provide more evidence that gaba alterations have an important impact on the early stages of psychosis. deficits in the gaba neurotransmitter system in schizophrenia have also been proven by postmortem and electrophysiological studies [5759 ]. postmortem studies have demonstrated gaba deficits in a subclass of fast - spiking interneurons expressing ca - binding protein parvalbumin in patients with schizophrenia. reduced message rna and expression of gad67 (the synthetic enzyme for gaba) and gat1 (the transporter that clears synaptic gaba) as well as an apparent compensatory upregulation in postsynaptic gabaa receptors in the dorsolateral prefrontal cortex [57, 61 ], acc, and prefrontal cortex [45, 59, 62 ] electrophysiological studies have provided more evidence for the dysfunctional action of gabaergic transmission in schizophrenia reflected by deficits in gamma oscillation, p50, and the cortical inhibition index [6367 ]. reduced auditory p50 suppression has reflected disrupted inhibitory gating of the brain in response to repeated auditory stimuli in drug - nave fes patients [63, 65 ]. fes patients showed reduced short - interval cortical inhibition (sici) and a prolonged cortical silence period (csp) compared with healthy control subjects, suggesting weakened gabaa - mediated inhibition [64, 66 ]. all these findings further support the hypothesis that dysfunctional gabaergic transmission exists in fes patients [6367 ]. in the present study, low medial prefrontal gaba levels were only observed in fes patients not in uhr subjects. a positron emission tomography (pet) study demonstrated the reduced binding potential of gabaa / benzodiazepine receptors in the right caudate in uhr individuals. a recent mrs study indicated an elevated gaba / water ratio in the mpfc in uhr subjects. in our previous study, we found that gabab - mediated cortical inhibition was impaired in uhr, whereas gabaa - mediated cortical inhibition was not altered yet. whether gaba dysfunction has occurred before the onset of psychosis remains unclear. there was interplay between two gaba - mediated neurotransmitter systems (gabaa - mediated and gabab - mediated ones) [70, 71 ]. thus, there might be a compensatory effect between two gaba subsystems, leading to gaba deficits in fes patients rather than uhr subjects [64, 69 ]. previous studies indicated that fes patients showed a reduced sici and a prolonged duration of csp, implying both gabaa and gabab dysfunction, whereas uhr subjects only had a reduced sici or prolonged duration of csp, suggesting partially impaired gaba functions [64, 69 ]. thus, medial prefrontal gaba reductions could be a candidate biomarker for the early stages of psychosis. in addition, we found significant correlations between glx and gaba levels in fes patients, in accordance with previous studies [17, 19 ]. kegeles. reported a positive correlation between gaba and glx levels in the mpfc in medicated and unmedicated patients with schizophrenia and hc subjects. de la fuente - sandoval. found a positive correlation between gaba and glx levels across uhr and hc subjects. glu serves as a precursor for the synthesis of gaba and glutathione [58 ]. we hypothesized that the reduction of gaba levels might be secondary to a reduction in the glutamatergic system. there was also a significant correlation between gaba and glx levels in uhr subjects, and both metabolites remained unchanged. these findings suggest that the interplay of gaba and glx systems might act in a mutually compensatory manner. both impaired gabaergic and glutamatergic systems may contribute to the development of psychosis [25, 27 ]. there are limitations of the present study ; thus, we should be cautious when interpreting our results. first, mrs data were only acquired in one region (mpfc), which made our results less comparable with previous studies on other regions, for example, the striatum, which will be improved in our further work. second, we only recruited drug - nave subjects, which made our sample size for each group relatively small. considering that the heterogeneity of fes patients and uhr subjects may affect metabolic levels, further studies with a large sample size in summary, we found both reduced medial prefrontal gaba and glx concentrations only in drug - nave fes patients but not in drug - nave uhr subjects, suggesting that gaba and glx alterations may be associated with the early stages of psychosis. | altered -aminobutyric acid (gaba), glutamate (glu) levels, and an imbalance between gabaergic and glutamatergic neurotransmissions have been involved in the pathophysiology of schizophrenia. however, it remains unclear how these abnormalities impact the onset and course of psychosis. in the present study, 21 drug - nave subjects at ultrahigh risk for psychosis (uhr), 16 drug - nave patients with first - episode schizophrenia (fes), and 23 healthy controls (hc) were enrolled. in vivo gaba and glutamate+glutamine (glx) levels in the medial prefrontal cortex were measured using proton magnetic resonance spectroscopy. medial prefrontal gaba and glx levels in fes patients were significantly lower than those in hc and uhr, respectively. gaba and glx levels in uhr were comparable with those in hc. in each group, there was a positive correlation between gaba and glx levels. reduced medial prefrontal gaba and glx levels thus may play an important role in the early stages of schizophrenia. |
biological research in affective disorders has focused primarily on the neurochemical basis of the disorder. radiological techniques that allow investigation of the living brain in health and disease have become widely available. later, the more precise methodology of magnetic resonance imaging (mri) was applied with the ultimate aim of describing the structural and functional neuroanatomy in mood disorder.[810 ] although still controversial, convergence of mri and other findings from studies utilizing post - stroke depression and patients with primary mood disorder have led to attempts at detailed exploration of the neuroanatomical basis of mood disorders. studies using high - resolution mri are now available to examine smaller brain structures with precision, and they have reported brain changes associated with major depression in the hippocampus (hc), amygdala, caudate nucleus, putamen and frontal cortex. interestingly, neurobiological substrate for this disorder has not reached finality. the circuitry underlying the representation and regulation of normal emotion and mood involves the prefrontal cortex, anterior cingulate, hc and amygdala. the abnormalities in the structure and function of these different regions are implicated in depression. the clinical manifestations of depressive disorder are considered to be mediated through changes in brain neurochemistry and structural / functional connectivity, irrespective of the etiology. apart from the newer modalities of research like mri, evidence from electroencephalographic (eeg) as well has unequivocally established that mental disorder has definite correlates with brain dysfunction. pathophysiological concomitants of psychiatric and developmental disorders have been provided by eeg and quantitative electroencephalography (qeeg). the exclusion of neurological conditions for confirmation of psychiatric disorder has led to the application of eeg and qeeg investigation. the percentage is as high as 6468% of eegs in psychiatric patients providing evidence of pathophysiology, and these results have additional utility beyond simply ruling out organic brain lesions. such eeg studies may also aid in differential diagnosis, treatment patient selection and evaluation. a voluminous literature attests to the robustness of conventional eeg studies and their clinical utility in disorders of brain function, which includes depressive disorders. the conventional eeg has contributed valuable information for the psychiatrist ; however, this method is essentially based on visual pattern recognition. while conventional eeg provides reliable diagnostic information especially sensitive to organic or neurological disorders, detecting features of wave shapes, frequency relationships and transitions of state seldom encountered in the healthy individual, qeeg, in addition, enables precise comparison of the individual patient 's record with normative and psychopathologic patient databases. across both eeg and qeeg studies, a broad consensus exists on the high proportion of abnormalities found in different psychiatric disorders and often on their electrophysiological profiles. however, the generalization of these findings and application in clinical practice is limited by the non - specific nature of the detected abnormalities found in psychiatric patients. also, there has been considerable controversy about the clinical utility of qeeg in psychiatric practice. the incidence of abnormal conventional eeg findings in mood disorders appears to be substantial, ranging from 20% to 40%. specific patterns noted in mood disorder patients include the controversial small sharp spikes (sss), 6/s spike and wave complexes, and positive spikes, seen especially in patients with suicidal ideation. evidence that the eeg is abnormal in depression has been coming of late from qeeg studies, and these have been in the form of increased alfa and/or theta power in a high percentage of depressed patients. hence, both eeg and qeeg studies report that a high proportion of patients with mood disorders display abnormal brain electrical activity. eeg studies report that sss and paroxysmal events are often found, especially on the right hemisphere, and that abnormal sleep studies are common. eeg findings in studies of genetic unipolar depressives show that depressed persons display a disorganized atypical sleep pattern that skips a level of deep sleep and prominent delta and theta waves (which are sleep waves) in the waking state. therefore, we may conclude that the depressed person 's brainwave activity in sleep is invaded by waking although biochemical, pharmacologic and brain imaging techniques have all been used to shed light on the neurobiology of mood disorders, knowledge of the underlying pathobiology remains sketchy. studies using high - resolution mri have reported brain changes associated with major depression in the hc, amygdala, caudate nucleus, putamen and frontal region, structures that are extensively interconnected and comprise a neuroanatomic circuit that has been termed the limbic cortical using mri, schaefer demonstrated gray matter volume differences in the dorsolateral prefrontal cortex, a region consistently implicated in functional neuroimaging studies of affective disorders. volume reductions has been seen in the frontal cortex ranging from 7% overall reduction in the frontal lobe volume to 48% in the subgenual prefrontal cortex. in some ways, the most provocative data linking the hc to major depressive disorder (mdd) have been from mri studies of the volume of the hc in patients with mdd. volume loss in the hc is the most robust finding and the only change consistently observed to persist past the resolution of the depression. the importance of the hc in the pathophysiology of mdd has also been supported by a substantial body of evidence from basic and clinical studies. post - mortem studies have shown moderate apoptosis in the dentate gyrus and the ca1 and ca4 regions of the hc of patients with mdd. in most of the studies that assessed depression in unipolar subjects and used high - resolution mri techniques, other studies have reported that patients with depression have smaller left hc volumes than control subjects. at the same time, at least one research also found reduction of right hc volume in depression as compared with controls table 1 ]. hippocampal volume in depressed subjects appears to be predicted by the length of the illness and other variables associated with past burden of illness. investigations of patients with recurrent episodes have consistently indicated that there are structural changes in the hippocampal formation. moreover, sheline., in a study of middle - aged depressed women, found that hippocampal volume reduction was related to total lifetime duration of depression. this finding was recently replicated in a larger sample of patients, where they reported that past illness predicted hc volume reduction ; others reported that volumetric reductions were greatest in patients with a chronic course and large number of weeks ill than in those who recovered fully with shorter overall illness duration. in one study, hippocampal atrophy was found in patients with chronic depression but not in patients with remitted depression. some studies have reported no changes when depressed patients are compared with healthy controls.[4347 ] predominantly, evidence is suggestive of bilateral reduction in hc volume,[4853 ] and it is negatively correlated with duration of depression [table 1 ]. in the studies of depression in which hippocampal atrophy has been found, the implication is that excessively high levels of cortisol associated with the stress - related disorder cause hippocampal cell death and result in the hippocampal atrophy seen on mri. cellular studies of the hc in depression have revealed that volume reductions of the hc might be the result of remodeling of key cellular elements, involving retraction of dendrites, decreased neurogenesis in the dentate gyrus and loss of glial cells. this dysregulation of glucocorticoid secretion with increased activity of excitatory amino acid neurotransmitters could result in both potentially reversible remodelling and irreversible cell death in the hc of patients with mdd. it has, therefore, been proposed that raised cortisol levels during depression might be associated with cognitive impairments, especially in functions subserved by medial temporal lobe structures. a large body of evidence has also established a link between stressful life events and development or exacerbation of depression. at the cellular level, evidence has emerged indicating neuronal atrophy and cell loss in response to stress and in depression. at the molecular level, it has been suggested that these cellular deficiencies, mostly detected in the hc, result from a decrease in the expression of brain - derived neurotrophic factor associated with elevation of glucocorticoids. cognitive deficits in mood disorders have been addressed in different function domains, some of them being attention, executive function (ef) and memory. it is interesting to observe that cognitive deficits are noted even during the euthymic / remitted states, which indicates that certain cognitive deficits may be associated with trait characteristics. impairment of working memory (wm), sustained attention, abstract reasoning and visuomotor skills, verbal memory, verbal fluency and visuospatial ability have all been reported, even in the euthymic phase of the illness. the deficits have been shown to correlate with both the number of affective episodes and the overall duration of illness. the types of executive deficits seen in depression include problems with planning, completing goal - directed activities, organizing, initiating, sequencing, shifting, information processing speed, inhibiting context - inappropriate responses and maintaining information in the working memory.[6769 ] the presence of ef in depression is associated with vocational disability and possibly poorer treatment response. studies have documented ef in depression using the wisconsin card sorting test (wcst) as well as other such tests of cognitive dysfunction. lesion studies in animals and neuropathological reports in humans have shown that defects in the medial temporal lobe region, including the hippocampal formation, are associated with a severe and global amnesia. aside from its well - documented contribution to learning and memory, the hippocampal formation plays a critical role in the regulation of motivation and emotion. this contribution of the hc to emotion and affective style has only recently begun to be gleaned from the available corpus of animal studies on its role in context - dependent memory. other studies have attempted novel emotional modification of the wisconsin card sorting test and brought out a possible role of hc in context - dependant memory. the wcst may actually check this context - dependant memory, with its substrate in the medial temporal lobe, and show the impairments in depression. the causes and associations of impaired cognitive function during depression remain uncertain. studies in animals and humans, involving both exogenous steroid administration and conditions such as cushing 's syndrome and stress, which raise endogenous levels, have shown a relationship between raised cortisol levels and neuropsychological dysfunction, especially memory impairment. both glucocorticoid and mineralocorticoid steroid receptors are present in high concentrations in the hc, and prolonged and raised cortisol levels can produce neuronal dysfunction. it has been proposed that raised cortisol levels during depression might be associated with cognitive impairments, especially in functions subserved by medial temporal lobe structures, and that persisting hypercortisolemia might cause hippocampal damage, explaining why impairments persist in depressed subjects, even when affective symptoms have resolved. although executive deficits have been reported in more severely depressed subjects with melancholic or psychotic features, scores may be affected even with relatively mild depression and ef may vary as a function of the severity of depression. significant association between depressive symptoms and magnitude of wisconsin card sorting test deficits in major depression have been seen. studied wisconsin card sorting performance in a large group of patients with major depression who had been without medication for at least 28 days. they found significant deficits on various indices of the wisconsin card sorting task in these patients in comparison with controls. in patients with major depression, hamilton depression scale scores were moderately correlated with the number of categories achieved, number of perseverative errors, number of perseverative responses and the percentage of conceptual - level responses. patients with major depression made more perseverative and non - perseverative errors, took longer to reach the first category, completed fewer categories overall and had fewer conceptual - level responses and lower learning - to - learn scores than healthy subjects. other authors have also described a positive correlation with duration of illness as well as with recurrent episodes of depression. the speed of information processing and response latency is another area of concern in patients of depression. a study done by taj on patients of bipolar disorder in remission noted significant impairments in attention, executive function and memory in the study group compared with a matched control group of normal subjects. in fact, memory deficit pattern also did not differ in both unipolar and bipolar cases of depression [table 2 ]. however, cognitive deficits did present in both types of depression. further patients with somatic syndrome had slower psychomotor speed and less mental flexibility compared with the non - somatic syndrome group. there appears to be internal consistency in the findings in the domain of cognition and depression. patients with depression are noted to be having various types of cognitive deficits ; primarily, the affected area may be executive functioning and memory. major depression is a mood disorder that is often accompanied by the impairment of cognitive function. the severity of illness, duration of illness and other structural changes appear to be natural concomitants influencing the outcome of depression. the large range of existing neuropsychological, electrophysiological and, lately, neuroimaging investigations have provided evidence of structural changes and cognitive deficits in cases of depression. the severity and type of depression in respect of cognitive changes and anatomical substrate have not yielded very consistent findings. the evidence is suggestive of common tenet of derangement of cognitive functions in depression irrespective of the type of depression. however, much more exhaustive, explicit and specific data analysis is required to yield a definite conclusion on structural changes leading to cognitive deficits in the cases of depression. | the neuropsychiatric and cognitive deficits have been shown to exist in various psychiatric disorders. an attempt has been made by authors to evaluate the evidence pertaining to electrophysiological, structural and neuropsychological domains in depression. renewal of interest in testing patients with depression on a broad range of neuropsychological tasks has revealed distinct pattern of cognitive impairment in cases with depression. the review focuses on structural and neuropsychological evidence of deficit in cases of depression. |
in his book, beyond good and evil, friedrich nietzsche once wrote, beware that, when fighting monsters, you yourself do not become a monster (1). in addition to the vagaries of human behavior, the phrase can also be applied to the dual effects of the human immune system that a system highly evolved to protect the host from marauding pathogenic monsters can also be the instrument of its own destruction. the host damage - response framework captures this duality by asserting that microbial pathogenesis occurs along a continuum, wherein host damage can be a consequence of microbial virulence or the host immune response (24). in the setting of immunodeficiency, microbial virulence predominates, whereas during an effective immune response or after immune reconstitution, exuberant inflammation may contribute to excessive host damage. within the spatial confines of the central nervous system (cns), the host is particularly vulnerable to a robust host response that may lead to cerebral edema, restriction of blood flow, and hypoxia, with resultant brain damage, progressing to coma and possibly death (5). among studies of humans with cns infectious diseases, recent studies have identified host cellular and cytokine / chemokine patterns compartmentalized within the cns which are specific to the particular infectious disease syndrome. consideration of patterns of pathogenesis may facilitate development of syndrome - specific clinical strategies that may help improve outcomes for these high - mortality conditions. conversely, extrapolating therapeutics from one syndrome to another syndrome with different host responses may be unwise. in addition, patterns of immune responses to pathogens, heavily constrained by evolutionarily pressures, may give insight into autoimmune inflammatory disorders. for example, multiple sclerosis is one of the most common neurologic inflammatory disorders leading to permanent disability in young adults (6). recently, similarities between the inflammatory response of non - hiv cryptococcal meningitis and the progressive form of multiple sclerosis led to validation of a cerebrospinal fluid (csf) soluble cd27 (scd27)-derived tissue inflammatory biomarker and helped to characterize this important form of multiple sclerosis as an inflammatory, rather than a neurodegenerative, disorder (79). pathogens associated with nonviral cns infections are among the leading infectious causes of death worldwide. hiv, tuberculosis (tb), and malaria result in a large number of cns infections, and along with bacterial meningitis due to respiratory pathogens such as streptococcus pneumoniae they are among the top 10 causes of death (10). hiv / aids - related cryptococcal meningoencephalitis in sub - saharan africa is the leading regional cause of adult meningitis, with deaths nearing that attributable to tuberculosis in some studies (11). viral meningitides are also a major source of infective mortality and have been the subject of a number of recent reviews (12, 13), but they will not be discussed here. in the present review, we summarize and compare the findings and impact of the host response on the pathophysiology and disease severity of these generalized cns infections. cryptococcus spp. cause a severe and often fatal meningoencephalitis in persons living with hiv / aids, accounting for 15 to 20% of aids - related deaths and resulting in approximately half a million deaths annually (11, 14, 15). the fungus is an encapsulated facultative intracellular pathogen that causes a deep tissue meningoencephalitis emanating from the meninges and the virchow - robin channels surrounding penetrating vessels within the brain parenchyma (16). this extensive tissue penetration beyond the superficial structures of the meninges potentially exposes the cns immune system to a large fungal burden. in hiv / aids, susceptibility to cryptococcus is a result of defects in adaptive immunity, centered around quantitative and qualitative t - cell defects (17). residual immune activation is likely the result of antigen - specific and compensatory responses supported by interleukin-7 (il-7) and il-15 (18, 19), but it is insufficient to control the infection. prior to treatment with antiretroviral therapy (art), cryptococcal meningitis is characterized by high fungal burdens, suggesting a predominant role for fungal virulence. pathogen virulence continues to play a role after institution of antifungal therapy, suggested by an inverse association between mortality and rates of csf fungal clearance (20). in contrast, low levels of th1-defining responses, such as gamma interferon (ifn-) production, as well as poorly expressed macrophage - associated tumor necrosis factor alpha (tnf-)(table 1) (21, 22), suggest minimal roles for immune - mediated damage in the art - naive, hiv - infected host. a predominance of pathogen - mediated damage in art - naive hosts may also explain lack of improvement with adjunctive potentially immune - suppressing corticosteroids that seeks to control host - mediated immune damage (23, 24). host damage response to cns infection syndromes on the other hand, treatment with art leads to immune recovery, with ciris occurring in 15 to 30% of hiv - infected persons with cryptococcosis (25). this immune reconstitution syndrome is defined for cns disease as a paradoxical clinical deterioration in the setting of negative fungal cultures or other explanations in a patient with previously diagnosed cryptococcal disease after initiation of art (25). the recovering immune system encounters a large intracerebral fungal burden which persists despite antifungal therapy (26). additionally, with antifungal therapy, fungicidal therapy releases intracellular and cell wall antigens for innate immune activation normally protected from immune surveillance in intact organisms by a thick immunotolerant cryptococcal capsule (27, 28). prior to art, viral and fungal antigens result in primed macrophages that have not received cd4 t - cell help via ifn- signaling to become fully activated, thus resulting in a decoupling of the innate and adaptive immune systems (29, 30). the pre - art lymphopenic environment is also believed to alter the function of the remaining cd4 t cells, rendering them more pathogenic as the population expands after immune recovery (31). supporting this are findings from a study by chang. (32), who found that the risk of ciris was accentuated by low cryptococcus - specific cd4 lymphocyte th1 ifn- responses prior to art. however, data showing that higher cd8 t cells predispose to ciris prior to art suggest that the lymphopenic risk factor is specific to cd4 t cells (33). after art, expansion and activation of th1 cd4 subsets drive macrophage activation and inflammatory cytokines with prominent ifn- and il-6 production (table 1). for example, boulware. reported an elevated th1-type response in ciris with a 2- to 3-fold elevated intrathecal ifn-, tnf-, granulocyte colony - stimulating factor (g - csf), vascular endothelial growth factor (vegf), and eotaxin (ccl11) response but low ccl2 (34) and elevated serum inflammatory markers, such as il-6 and c - reactive protein (35). worsley. also reported a robust csf ifn- response during cryptococcal iris (36). these findings with ciris are similar to the t - cell activation of iris associated with a range of other conditions, such as tuberculosis - associated immune reconstitution inflammatory syndrome (tb - iris), in which increased frequencies of effector memory, hla - dr, and ki67 cd4 cells and higher serum ifn- production are reported (37). increased recruitment of mononuclear immune cells to the intrathecal space has also been implicated in ciris, based on elevations in monocyte recruitment, growth factors, and chemokines such as g - csf, vegf, and ccl11 (22, 33, 34). these responses are echoed by iris related to other infections, such as pulmonary and disseminated tuberculosis, with increased frequencies of cd14 cd16 classical monocytes in blood, associated with increased plasma levels of cytokines, including tnf- (38). more recent data for ciris have described cd14 cd16 classical subsets of monocytes in csf samples at baseline developing into a more proinflammatory intermediate phenotype (cd14 cd16) that produced radical oxygen species concurrent with ciris onset (39, 40). predisposition to ciris is also associated with higher csf ratios of monocyte - recruiting chemokines, such as ccl2::cxcl10 and ccl3::cxcl10 ratios, suggesting that more intact cell trafficking of partially activated macrophages prior to art initiation may predispose to future ciris once cd4 t - cell - mediated activation occurs (33). early initiation of art after treatment of cryptococcal disease results in higher levels of csf macrophage cytokines, such as scd14 and scd163, suggesting macrophage activation with art - mediated immune reconstitution (40). macrophage activation in ciris thus acts in concert with t - cell - mediated damage and results in multiple, potentially damaging inflammatory effects. in summary, these findings suggest that the coordinated response of a recovering immune system, confronting a large antigen load, results in activated t cells and monocyte axes (high th1-m1 in synchrony), leading to inflammatory damage (fig. 1, top panel). mechanisms of immune damage in the cns are an area of active study and may include induction of cerebral edema (41), direct neurotoxic effects from macrophages (42), or metabolic programming of neurons by adjacent inflammatory signals (43). these findings also support the adjunctive role of immunosuppressive therapies, including corticosteroids, during ciris (high csf ifn- state) (44), while immune - stimulating strategies, such as use of recombinant ifn--1b, are most likely to be useful for microbiologically refractory disease in hiv - naive individuals (especially those with low csf ifn- levels), while it is likely to exacerbate ciris (45, 46). the host - damage framework applied to activation of the antigen - presenting cell t - cell macrophage activation pathways. (left) panels illustrate the predominant cellular response ; (right) panels illustrate the potential contribution to host cell damage by the immune response. in the setting of cryptococcal meningoencephalitis, antifungal therapy followed by antiretroviral therapy results in pathogen activation of dendritic antigen - presenting cells through activation of tlrs, mannose receptors (mann r), and -glucan receptors (-glucan r), resulting in a robust concordant th1-m1 intrathecal response (ciris), whereas cryptococcal postinfectious inflammatory response syndrome (piirs) displays a discordant th1-m2 csf response (red arrow) with activated t cells causing increased inflammation but poor macrophage - mediated pathogen / antigen clearance. tuberculous meningitis has an intermediary immunophenotype with moderately constrained inflammation and pathogen / antigen clearance. although hiv - related cryptococcal disease is declining in high - income countries due to art access, non - hiv - related cryptococcosis represents an ever - increasing proportion of cases (47). the first are those with preexisting conditions, such as cushing s syndrome or immune suppression by immunotherapy, cancer chemotherapy, or transplant conditioning. a second category of disease afflicts previously healthy, apparently immunocompetent individuals (15). previous clinical research in non - hiv disease emphasized microbiological clearance of live organisms as a key to the resolution of pathophysiology, similar to art - naive cryptococcosis, with cerebrospinal fluid culture negativity at 2 weeks an important prognostic marker (48). in addition, an assumption of t - cell defects based on hiv - related susceptibilities has prompted recommendations for th1-biasing immune therapy, such as ifn- for refractory cases among non - hiv - infected patients (44). however, reconsideration of the pathophysiology in these patients has occurred recently, with greater attention to a role for the host damage response. for example, in transplant - related cryptococcosis, clinical failure has been related to adjunctive reductions in immune suppression, potentiating an iris - like syndrome (49). such reductions in immune suppression are commonly undertaken in immunosuppressed hosts during therapy to potentiate the immune response against pathogens. yet, augmenting the immune response in patients with cns infection may not be beneficial considering the damage - response framework. in contrast, other investigators have suggested a role for corticosteroids in non - hiv - related cryptococcosis (50). in addition, some previously healthy individuals with cryptococcus infection have appeared to have defective macrophage signaling, suggested by stat5-blocking antibodies to gm - csf, with retention of normal t - cell activity (51, 52). similarly, g - csf given to lymphopenic, hiv - uninfected hosts resulted in unmasking of clinical symptoms of infection (53). thus, mechanisms related to immune - mediated host damage in this population have remained unclear. to help clarify these issues, a prospective immune analysis of previously healthy patients with active cns cryptococcosis previously healthy patients were chosen, both because they represent an important subpopulation of susceptible hosts and also because this provided greater patient sample uniformity due to a lack of confounding by variable levels of immunosuppression present in other at - risk populations, such as solid organ transplant patients or those receiving corticosteroids. all patients were severely ill with severe mental status changes despite antifungal therapy and their apparent immunocompetent state. a majority required ventricular - peritoneal shunting to relieve csf obstruction from choroidal inflammation. surprisingly, all had negative csf cultures after standard courses of antifungal therapy, suggesting clinical deterioration despite effective microbiological control of their disease. this suggested that, unlike the art - naive hiv cryptococcus - infected patients, pathogen virulence was not the predominant cause of their refractory illness. however, much like ciris, the patients with cns disease exhibited a robust intrathecal cellular th1 response, with both cd4 and cd8 activation, as measured by hla - dr positivity with production of high levels of th1-biasing ifn- and inflammatory cytokines, such as il-6, and a relative lack of th2 cytokines, such as il-4 and il-13 (table 1). activation of t cells was also suggested by ex vivo study results, which demonstrated high levels of both cd4 and cd8 production of ifn- when t cells from csf samples were cocultured with cryptococcal antigen - exposed dendritic cells. in addition, specific biomarkers of tissue inflammation, such as scd27/t - cell ratios, suggested that inflammation was not restricted to the csf alone but was also present within the meninges or brain parenchyma (8). analysis of brain biopsy specimens and of a second set of autopsy specimens confirmed extensive meningeal and virchow - robin channel macrophage and t - cell infiltration, suggesting an immune etiology for the cerebral edema accompanying non - hiv - related cryptococcosis (9). this inflammatory response was also accompanied by elevated levels of csf neurofilament light chain (nfl), a marker of axonal damage (55), suggesting ongoing host neurological damage. this cryptococcal postinfectious inflammatory response syndrome (piirs, pronounced prs, or cpiirs), associated with host cell damage, was present whether the original infecting organism was cryptococcus neoformans or cryptococcus gattii, the latter of which is typically believed to cause greater inflammation (56). however, both among persons with anti granulocyte - macrophage colony - stimulating factor (gm - csf) autoantibody and those without, tissue macrophage recruitment to the site of cns infection was intact but brain histopathology demonstrated an m2 macrophage polarization (cd68 cd200r1) and poor phagocytosis of fungal cells, as identified by calcafluor white staining (54). this finding was supported by significantly high il-10 and low tnf- levels intrathecally (the latter predominantly produced by tissue macrophages). il-10 production by alternatively activated m2 macrophages has been associated with other diseases for which there is poor microbial / antigen clearance, such as lepromatous leprosy (57). the known plasticity of monocytes coupled with this apparent th1-m2 discordance suggests that those non - hiv - infected patients with severe cryptococcal meningoencephalitis may have a downstream monocyte defect in the efferent arm of the immune response (fig. 1, second panel). this th1-m2 dissociation in cpiirs thus results in a damaging t - cell host response but poor antigen clearance by macrophages, resulting in a prolonged clinical course of 1 to 2 years in many of the severe cases. clearly, with up to 30% mortality in non - hiv - related cryptococcosis, clinical identification of patients with cpiirs is essential to rational therapy. novel approaches taking into account immune - mediated host damage may reduce mortality in these refractory clinical cases. according to the world health organization (who), tuberculosis affects 1/3 of the world s population, with 1.5 million deaths annually ; it is the second leading infectious cause of death after hiv (58). although tuberculous meningitis (tbm) occurs in approximately 1% of tuberculosis cases, it is most frequent and severe in children (59), with an estimated mortality rate of 15 to 75% (60) and adverse sequelae in 10 to 85% of patients (61). steroid responsiveness in tbm suggests that immune - mediated host damage may play a significant role in disease pathology (6264). further immune studies of tbm in children suggested an intermediately activated t - lymphocyte / monocyte immunophenotype (fig. 1, third panel). it is characterized by intrathecal th1 markers, such as ifn-, accompanied by th2 and m2 markers, such as il-13 and il-10, respectively, suggesting a mixed t - cell and macrophage polarity (table 1) (65). in addition, elevated levels of cathelicidin ll-37, vegf, and ccr5 suggest intact macrophage recruitment but deficient tnf- expression, indicating a lack of effective macrophage activation, similar to cryptococcal piirs (66). cathelicidin ll-37 is an antimicrobial peptide found in the lysosomes of macrophages and neutrophils and is important in the vitamin d receptor pathway. indeed, vitamin d deficiency has been associated with tuberculosis progression (67), but vitamin d has not been associated with other cns infections, such as cryptococcosis (68). with such a robust csf ifn- response in tbm, similar to that in ciris and cpiirs, it is interesting that the adjunctive corticosteroid dexamethasone reduced mortality by 31% through 9 months (64). corticosteroids, however, did not reduce chemokine expression in tbm (69), suggesting that clinical effectiveness could be more related to control of associated noninflammatory parameters, such as cerebral edema, which is the primary target of moderate doses of corticosteroids (1 to 1.5 mg / kg of body weight of prednisone equivalent) used in these studies (70). in contrast, high doses of corticosteroids of 18 mg / kg / day are typically used for severe inflammatory states, such as cerebral vasculitides (71). indeed, alternative pathways of therapy are indicated by recent data suggesting that corticosteroids at achievable doses may reduce cerebral edema by coordinate regulation of angiopoietin-1 and vegf, which are direct modulators of vasogenic brain edema and the blood - brain barrier, independent of inflammation (72). thus, excess, damaging inflammation may fulfill the role of a prognostic marker of poor outcome because of its association with cerebral edema, but control of inflammation apart from cerebral edema may or may not affect outcome. this concept of the independence of a prognostic marker from a treatment surrogate is an important therapeutic principle that, if ignored, may lead to a false linkage of pathophysiology to treatment expectations (73, 74). the failure of a recent drug trial based on a mistaken belief that a microbiological prognostic marker of mycobacterium spp. pathogen clearance would provide a good treatment surrogate is a good demonstration of this concept. that study hypothesized that increasing clearance by using a higher dose of antibiotic would lead to better outcomes. however, although pathogen clearance was increased, there were more deaths because of untoward effects of the higher dose of antibiotic (75). currently, in cases of neurological infections, higher doses of corticosteroids or additional immunosuppressants have been proposed as adjunctive therapy in tbm, to reduce both innate and adaptive immune responses through the extracellular signal - regulated kinase 1/2 and nf-b pathways (76). however, the threat of adjunctive immunosuppressive therapies to effective microbiological control is a clear danger in diseases such as tbm and cryptococcosis and could result in adverse outcomes. clearly, precise immunophenotyping analyses, to stratify patients and monitor therapeutic interventions, will be required for rational design and selection of adjunctive therapies. because of the scale - up in art and large numbers of tb - infected individuals globally, tb - related immune reconstitution inflammatory syndrome (tb - iris) after art initiation is a significant contributor to the health care burden, especially in high - tb - hiv coinfection incidence populations (77). cns involvement is the most severe form of tb - iris with a high associated mortality (78). interestingly, in addition to a monocytic infiltration typical of tbm, tbm - iris results in an additional neutrophilic csf infiltration (79), distinguishing it from non - hiv - associated tbm in children and ciris in adults (80) (fig. 2, top panel). similar to ciris, tbm - iris has been associated with elevations in th1 cytokines, such as ifn-, and a delayed - type hypersensitivity response (81) (table 1). more recently, marais. studied compartmentalized csf immune responses from hiv tbm patients at tbm diagnosis, start of art, and at iris diagnosis. tbm - iris was associated with elevated th1 markers (ifn-, cxcl10) and inflammatory markers such as il-6, similar to that in ciris, with the addition of neutrophil recruitment markers s100a8/a9 (calprotectin), matrix metalloproteinase 9 (mmp-9), and its inhibitor, tissue inhibitor of metalloproteinases 1 (80). production of il-8 by endothelial cells and macrophages is important for neutrophil influx in pulmonary tb (82), and il-8 is also elevated in tbm (83), although il-8 has not been studied specifically in tbm - iris. in addition, based on a murine tb model showing that il-17a - induced s100a8/a9 was key in neutrophil accumulation and lung infiltration (84), csf il-17a levels were measured and found to increase with development of tbm - iris, supporting previous suggestions that il-17 may be important in host cell - mediated immune damage (85). a predilection for tbm - iris is also related to preceding high csf ifn- and tnf- levels with neutrophilia, in contrast to the lymphopenia and low csf ifn- levels predictive of ciris (79). however, in tb as in ciris, pathogen antigen load is a major driver of this paradoxical pathological immune response process. host - damage framework in diseases with an additional csf neutrophilic response. in iris related to tuberculous meningitis and pneumococcal meningitis, activation of antigen - presenting cells through tlrs leads to t - cell activation and cytokine release (il-6, ifn-, tnf-, gm - csf) and a macrophage response leading to further tnf- and il-12 production as well as cross talk with neutrophils. further activation of neutrophils follows pathogen phagocytosis and, in pneumococcal meningitis, to complement and il-1 activation. in both cases, the addition of neutrophil activation leads to additional pathogen clearance after therapy but at the cost of host - damaging intrathecal inflammation. corticosteroids have been shown to be useful in tb - iris in a randomized, placebo - controlled trial, although patients with cns involvement were excluded (86), but symptomatic improvement in tbm - iris has been reported with steroid use (80). however, csf inflammation (e.g., ifn- but not tnf- changes) persisted following art in tb - iris, despite adjunctive corticosteroids, again suggesting that moderate doses of corticosteroids may act primarily by control of cerebral edema, similar to the experience in tbm (69, 80). however, effects on neutrophil markers such as mmp-9 have been variable, with some investigators showing decreases with steroid treatment (87) and others not showing reductions (80). these findings have also suggested to some that immunomodulatory treatment options more potent and specific than corticosteroids need to be explored for the prevention and management of tbm - iris (80), but the risks again must be balanced, as described above. pneumococcal infection causes approximately 2 million deaths and requires medical outlays of hundreds of billions of dollars per year (88). streptococcus pneumoniae frequently colonizes the nasopharynx but can spread from the airway to the lower respiratory track, sinuses, middle ears, or to the cns. t helper cell (th-17) t - cell responses are key to controlling colonizing bacteria (89), and they are mediated by recruitment of macrophages in naive hosts and of neutrophils in previously exposed individuals. the organism is the most frequent bacterial cause of meningitis in adults (excluding africa) and can lead to host damage through a variety of mechanisms, resulting in the highest case fatalities and neurological disability rates of the bacterial meningitides (90, 91). the virulence factor / toxin pneumolysin plays a pivotal role in both direct host damage and immune recognition / inflammation (92). pneumolysin has the capacity to form membrane pores to lyse host cells, but it also activates innate immunity, as it stimulates caspase-1-dependent processing of il-1, dependent on the nucleotide - binding oligomerization domain (nod)-like receptor p3 inflammasome (93). adaptive immunity is also stimulated by pneumolysin, which is recognized by toll - like receptor 4 (tlr4) on antigen - presenting cells, which, in turn, induces production of inflammatory cytokines such as ifn- and il-17a by t cells (94). interestingly, tlr4-deficient mice did not differ from wild - type mice in their host response, while tlr2/4 double deficient mice showed a marked reduction in inflammatory mediators and improved outcome compared to wild - type mice or those with single tlr deficiency with pneumococcal meningitis, suggesting a role for tlr signaling in cns - related host damage (95). in a study of 28 patients with pneumococcal and meningococcal meningitis, csf ifn- levels were significantly higher and borderline higher il-2 levels were observed in pneumococcal compared with meningococcal patients across time, implying a greater th1 bias for pneumococcal meningitis (table 1). in another study of 45 patients, those with pneumococcal meningitis had significantly higher csf levels of ifn-, ccl2 (mcp-1), and mmp-9 than those with meningococcal or haemophilus influenzae meningitis (96). high mmp-9 expression has been associated with blood - brain barrier damage and neurologic sequelae (97). in vitro coculture of astrocytes and pneumococcal cell walls with microglia also led to these resident brain macrophages producing nitric oxide and causing neuronal toxicity, which was suppressed by dexamethasone (98) ; microglia are key in the recruitment of effector immune cells from the periphery following infection and may undergo a caspase - induced apoptosis leading to cytokine release in response to pneumococcus (99). activation of a strong t - cell response in bacterial meningitis thus has some features common to tbm and cryptococcus infection of hiv - infected and uninfected individuals. however, in bacterial meningitis, robust recruitment of neutrophils to the intrathecal space is a prominent feature, as in tbm - iris (fig. 2, bottom panel), and requires the 2 integrin mac-1, resulting in the generation of neutrophil serine proteases cathepsin g and neutrophil elastase (100102). despite the key role of neutrophils in controlling bacteria the production of nadph oxidase - dependent reactive oxygen species contributes to collateral host cell damage in tissues (103) but, paradoxically, is not required to kill s. pneumoniae. in the cns, neutrophil - produced myeloid - related protein 14 (mrp14) was found in a mouse model to exacerbate meningeal inflammation even after treatment with antibiotics in a tlr4-, cxcl2-dependent manner, again implicating tlr signaling in cns host damage (104). interestingly, treatment with the mrp14 antagonist paquinimod reduced inflammation and disease severity in mice, suggesting that identification of key host damage pathways may result in effective adjunctive therapies (104). in addition, moderate - dose adjunctive corticosteroids (e.g., dexamethasone equal to prednisone at 1 mg / kg four times daily for 4 days) has been used successfully in bacterial meningitis with significantly decreased mortality in human clinical trials (105, 106). a particularly exciting development is the finding that adjunctive treatment with anti - complement c5 antibodies reduced mortality in pneumococcal meningitis in humans (107). recently, an additive effect of dexamethasone and anti - c5 antibodies as adjunctive treatment has been shown in experimental pneumococcal meningitis (108). this provides a strong precedent that addition of specific inflammatory inhibitors in neuroinflammatory infections may potentiate the effects of corticosteroids on cerebral edema. complement components are expressed in the csf by microglia as well as injured astrocytes and neurons in response to inflammatory cytokines (109, 110). the complement cascade is activated through classical and alternative pathways after specific pathogen - cell interactions, although s. pneumoniae expresses several anticomplement strategies, such as pneumococcal surface protein c (pspc), which binds human factor h and blocks c3 convertase. however, proinflammatory cytokines such as il-6 have also been demonstrated to upregulate expression of c5ar in both liver and lung tissue, and anti - il-6 antibody reduces complement activation during sepsis (112). newly described roles for inflammatory pathways in complement activation, independent of the organism, may thus suggest studies of complement in inflammatory syndromes whose pathogens typically do not activate complement strongly, such as infection with encapsulated cryptococcus (113). in countries where malaria is endemic, cerebral malaria caused by plasmodium falciparum affects primarily children and malaria - naive visitors, with case fatality proportions of 15 to 25% (114). the pathogenesis of cerebral malaria is incompletely understood, but it is distinguished from the infections described above by a predominance of endothelial activation by parasites sequestered in the brain microvasculature accompanied by only a modest inflammatory response (fig. demonstrate intravascular parasites, vascular congestion and obstruction (116119), and endothelial cell activation (120). in children this has been associated with a breakdown of the blood - brain barrier, ring hemorrhages, and cerebral edema, as evidenced by increased brain weight (121). in a recent study of 168 african children with cerebral malaria, 84% of the 25 children that died had brain swelling / volume findings on magnetic resonance imaging (mri), whereas only 27% of the survivors had such findings, which eventually improved (122). an important confounder in the mri study was that over 80% of the cerebral malaria patients had a history of seizures. host - damage framework applied to cerebral malaria with a predominance of endothelial injury and limited inflammation. parasite infection of rbcs results in binding to the endothelial receptors icam and epcr, followed by microvascular sequestration. released parasite products (black dot) and rbc arginase activate tlrs and lead to no inhibition. activated endothelial cells release intravascular il-8 and il-1ra, leading to a monocyte inflammatory response that results in thrombin and fibrin production, potentiating endothelial injury and sequestration. notably absent are reports of autopsy samples with lymphocytes or neutrophils localizing to parenchymal areas of parasite sequestration or the csf (116, 118, 121). in contrast, intravascular monocytes, fibrin, and platelets have been reported to localize with sequestered parasites in children, particularly those who are hiv (111, 123). thus, a lack of a marked leukocyte infiltration has focused attention on endothelial cells and the local vascular environment as mediators of the pathology that leads to impaired consciousness and provides a sharp contrast to the other cns infections we have described. cerebral edema associated with cerebral malaria is also unusual in that it was not found to be responsive to steroids in two randomized controlled trials (119, 124). in addition, cerebral edema may differ in children from that of adults, as brain weights in adults have been reported not to increase and expression of the brain water channel, aquaporin 4 (or vegf), did not show elevations in adult patients dying of cerebral malaria (125, 126). a significant increase in endothelial regulator angiopoietin 2 (ang2) and a decrease in angiopoietin 1 (ang1) have been reported in both adult and pediatric cerebral malaria patients (127130) ; ang2 antagonizes ang1, resulting in an increase in vascular permeability, nf-b activation, and endothelial receptor upregulation (131). it is thus possible that high ang2/ang1 ratios could have a greater impact in children (125). in addition, prolonged seizures, more typical of cerebral malaria in children, may contribute to cerebral edema and abnormal mri findings, suggesting cerebral malaria (122, 126). the specific induction stimulus for ang2 is unknown but could include hypoxia, thrombin, and low nitric oxide (no) levels, which have been associated with areas of parasite sequestration (132134). a role for ang2 is also consistent with the lack of steroid responsiveness, as in vitro studies have shown that dexamethasone does not decrease ang2 expression in human brain microvascular endothelial cells (72). clearly, much remains to be understood regarding the pathogenesis of cerebral malaria and the impact of possible edematous states. parasite cytoadherence to endothelial cell surface receptors is thought to be mediated primarily by a p. falciparum - specific, polymorphic, parasite - produced protein, pfemp1, that is exported to the red blood cell (rbc) surface (135). in the brain microvasculature, intercellular adhesion molecule 1 (icam-1), which binds to a subset of pfemp1 molecules, has been found upregulated in areas of parasite sequestration (118, 132). recently, expression of group a pfemp1s that bind to the endothelial protein c receptor (epcr) (136) have been associated with severe malaria (137139). intriguingly, epcr expression levels have been reported to be reduced in the microvasculature, with sequestered parasites and soluble epcr levels increased in the csf of cerebral malaria patients, suggesting that parasite binding stimulates receptor shedding (132). the effects of pfemp1/epcr or icam-1 binding are still under investigation, but it has been proposed that pfemp1 binding to epcr disrupts the production of activated protein c, inhibiting its anticoagulant and cytoprotective effects (132, 140). the low arginine and no levels associated with cerebral malaria (141143) as well as the ability of parasite material released during infected rbc rupture to stimulate tlrs (144) could also contribute to local endothelial activation, vascular obstruction, and local areas of hypoxia (133, 140). increasing no bioavailability as an adjunct to antimalaria chemotherapy has been tested in several recent clinical trials (145). one trial found the administration of l - arginine to adult severe malaria patients in addition to intravenous artesunate to be safe, but there was no alteration in endothelial no bioavailability as measured by reactive hyperemia peripheral arterial tonometry (146). the plasma arginine levels achieved were lower than predicted and could have contributed to the lack of efficacy. direct no inhalation has also been tested in conjunction with antimalaria chemotherapy in pediatric cerebral malaria patients. again, the intervention was safe but did not significantly alter morbidity or mortality (147), suggesting that alternative strategies are needed to improve treatment of cerebral malaria. the association of specific cytokines with cerebral malaria has been more difficult to define, as results vary between groups, possibly due to underlying coinfections or individual genetic variation (115). several groups have found both il-8 and interleukin 1 receptor antagonist (il-1ra) to be significantly increased in csf samples from patients with cerebral malaria compared to samples from those with severe malaria (table 1) (148, 149). interestingly, both these cytokines can be produced by endothelial cells (150, 151), and the finding that il-8 levels are higher in csf than in serum during cerebral malaria is also consistent with production in the cns (149). in vitro, the incubation of p. falciparum - infected rbcs with human brain microvascular endothelial cells has also been shown to activate the nf-b pathway, leading to the production of a number of chemokines, including il-8 (152). it is possible that il-8 plays a role in recruiting the monocytes observed in pediatric patients, as well as enhancing angiogenesis (151), while il-1ra could act to downregulate the inflammation response by directly competing with il-1 and il-1 receptor stimulation (153). together, the data to date suggest that parasite sequestration in the brain microvasculature coupled with the subsequent rupture of infected rbcs during parasite release leads to marked endothelial cell activation and vascular obstruction with minimal lymphocyte infiltration or activation in the cns. the resulting neuronal damage is likely due to hypoxia and metabolic disruption and, in children, is exacerbated by cerebral edema and seizures. in summary, the host damage - response framework is exemplified in the generalized cns infectious syndromes considered here. defined repertoires of host cytokine / chemokine immune response profiles in these syndromes lead to a common endpoint of host neuronal damage. an interesting finding from our review is that neuroinfections characterized by significantly high csf ifn- and t - cell inflammation may be more likely to benefit from adjunctive corticosteroid use, i.e., cryptococcal iris and piirs, pneumococcal meningitis, tbm, and tbm - related iris, versus cerebral malaria, where parenchymal and intrathecal inflammation is minimal. however, with regard to treatment, cns inflammation may only be a marker of other pathologies, such as cerebral edema, since moderate doses of corticosteroids that are clinically beneficial have little effect on intrathecal inflammation in diseases such as tbm and tb - iris. however, caution must be exercised in the selection of immune - modulating therapies, as exemplified by the finding that adjunctive immune stimulators such as recombinant ifn-1b benefited only small subsets of patients who had low csf ifn- levels and poor microbiological control in the case of art - naive hiv - related cryptococcal disease (45, 46) and could be detrimental in inflammatory states such as piirs, where a high - ifn- inflammatory state is mistaken for refractory disease (54). thus, an appreciation of the potential for host - mediated immune damage and development of validated biomarkers that quantify the csf immune response profile may inform judicious therapeutic selection and management to mitigate host damage in cns infectious disease syndromes. further dissection of associated inflammatory pathways may thus identify targeted interventions for prevention and/or treatment of these cns infectious inflammatory syndromes. | abstractthe host damage - response framework states that microbial pathogenesis is a product of microbial virulence factors and collateral damage from host immune responses. immune - mediated host damage is particularly important within the size - restricted central nervous system (cns), where immune responses may exacerbate cerebral edema and neurological damage, leading to coma and death. in this review, we compare human host and therapeutic responses in representative nonviral generalized cns infections that induce archetypal host damage responses : cryptococcal menigoencephalitis and tuberculous meningitis in hiv - infected and non - hiv - infected patients, pneumococcal meningitis, and cerebral malaria. consideration of the underlying patterns of host responses provides critical insights into host damage and may suggest tailored adjunctive therapeutics to improve disease outcome. |
glaucoma is a relatively common disease in which the death of retinal ganglion cells causes a progressive loss of sight, often leading to blindness. there have been several new developments in automated threshold perimetry, which has been shown to be a rapid and effective method of detecting glaucomatous visual field loss. however, the accuracy and reliability of measurements of the visual field with these standard perimeters depends upon the cooperation of the patient.1,2 pupil perimetry uses the pupillary light reflex to make possible an objective analysis of the visual field. this method requires little effort and attention on the part of the patient, and may reflect damage at an earlier stage of the relevant disease.3 in the past, several attempts that used the pupillary light reflex have been made.411 however, this method also has disadvantages, which include doubts as to whether the pupil field defects actually reflect the visual field loss. the main problem related to the worsening of detection defects was that the normal values for differentiating an artifact from pupil field defects could not be established in past pupil field study. at present, the concept of pattern deviation has been widely accepted as a viable type of analysis for identifying abnormal points using the humphrey field analyzer (carl zeiss meditec, dublin, ireland). pattern deviation is expressed as a value representing the difference between an age - matched norm (median value) and the threshold for each test point after correction for the degree of sensitivity of the entire visual field. our recent study demonstrated that age - matched normal pupil constriction data can be obtained using innovated pupil perimetry, and confirmed the individual variations of pupillary responses at each test point by pupil perimetry in a large group of healthy subjects.12 based on these background studies, the purpose of this study was to calculate the pattern deviation of pupil perimetry as a new objective assessment, and also evaluate the gray scale for distinguishing between a glaucomatous pupil field loss point and a normal pupil field point. there were 14 patients (10 men, 4 women) enrolled in this study, ranging in age from 51 to 80 years (mean age, 61.4 years). the patients comprised cases of normal - tension glaucoma (ntg, 6 eyes) and primary open - angle glaucoma (poag, 8 eyes). the inclusion criteria required a corrected visual acuity of 1.0 (= 0 logmar) or better and a pupil size of at least 2.5 mm without dilation. among the exclusion criteria of the patients were severe cataracts (grade iii to v in the emery - little classification) and drugs affecting the pupil, particularly pilocarpine. also, this study examined patients without any systemic or ophthalmic diseases likely to affect the visual field (apart from glaucoma). prior to the study, all patients were examined with a humphrey field analyzer (30 - 2, full threshold program). the mean deviation (md) value ranged from 6.50 db to 20.18 db. patients with more than 20% false - positive or false - negative responses were excluded. a diagnosis of poag was based on anderson and patella s criteria,13 as well as on a glaucomatous optic disc, and an intraocular pressure (iop) of more than 21 mmhg measured with the applanation tonometer. some ntg patients had visual field defects and optic disc changes as well as a diagnosis of poag, but none had an iop of more than 20 mmhg. the definition of a visual field defect used the pattern deviation plot obtained with the above - mentioned program. a minimum scotoma diagnosis required at least three adjacent points depressed at p we used pupil perimetry, which combined automated static perimetry and infrared pupillography (kowa and hamamatsu, japan). this pupil perimeter was developed by yoshitomi and enabled the automatic rejection of artifacts such as blinking and fixation. after dark adaptation for 10 minutes, each patient was sat in a chair comfortably and was asked to fixate on a red light point at the center of the stimulus background. a light stimulus of 1.7 (goldmann v) with an intensity of 1000-apostilb the pupil response was obtained from the pupil diameter tracings before light stimulation (baseline pupil diameter, a), and the minimum diameter during light stimulation (b). the following calculation was then performed : percentage of pupil constriction (%) = (a b)/a 100. visual fields were confirmed with the above - mentioned 30 - 2 program on the humphrey field analyzer, and the visual field defects. a database based upon 90 normal subjects included the median percentage pupil constriction for each of the 30 - 2 test points (blind spot, 3 above and below, excluded) in the stimulated area12 (figure 1). to assess the ability of pupil perimetry to detect glaucomatous field loss (ie, abnormal points) and normal field points, the results from our 14 patients were analyzed in several ways. of the 76 test points, the 22 surrounding points and the 3 points corresponding to the blind spot (3 above and below, and 9 below) were excluded, and among the remaining 51 points, the sensitivity of the seventh highest point when compared with the norm (ie, the 85th percentile value) was used as the standard degree of sensitivity for the entire visual field.14 probability plots of pattern deviation on the humphrey field analyzer were expressed in the following five grades : (dot), (p < 5%), (p < 2%), (p < 1%) and (p < 0.5%). as with detecting defects of pupil perimetry, the rate of detection of probability plot abnormalities (p < 5% or less) on pattern deviation and pupil field abnormalities independent of the gray scale display in each value of deviation (from 1 to 10) compared with the healthy subjects database was investigated. we also used this technique to identify the value for distinguishing glaucomatous field loss from the normal pupil field (one point being indicated by one square in figures 3 to 5). the following calculations were performed : ratio of abnormal points to abnormal plots (%) = number of abnormal pupil field points / number of abnormal probability plots. the proportion of the normal visual field was also calculated : ratio of normal points to normal plots (%) = number of normal pupil field points (indicated by white)/number of normal probability plots (indicated by dots [total 74 test points number of abnormal probability plots ]). based on the above results, pupil perimetry gray scales were assessed in the following five grades so as to correspond with the probability plots of the hfa : white, gray 1 (25% of black), gray 2 (50% of black), gray 3 (75% of black) and black. there were 14 patients (10 men, 4 women) enrolled in this study, ranging in age from 51 to 80 years (mean age, 61.4 years). the patients comprised cases of normal - tension glaucoma (ntg, 6 eyes) and primary open - angle glaucoma (poag, 8 eyes). the inclusion criteria required a corrected visual acuity of 1.0 (= 0 logmar) or better and a pupil size of at least 2.5 mm without dilation. among the exclusion criteria of the patients were severe cataracts (grade iii to v in the emery - little classification) and drugs affecting the pupil, particularly pilocarpine. also, this study examined patients without any systemic or ophthalmic diseases likely to affect the visual field (apart from glaucoma). prior to the study, all patients were examined with a humphrey field analyzer (30 - 2, full threshold program). the mean deviation (md) value ranged from 6.50 db to 20.18 db. patients with more than 20% false - positive or false - negative responses were excluded. a diagnosis of poag was based on anderson and patella s criteria,13 as well as on a glaucomatous optic disc, and an intraocular pressure (iop) of more than 21 mmhg measured with the applanation tonometer. some ntg patients had visual field defects and optic disc changes as well as a diagnosis of poag, but none had an iop of more than 20 mmhg. the definition of a visual field defect used the pattern deviation plot obtained with the above - mentioned program. a minimum scotoma diagnosis required at least three adjacent points depressed at p we used pupil perimetry, which combined automated static perimetry and infrared pupillography (kowa and hamamatsu, japan). this pupil perimeter was developed by yoshitomi and enabled the automatic rejection of artifacts such as blinking and fixation. after dark adaptation for 10 minutes, each patient was sat in a chair comfortably and was asked to fixate on a red light point at the center of the stimulus background. a light stimulus of 1.7 (goldmann v) with an intensity of 1000-apostilb was presented at each of 76 test locations with a background luminance of 6-apostilb. the pupil response was obtained from the pupil diameter tracings before light stimulation (baseline pupil diameter, a), and the minimum diameter during light stimulation (b). the following calculation was then performed : percentage of pupil constriction (%) = (a b)/a 100. visual fields were confirmed with the above - mentioned 30 - 2 program on the humphrey field analyzer, and the visual field defects. a database based upon 90 normal subjects included the median percentage pupil constriction for each of the 30 - 2 test points (blind spot, 3 above and below, excluded) in the stimulated area12 (figure 1). to assess the ability of pupil perimetry to detect glaucomatous field loss (ie, abnormal points) and normal field points, the results from our 14 patients were analyzed in several ways. of the 76 test points, the 22 surrounding points and the 3 points corresponding to the blind spot (3 above and below, and 9 below) were excluded, and among the remaining 51 points, the sensitivity of the seventh highest point when compared with the norm (ie, the 85th percentile value) was used as the standard degree of sensitivity for the entire visual field.14 probability plots of pattern deviation on the humphrey field analyzer were expressed in the following five grades : (dot), (p < 5%), (p < 2%), (p < 1%) and (p < 0.5%). as with detecting defects of pupil perimetry, the rate of detection of probability plot abnormalities (p < 5% or less) on pattern deviation and pupil field abnormalities independent of the gray scale display in each value of deviation (from 1 to 10) compared with the healthy subjects database was investigated. we also used this technique to identify the value for distinguishing glaucomatous field loss from the normal pupil field (one point being indicated by one square in figures 3 to 5). the following calculations were performed : ratio of abnormal points to abnormal plots (%) = number of abnormal pupil field points / number of abnormal probability plots. the proportion of the normal visual field was also calculated : ratio of normal points to normal plots (%) = number of normal pupil field points (indicated by white)/number of normal probability plots (indicated by dots [total 74 test points number of abnormal probability plots ]). based on the above results, pupil perimetry gray scales were assessed in the following five grades so as to correspond with the probability plots of the hfa : white, gray 1 (25% of black), gray 2 (50% of black), gray 3 (75% of black) and black. figure 2 shows the ratio of abnormal to normal points identified by respective numerical values for distinguishing glaucomatous field loss from the normal pupil field. the value to identify the test points with abnormal and normal values was 4 of deviation. this value was associated with the ratio of abnormal points to abnormal plots (66.0 25.5%) and the ratio of normal points to normal plots (62.6 22.2%). the difference between these two ratios was the least significant (p = 0.78). on the basis of this result, pupil perimetry gray scale values were assessed in the following five grades : white (negative deviation of less than 4 : < 4), 25% gray (from 4 to 8), 50% gray (from 9 to 13), 75% gray (from 14 to 18) and black (negative deviation of more than 19). the results of three patients are presented here, since they were typical of our patients. the visual field results obtained with the humphrey field analyzer (gray scale and pattern deviation plot) together with the pupil perimetry results (gray scale and pattern deviation values compared with our healthy subject database) are presented for each patient. patient 1 (a 52-year - old man, ntg, md 14.02 db, figure 3) showed glaucomatous field losses almost completely distinguished from the normal pupil field. the ratio of abnormal points to abnormal plot detection defects and the ratio of normal points to normal plot normal pupil field detection were 91.4% (31/35) and 66.7% (28/39), respectively. patient 2 (female, 68 years old, ntg, md 7.63 db, figure 4) had visual field loss with diffuse defects with humphrey standard automated perimetry. this patient had incomplete detection (especially, abnormal test points) between these two perimeters. these above - mentioned two ratios were 38.1% (8/21) and 86.8% (46/53). despite his age (78 years), patient 3, who had poag with md 16.86 db had a generally well - detected visual field and pupil field defects (figure 5). these ratios of abnormal to normal points were 76.6% (36/47) and 70.4% (19/27), respectively. in past pupil field study, pupil perimetry for glaucoma patients has provided details on the variability and difficulty, which limits its use in a clinical setting. the problems involved anatomical differences in the characteristics of the retinal ganglion cells and the visual and light reflex pathways. additionally, standard pupil constriction values can not be established because the pupil field varies considerably between individuals, and because of variations within the same individual also.6,15,16 furthermore, the pupil fields exhibited a maximal percentage of pupil constriction as the white portion of the gray scale. when artifacts were included, the gray scale maps indicated pupil field deficits even in the normal test points. with regard to pupil perimetry data analysis, schmid proposed that each matching defect in the pupil and visual fields be evaluated independently by three doctors as a good or a poor match without applying statistical criteria. therefore, not only was it impossible to compare the results with those of other subjects, but also, in clinical settings, it became necessary to depend upon subjective data obtained from raw pupillary traces and percentage pupil constriction. in this study, we evaluated the pattern deviation of pupil perimetry, and also determined the gray scale display for differentiating a glaucomatous pupil field loss from a normal pupil field in each patient. the results of our study demonstrate the accuracy of the clinical assessments that can be successfully performed and used as values of gray scale in pupil perimetry. however, not every patient showed good correspondence between results for pupil field and light threshold. although pupil perimetry generally well detected visual field defects in glaucoma with absolute scotoma, diffuse visual field defects exhibited various patterns. moreover, our results also show that in all patients, there was a pronounced decrease of pupil constriction from the center to the periphery. the question that is raised by these results is whether or not neuronal cell loss,17 increased cataract18 or decreased pupil size19,20 are responsible for the reduction of the pupil response that occurs with age. with regard to the conditions of light stimulation, it is possible that the use of a small or dim light stimulus might be below the minimum level required to cause pupil constriction. conversely, the use of brighter or bigger stimuli increased the amount of stray light, so that the maximal level of stimulus brightness that can be used is limited. furthermore, other characteristics such as the stiles - crawford effect on the pupil field might possibly be involved, thereby limiting the data that can be obtained. previous studies on the visual field have reported that with ageing there was a steeper decline of the light sensitivity towards the periphery,21,22 and that this decline was due to the loss of peripheral photoreceptors and ganglion cells as compared to the central cones.23,24 thus, further studies using improved measuring apparatus that is capable of evaluating the field periphery are required. moreover, unfortunately, the design of this study does not permit claims of true test sensitivity and specificity to be made. to overcome this problem, further studies establishing clinical criteria similar to those of anderson and patella13 for diagnosing glaucoma using pupil perimetry are needed. most patients stated that they preferred undergoing this automated perimetry examination because it was free of decision - making and performance pressure. the method requires no special technical expertise, and the patient need only fixate the eye on a central red point. also, the time required for measurement (almost 4 minutes) is considerably shorter than that for automated perimetry. our pupil perimetry glaucoma detection and pattern deviation analysis methods (pupil perimetry glaucoma detection analysis, ppgda), can therefore be used for reliable detection and confirmation of visual field loss in glaucoma patients. our results demonstrate that, with the use of our technique of pattern deviation and the pupil perimetry gray scale, the glaucomatous pupil field losses can generally be distinguished from the normal pupil field in glaucoma patients, and also that there is a need to establish the optimal conditions and test points for pupil field periphery detection. | objective : to calculate the pattern deviation for identifying abnormal points of pupil perimetry, and also to evaluate the grayscale display for distinguishing glaucomatous pupil field loss (abnormal test points) from normal pupil field (normal test points).methods : fourteen patients ranging in age from 51 to 80 years, who had normal - tension glaucoma (6 eyes) and primary open - angle glaucoma (8 eyes) were tested. pupil perimetry (kowa & hamamatsu, japan) was used to objectively measure the visual field. also, to obtain a subjective visual field, the analysis was performed with a humphrey field analyzer (30 - 2, full threshold program, carl zeiss meditec, dublin). of the 76 test points, the 22 surrounding points and the 3 points corresponding to the blind spot are excluded ; and among the remaining 51 points, the 85th percentile value of pupil perimetry was calculated. the abnormal and normal test points were recorded, and the amount of positive or negative deviation of each test point from the normal median value for the corresponding test points was determined. we also used this technique to identify the value for distinguishing glaucomatous pupil field loss from the normal pupil field.results:this study could be improved by calculating the sensitivity and specificity of a certain cut - off value between the normative data and the glaucoma patients. the value for identifying both abnormal and normal test points was a negative deviation of 4. based on these results, pupil perimetry gray scales were determined : white (19). glaucomatous pupil field losses were generally distinguished from the normal pupil field by use of a gray scale.conclusion:our studies demonstrated that, when a deviation of > 4 was regarded as an abnormal value, the detection of pupil perimetry exhibited improvement in glaucoma patients. |
the supercolonies formed by so - called ' unicolonial ' ant species are huge cooperative groups networks of interconnected nests that exchange individuals and share territory peacefully over extensive areas. in extreme cases, a whole population can comprise a single supercolony ; for example, the largest supercolony discovered, the mediterranean argentine ant supercolony, covers 6,000 kilometers of southern european coastline, and individuals accept each other as colonymates all the way from italy to the spanish atlantic coast. a feature of supercolonies compared with the more usual family - based colonies of social insects is the free movement of individuals between nests, and the fact that each nest contains several queens. consequently, the relatedness between nestmates approaches zero in the argentine ant and many other species, both invasive and non - invasive. in other words, individuals that share a nest are no more similar genetically than would be individuals chosen at random from the whole population. the evolution and persistence of unicoloniality poses a dilemma for the widely accepted theory of kin selection as an explanation for social evolution. in all colonial insects, including the unicolonial ants, the success of the colony depends on so - called ' altruistic ' behavior of non - reproductive individuals the workers who build the nest, collect food for the sexually reproducing queen and care for the larvae (brood rearing) without any apparent benefit to themselves in terms of maximizing their own reproduction, their ' fitness '. kin selection theory proposes that such altruism can evolve and persist if the altruists, such as the ant workers, direct help only to individuals that are genetically related to them. in family - based social insect colonies, which usually have a single queen per colony, so, even though the workers do not reproduce, their cooperative altruistic behavior increases the chance that genes identical to their own will be transmitted to the next generation ; in other words, altruism increases the ' inclusive fitness ' of the individual worker the overall reproductive fitness of itself and its close relatives. in such situations, production of both queens and workers, in addition to males, is in the interest of the workers, queens, and the developing larvae. by this logic, altruism should not persist in a supercolony, where the receivers of help the nestmate queens may be no more closely related to the workers in that nest than they are to the rest of the supercolony. one might expect a complete breakdown of co - operation in such circumstances, with individuals behaving antagonistically towards non - relatives, and natural selection no longer favoring worker traits. furthermore, in the absence of genetic relatedness, ' selfish ' queens could maximize their fitness by producing larvae that develop into females and males rather than workers in which case the supercolony, and the trait of unicoloniality, would eventually die out. the paradox has been investigated by laurent keller and colleagues in a series of papers on native european wood ants of the genus formica [3 - 6 ]. in their most recent paper, published in bmc evolutionary biology (holzer.), keller and colleagues have looked more closely at the genetic structure of supercolonies of the wood ant formica paralugubris. they find that supercolonies that appear to behave as single units in fact harbor genetic substructure and consist of several extended families living side by side. such structuring may help to explain the evolutionary maintenance of unicoloniality, while the peaceful coexistence of potentially competing families can also teach us lessons about behavioral adaptations. even if supercolonies are problematic for kin selection theory when they have grown to cover whole populations, the behavioral rules that underlie their development can be understood through inclusive fitness principles. a super - colony starts from a family group that extends its network of nests and retains more and more queens (figure 1), competing against other such colonies. this is often a successful strategy for monopolizing resources over a large area, and can be favored by kin selection. but when this successful family - based strategy continues for a long time and the colony grows larger and larger, it paradoxically dilutes the relatedness between individuals and eventually leads to a situation where individual workers may no longer be helping kin. worker behavior is no longer favored by selection, but because workers lack the means to assess the decline in relatedness they continue to behave according to the previously successful rules. formation of supercolonies with extremely low relatedness is especially easy for foreign invasive species, whose colonies grow unhindered by conspecific competitors and native parasites, and can extend over hundreds of square kilometers. but in evolutionary terms, supercolonies are thought to be evolutionary dead - ends as the link between genetic relatedness and behavior has been lost. relatedness between individuals is high as a result of the small number of founder queens. but even if relatedness between individuals who do not behave aggressively to each other drops to zero, relatedness to individuals who benefit from worker altruism remains above zero. factors that increase relatedness between individuals in altruistic interactions include : limited dispersal of sexuals, context - specific discrimination and variation in kin structures over worker life - time. in contrast to the argentine ant, supercolonies of indigenous european ants, such as certain species of formica wood ants, are found in stable habitats such as boreal or alpine forests and are non - invasive. but like their invasive counterparts, these supercolonies cover large areas, and workers from distant parts of the supercolony behave unaggressively, like nestmates, towards each other. holzer. made a detailed genetic and behavioral study of three f. paralugubris supercolonies in the swiss jura mountains, which revealed that the supercolonies are not as genetically heterogeneous as expected, but consist of cryptic genetically related clusters of nests. these clusters define groups of individuals that are genetically similar at a level comparable to cousins, with the proportion of genes shared approximating to 0.125. holzer. suggest that such family substructures might enable workers to direct help to their relatives, and that this might help to maintain the trait of unicoloniality, in line with kin selection theory. however, the presence of genetically differentiated subunits raises the question of why there is no aggression within the supercolony, and why the clusters keep exchanging individuals. in theory in other supercolony - forming species studied, within the supercolony aggression seems to be minimized by workers being unable to recognize whether an ant is close kin or not, but f. paralugubris workers behave unaggressively in artificial bioassay encounters even though they do seem able to recognize non - nestmates. in contrast to most supercolonial ants, aggression is rare in f. paralugubris, even towards individuals from other super - colonies. the workers seem to treat most conspecifics amicably, even if recognized as ' outsiders '. does this mean that the potential inclusive fitness payoffs from the family structuring are not in fact realized ? whether lack of discrimination is adaptive depends on the inclusive fitness costs of accepting strangers, compared with the costs of aggression and of erroneously behaving aggressively to group members (figure 2). in f. paralugubris, it seems that workers are prone to making mistakes in recognition, probably because the large number of queens in each family cluster increases genetic diversity within, and decreases differentiation between, the clusters. this is likely in f. paralugubris, because the population structure found by holzer. of particular importance in this regard, keller and colleagues previously found that the dispersal of queens in f. paralugubris is limited. this would help to guarantee that brood care by the young workers is directed towards relatives, even if the workers later disperse. workers in f. paralugubris increase their inclusive fitness not by territorial aggression, but by rearing related queens and males that disperse and compete with queens and males from less related nests in other clusters over recruitment as a breeder. holzer. found that especially males are prone to disperse and spread genes across the clusters. in other words, relatedness will be above zero in the brood - rearing context, which is most important for inclusive fitness, even where territorial aggression is lacking (figure 1). the current study by holzer. confirms previous findings by keller and colleagues that family structures are more obvious in queens and the brood than in old workers in another supercolonial formica ant. this suggests that young workers take care of related broods, even if they drift away from their relatives later in their lives. whether similar patterns of genetic structure and behavior also occur in invasive supercolonies remains to be seen. it is also worth noting that acceptance of non - kin in the context of artificial aggression bioassays does not necessarily mean that they are always accepted in natural contexts more directly connected to inclusive fitness. favoring individuals that are likely to be relatives is adaptive only in contexts where the inclusive fitness benefits of discrimination are large. if competition for breeding places occurs mainly by the recruitment of sexuals into the breeding population, workers should be most prone to discriminate in contexts more directly linked to their inclusive fitness, such as behavior towards queens and males, and brood care. thus, worker ants in formica supercolonies might be able to direct the benefits of brood care mainly to the ' right ' individuals as dictated by kin selection theory. the other side of the non - aggression coin is whether it is harmful to the rearing of related reproductives to allow strangers into the territory. as long as this does not harm the production of reproductives in your own nest, the risk of aggression is perhaps not worth taking. this might be the case when resources are abundant and the benefits of territorial defense are subsequently small. wood ants tend aphids, a resource that thrives when ants are abundant, so resource limitation may not be too severe in their case. it will be very interesting to see whether the putative family clusters in wood ants also correspond to foraging areas and the flow of resources, and whether ants are more prone to discrimination at food sources, especially when resources are scarce. study of nutrient flow within a supercolony of the argentine ant has also shown that a seemingly uniform supercolony might actually consist of separate nest networks across which food resources are not shared. ideally, relatedness should be measured at the level of such functional units, in both native and invasive supercolonies. the problem of indiscriminate altruism demonstrates how complicated assessing the adaptive benefits of a behavioral strategy can be. in particular, we need to consider all the contexts in which a behavioral rule is applied. the ability to readily recognize and respond to environmental information is an important constraint on adaptation, and organisms are not always able to adjust their behavior to make an optimal response to a given situation. instead, they might need to rely on rules that work on average. in the case of ant workers in a supercolony, the payoffs of behavioral rules need to be assessed across all the contexts in which they are used, over seasons, over the lifetime of the workers, but also over the lifetime of the supercolony. there may be cases where workers in a supercolony seem to be in a dilemma. they are obeying the behavioral rules that made their family big and strong, but this has brought them to a situation where the rules no longer pay off. but this is only a dilemma for evolutionary theory if we assume that adaptation must always be perfect. even if the rules seem maladaptive in some cases, the evolutionary benefits may have already been reaped in another. i thank joan strassmann, dave queller, jes s pedersen and tobias uller for inspiring discussions on unicoloniality, and tobias uller for comments on the manuscript as well. | vast supercolonies of interconnected nests formed by unicolonial ant species are the largest cooperative groups of animals known. research published recently in bmc evolutionary biology reveals that a supercolony can be more genetically structured than previously thought, comprising several extended families. surprisingly, the families coexist peacefully, even though they seem to recognize each other as non - kin. |
the terms phase iii and multiple sclerosis were used for a pubmed search with no time restriction in march 2014. studies were eligible for inclusion if they (1) were phase iii trials of dmds in pwrms and (2) reported the rate of neoplasms and cancers observed. conference abstracts were excluded as were studies investigating only an acute dose effect or follow - up studies of previously published phase iii trials. the proportion of pwrms who developed cancer was extracted from each study. in trials where multiple doses were tested, given the relatively short and very similar duration of analyzed studies, the proportion of pwrms in whom cancer occurred was used as an estimate of the rate of cancer. the cancer rate in the treatment group of clarity was first compared to the combined cancer rate of all treatment groups of placebo - controlled phase iii trials of other dmds. the cancer rate in the placebo group of clarity was then compared to the combined cancer rate of the placebo groups of all other phase iii trials. study heterogeneity was calculated using test and publication bias was assessed using a funnel plot. statistical analysis was performed using graphpad prism, revman, and stata 13.1 (statacorp, college station, tx). analysis was similarly done using the cancer rates reported in a recently completed phase iii trial on the effect of oral cladribine on time to conversion to clinically definite ms in patients with a first demyelinating event (oracle ms). furthermore, meta - analysis pooling using a random - effects model was performed based on risk difference to assess any excess risk from cladribine. risk difference was selected as a measure rather than the more commonly used measures of relative risk or relative odds because when incidence rates are low and the comparator arm has zero cases (as in clarity), these latter measures are not reliably estimated. the terms phase iii and multiple sclerosis were used for a pubmed search with no time restriction in march 2014. studies were eligible for inclusion if they (1) were phase iii trials of dmds in pwrms and (2) reported the rate of neoplasms and cancers observed. conference abstracts were excluded as were studies investigating only an acute dose effect or follow - up studies of previously published phase iii trials. the proportion of pwrms who developed cancer was extracted from each study. in trials where multiple doses were tested, given the relatively short and very similar duration of analyzed studies, the proportion of pwrms in whom cancer occurred was used as an estimate of the rate of cancer. the cancer rate in the treatment group of clarity was first compared to the combined cancer rate of all treatment groups of placebo - controlled phase iii trials of other dmds. the cancer rate in the placebo group of clarity was then compared to the combined cancer rate of the placebo groups of all other phase iii trials. study heterogeneity was calculated using test and publication bias was assessed using a funnel plot. statistical analysis was performed using graphpad prism, revman, and stata 13.1 (statacorp, college station, tx). analysis was similarly done using the cancer rates reported in a recently completed phase iii trial on the effect of oral cladribine on time to conversion to clinically definite ms in patients with a first demyelinating event (oracle ms). furthermore, meta - analysis pooling using a random - effects model was performed based on risk difference to assess any excess risk from cladribine. risk difference was selected as a measure rather than the more commonly used measures of relative risk or relative odds because when incidence rates are low and the comparator arm has zero cases (as in clarity), these latter measures are not reliably estimated. including clarity, a total of 11 phase iii trials in pwrms were eligible for inclusion in the analysis. dmds investigated in these studies were cladribine, dimethyl fumarate, fingolimod, teriflunomide, natalizumab, alemtuzumab, and glatiramer acetate. the combirx trial (interferon [ifn]--1a plus glatiramer acetate) was excluded because malignant and nonmalignant neoplasms were not reported separately. seven of the included studies compared dmd with placebo ; 4 compared dmd with ifn--1a. one trial compared natalizumab as an add - on to ifn--1a with placebo added to ifn--1a. study characteristics, references, and annualized cancer rate of dmds included in the analysis are provided in table e-1 at neurology.org/nn. the cancer rate in the clarity treatment group was 0.34% and thus not different from all other treatment groups of placebo - controlled trials (0.6%, p = 0.4631). among these treatment groups, the cancer rate in clarity was the third lowest observed (figure 1). among placebo groups, the cancer rate of zero in clarity was the lowest observed and significantly lower than the combined cancer rate of all other placebo groups (1.19%, p = 0.0159 ; figure 2). forest plot of malignancy rates in treatment groups from phase iii trials with a placebo group. forest plot of malignancy rates in placebo groups of phase iii trials. comparing all trials (i.e., placebo - controlled and active comparator ifn--1a controlled), no difference was detected in the cancer rate of clarity treatment arms vs all other trial arms (0.67%, p = 0.3669 ; figure 3). no difference in cancer rate was detected between treatment arms of clarity and oracle ms (0.34% vs 0.49%, p = 0.6546) ; both trials used identical doses of cladribine. comparison of the total neoplasm rate (including both malignant and nonmalignant neoplasms) in clarity treatment arms (1.13%) vs oracle ms treatment groups (0.98%) did not reveal any difference (p = 1). the clarity placebo group had a lower neoplasm rate (0%) than the oracle ms placebo group (2.91%, p = 0.0012). random - effects pooling based on risk difference indicated a cancer risk profile from the clarity trial comparable to that of other dmds (cladribine had a lower risk difference compared to 4 other trials ; figure 4). we further noted that the clarity trial did not indicate a higher risk difference than the other induction dmd, alemtuzumab, in previously untreated pwrms in the cohen 2012 trial. given the heterogeneity of treatments, significant heterogeneity between trials was detected (i = 56.3%, p = 0.009). alem = alemtuzumab ; ci = confidence interval ; clad = cladribine ; dime = dimethyl fumarate ; fing = fingolimod ; nata = natalizumab ; teri = teriflunomide. the main findings of this study are that (1) the rate of cancer was similar between the only phase iii trial of cladribine and all other phase iii trials of dmds in pwrms, and (2) a significant difference emerged when comparing the placebo groups of clarity (no cancers) with the placebo arms of all other phase iii trials included in this study. these findings were corroborated by a cancer rate difference in clarity that was comparable to that of all other phase iii trials for dmds for pwrms. focusing on cancers observed in pwrms treated with cladribine in clarity (and oracle ms) might lead to the suspicion that cladribine plays a causative role, given the fact that 3 pwrms on cladribine developed cancer whereas none on placebo did. however, comparison with other dmds shows that the number of malignancies in clarity was not increased. indeed, the cancer rate in clarity was among the lowest observed for any treatment group, suggesting that the cancers developed on cladribine are a reflection of the background risk in the population. moreover, the short latency between initiation of treatment and cancer diagnosis in clarity (18 months) renders a causal relationship unlikely. we also note that in contrast to the trials of alemtuzumab, cladribine was compared with placebo (rather than an active comparator). investigations of the general risk of cancer among pwrms in large cohorts reported conflicting results. in a danish study using population - based registers of ms and cancer, there was no evidence of an overall increased risk of cancer after a diagnosis of ms ; however, they did report a small excess risk of breast cancer among women. a recent systematic review of the incidence and prevalence of cancer in ms reported substantial variation in reported estimates between studies. overall, the risk of any cancer was most commonly reported to be lower in pwrms compared to the general population. cancer risk appeared to be higher for malignant brain tumors and for cancers affecting the urinary tract system. however, potential surveillance bias and variation of age and sex of pwrms across studies stand in the way of more definitive conclusions. the data from our meta - analysis of dmds in pwrms are supported by the long - term outcome of people with leukemia treated with cladribine. in a 20-year follow - up report of 88 patients diagnosed with hairy cell leukemia before the age of 40, no increase in the incidence of secondary malignancies was detected. according to the preliminary report of the 96-week phase ii oral cladribine as add - on to ifn- therapy in patients with active multiple sclerosis (onward) trial, in which oral cladribine (dose 3.5 mg / kg) was compared vs placebo as an add - on to interferon - beta treatment, 1 of 124 patients, a 57-year - old woman with a 25-year smoking history, developed squamous cell carcinoma about 2 years following first cladribine exposure. cladribine administered as an injection or infusion has previously been investigated in placebo - controlled phase ii trials of varying dosages, with results demonstrating reduced disease activity (clinically and on mri) with no reported cancer signal. limitations of our study include the inevitable variation between trials in terms of sample size, inclusion criteria, follow - up period, and dosing regimens ; the fact that not all phase iii trials for approved dmds in pwrms reported data on cancer rates ; and the fact that all cancers were considered together, i.e., regardless of affected organ(s). furthermore, clarity was 2 months shorter than most of the other trials included, which lasted 2 years. mathematically, a slightly lower number of cancers would therefore be expected in clarity due to the shorter observation period. however, there is no indication that this difference had any material impact on the results of our meta - analysis. we also acknowledge that in a randomized trial such as clarity, if the cancer rate is truly low, it should also have been low relative to the placebo group in its own trial. however, the apparently high risk in clarity relative to placebo may be due to the use of the risk ratio rather than the risk difference ; the former is inappropriate in this context because of the zero cases in the clarity placebo group. our meta - analysis of risk differences, which does not require arbitrary substitutions for the zeros, suggests that the risk difference for several other comparable drugs is higher than that in clarity. our data suggest that although current evidence can not rule out an increased risk of cancer on cladribine, it also can not confirm that such a risk exists. a more definitive assessment of cancer risk will only be feasible through long - term follow - up, be it of people treated with cladribine (prospective observational long - term safety registry of multiple sclerosis patients who have participated in cladribine clinical trials the results of our meta - analysis should encourage further investigation into the potential use of cladribine as a dmd for pwrms. k.s. has been supported by a higher education funding council for england (hefce) clinical senior lectureship. altmann consulted for merck & co and received research support from multiple sclerosis society of great britain and northern ireland. turner is on the scientific advisory board for biogen, novartis, and sanofi aventis. m. marta received travel funding from biogen and abbott laboratories and received research support from merck - serono. d. baker has a patent filed concerning the treatment of multiple sclerosis with cladribine and other compounds ; has consulted for canbex therapeutics ; received research support from genzyme sanofi, national multiple sclerosis society, and academic entities ; has stock options from canbex therapeutics ; and provided legal consult for teva. j. chataway is on the scientific advisory board for novartis ; received travel funding and/or speaker honoraria from novartis, teva, and sanofi ; and received research support from novartis, uk mrc, and uk national institute of health research (nihr) university college london hospitals / ucl biomedical research centres funding scheme. k. schmierer is a principal investigator of trials sponsored by novartis, roche, and teva. he has received speaking honoraria from, and served on advisory boards for, biogen, novartis, teva, merck - serono, and merck inc ; and received research support from novartis, the multiple sclerosis society of great britain & northern ireland, the national ms society, the royal college of radiologists, and barts charity. | objective : to compare the cancer risk of cladribine and other disease - modifying drugs (dmds) in trials of people with relapsing multiple sclerosis (pwrms).methods : meta - analysis of phase iii trials of licensed dmds for pwrms and a phase iii trial of cladribine (clarity). cancer rates were compared using fisher exact test.results:eleven trials were included. investigated treatments included cladribine, dimethyl fumarate, fingolimod, teriflunomide, natalizumab, alemtuzumab, and glatiramer acetate. the cancer rate in the clarity treatment group (0.34%) was not increased compared to all other treatment groups, whether including placebo - controlled trials only (0.6%, p = 0.4631) or all trials, i.e., including those with an active comparator arm (0.67%, p = 0.3669). no cancer was reported in the clarity placebo group, whereas the combined cancer rate of all other placebo groups was 1.19% (p = 0.0159). the cancer rate of zero in the clarity placebo group was also lower than that in the phase iii trial of cladribine in people with clinically isolated syndrome (oracle ms, 2.91%, p = 0.0012). in fact, no difference was detected between cancer rates in the treatment groups of clarity (0.34%) and oracle ms (0.49%) (p = 0.6546).conclusions : our study does not support an increased cancer risk from cladribine in the doses used in clarity and oracle ms, which previously contributed to refusal of market authorization of cladribine in europe. longer - term follow - up is required to assess the safety profile of cladribine, as well as currently licensed dmds, to definitively assess cancer risk. |
hypertension is a major risk factor for the atherosclerotic vascular disease in coronary, cerebral, and peripheral arteries. examination of atherosclerotic lesions has revealed that each lesion contains significant amounts of three cellular elements : smooth muscle cell proliferation ; large amounts of connective tissue matrix, including collagen, elastic fibers, and proteoglycans ; and an accumulation of intracellular and extracellular lipid. these atherosclerotic plaques arise because altered endothelial permeability allows certain reactive macromolecular plasma proteinsd (plansma low - density lipoproteins and fibrinogens, which are normally largely confined to the circulation) to penetrate endothelium and interact with charged components of the connective tissue gel of the arterial wall or other tissues. with recent advances in our knowlege of lipoprotein metabolism apob / a ratio, especially, are now considered to be good discriminators of atherosclerosis as well as high - density lipoprotein cholesterol (hld - cholesterol) and low - density lipoprotein (ldl), there have been several studies on plasma lipid in the hypertensive patient, but few on apolipoproteins in the hypertensive patient, carried out in this country. we measured the plasma apoa and apob as well as the total cholesterol, triglyceride, hld cholesterol, and beta - lipoprotein (lpb) in hypertensive patients and compared them with those of the normal subjects in the control group to see whether the risk indices for coronary artery disease are increased in hypertensive patients. we also tried to compare the plasma lipid and apolipoprotein levels between the blood pressure controlled and blood pressure uncontrolled subgroups, and between the patients with complications and those without complications. we studied 115 hypertensive patients, who visited or were admitted to internal medicine service at koryo general hospital and a control group of 100 normal subjects. in the hypertensive group, there were 68 men and 47 women, and in the control group, 52 men and 48 women. we divided the 115 hypertensive patients into subgroups, a and b, with a representing a blood pressure - controlled (systolic blood pressure under 160 mmhg and diastolic blood pressure under 95 mmhg) hypertensive subgroup, and with b representing uncontrolled (systolic blood pressure over 160 mmhg or diastolic blood pressure over 95 mmhg) hypertensive subgroup. subgroup a included 16 patients, 8 males and 8 females, and subgroup b, 99 patients, 60 males and 39 females. then, we divided the same 115 hypertensive patients into subgroup c and subgroup d. subgroup c included patients without complications according to a fundoscopic examination, a chest x - ray and an electrocardiogram, and subgroup d included those in whom at least one complication was found on the above tests : retinopathy on the fundoscopic examination, evidence of hypertensive cardiovascular disease or atherosclerotic heart disease on the chest x - ray, or strain, ischemia, or infarction on the 12-lead electrocardiogram. subgroup c included 41 patients, 27 males and 14 females ; and subgroup d included 74 patients, 41 males and 33 females. blood samples were taken in the morning after the subjects had fasted for 12 hours. the plasma apoa and apob levels were measured by the radial immunodiffusion method ; total cholesterol and triglyceride levels by the enzyme method ; hld cholesterol by the heparin - mn precipitation method ; and lpb by the immunoturbidity method. the apolipoproteins and lipid values for men and women in the normal control group are shown in table 1. mean plasma apoa and apob levels of the normal control group were 1.8770.447 and 1.2720.268 (gm mean plasma total cholesterol and hld cholesterol levels in the normal control group were 183.836.6 and 52.412.2 (mg / dl), respectively. those of the female control group were significantly higher than those of the male control group (p.005, respectively). the mean plasma triglyceride level in the hypertensive group was 193.1115.5 (mg / dl), which was significantly higher than that in the control group (p.005, respectively). the mean plasma triglyceride level in the hypertensive group was 193.1115.5 (mg / dl), which was significantly higher than that in the control group (p<.005). the apob / a ratio in the hypertensive group was 0.7370.212, which was not significantly different from that of the control group. the total cholesterol / hld cholesterol ratio in the hypertensive group was 3.9861.003, which was significantly higher than that in the control group (p<.05). the comparisons of apolipoproteins and lipid levels between the hypertensive subgroups and the control group are summarized in table 2. in subgroup a, the mean plasma apoa, apob, hld cholelsterol, and total cholesterol levels and the total cholesterol / hld cholesterol ratio were higher than those in the normal control group : these differences, however, were not significant. the mean plasma triglyceride and lpb levels were 211.6142.7 (mg dl) and 463.1142.7 (mg dl) which was significantly higher than those in the normal control group (p<.005, p<.05, respectively). the apob / a ratio was lower in subgroup a than in the control group, but still without significance. in subgroup b, / l), and the total cholesterol 199.140.5 (mg / dl), triglyceride 190.1111.1 (mg / dl), lpb level 469.1134.5 (mg dl) and the total cholesterol / hdl cholesterol ratio was 3.9821.023. all of these levels were significantly higher than those in the control group (p<.05 or 0.005). in subgroups c and d, the mean plasma apoa levels were slightly higher than those in the normal cotrol group, but there were no significant differences between each subgroup and the control group. the mean plasma apob levels in subgroups c and d were significantly higher than those in the normal control group (p<.005). the mean plasma total cholesterol levels in subgroup d was significantly higher than in the control group (p<.005), but there was no significant difference between subgroup c and the control group. the mean plasma triglyceride and lpb levels in subgroups c and d were significantly higher than those in the control group (p<.005). in subgroups c and d, the apob / a ratio tended to be higher than that in the control group, without significance. the total cholesterol / hld cholesterol ratio of subgroup d was significantly higher than that of the control group (p<.05), but not that of subgroup c. between subgroups a and b, and subgroups c and d there were no significant differences in all kinds of levels and ratios. the chief cause of the excess morbidity and mortality rate in the hypertensive subject is its increased propensity to atherosclerotic disease. when atherosclerosis has developed, treatment of hypertension alone may lower the incidence of stroke, but will have little effect on the mortality from coronary heart disease. antihypertensive agents, such as beta - adrenergic recepter blocker and diuretics may cause hyperlipidemia. apob. ldl. triglyceride. and total cholesterol are closely related to atherogenesis but apoa i and hld cholesterol are inversely related to it. apoa is the main apolipoprotein of hld and the amino acid sequence of apoa i and apoa ii are known. apoa i is an activator of lecithin : cholesterol acyltransferase (lcat) and acts as a protector against atherogenesis. apob is a structure protein of chylomicron, vldl, and ldl, and interacts with the ldl receptor of the peripheral cells such as the skin fibroblast, the intimal endothelial cells, and the smooth muscle cells of the arterial walls, as well as with the corresponding receptors of hepatocytes, and with ldl internalization and degradation. the mean plasma apoa level in the normal male and female control groups were 1.8250.473 and 1.934.416 (gm / l) and there were no significant differences between the groups. according to curry., the mean plasma levels of apoa i and apoa ii were 1.430.24 and 0.780.17, respectively, in men, and 1.460.78 and 0.410.46 (gm / l), respectively, in women, which were higher than our results in the control group. observed that the mean plasma levels of apoa i and apoa ii were 1.130.016 and 0.350.38 in men, and 1.240.068 and 0.410.046 (gm reported that the mean plasma apoa i and apoa ii levels were 1.1780.098 and 0.3380.079 (gm / i). we observed that there was a positive correlation between apoa and hld cholesterol (r=0.56, p<.005) in the control group. in the hypertensive group and all of its subgroups, those subgroups with and without complications and the controlled and uncontrolled hypertensive subgroups, the mean plasma apoa levels were higher than in the control group, but the differences did not reach statistical significance. our data showed that the mean plasma apob level in the control group was 1.2520.253 (gm 278 (gm / l) in women and avogaro. reported that the mean plasma apob level was 1.2630.03 (gm mean plasma apob level was 1.070.22 in men and 0.90.2 (gm / l) in women, which was lower than our result in the control group. in the control group, there were positive correlations between apob and the total cholesterol (r=0.723, p<.005), and between apob and lpb (r=0.67, p<.005) in our study. in the hypertensive group and in three of its subgroups (those subgroups with and without complications, and the subgroup of uncontrolled hypertensive patients) the mean plasma apob levels were significantly higher than those in the control group (p<.005), but not higher than those in the controlled hypertensive subgroup. no other data were available concerning the levels of the apoa and apob relating to hypertension. in this study, the mean plasma hld cholesterol level in the normal control group was 52.412.2 (mg / dl), which was significantly higher in the female than in the male control group (p<.005). yim. observed that the mean plasma hld cholesterol levels were 48.813.5 and 46.05.2 (mg / dl), respectively, which were lower than our results for the control group. the mean plasma hld cholesterol levels were 48.211.4 (mg / dl) in men and 52.413.2 in women, which were similar to our results. in the hypertensive group, the mean plasma hld cholesterol level in our study was 51.810.4 (mg / dl), which was similar to that in the control group, which was in accord with the reports by lee. and we observed that the mean plasma total cholesterol level in the normal control group was 177.529.8 (mg / dl) and that women tended to have a higher total cholesterol level than men ; this difference was not statistically significant, however, lee., yim., and sohn. reported that the mean plasma total cholesterol levels were 170.335.4, 172.123.3, and 172.278.9 (mg / dl), respectively, which results also represented no significant difference between that for the male and that for the female control groups. in the hypertensive group of our study the mean total plasma cholesterol level was significantly higher than that of the control group (p<.01)., yim., ryoo., and sohn. observed also had a significantly higher value for the total cholesterol level than their control group had. our study showed that the hypertensive subgroup with complications and the uncontrolled hypertensive subgroup have significantly higher values of total cholesterol than the control groups, but the hypertensive subgroup without complications and the controlled hypertensive subgroup have not. in our study, in the control group, the mean plasma triglyceride level was 13057.7 (mg / dl), and men tended to have a higher value for the triglyceride level than women ; this difference was not statistically significant, however. according to sohn men also tended to have a higher value for the triglyceride level than women, in their studies. in the hypertensive group, our data showed that the mean plasma triglyceride level was 190.1111.1 (mg / dl), which was significantly higher than in the normal control group (p<.005). lee., ryoo., and sohn. also observed that hypertensive patients have a significantly higher value of triglyceride level than a control group. we observed that all of the hypertensive subgroups : those subgroups with and without complications and the controlled and uncontrolled hypertensive subgroup had a significantly higher value for the triglyceride level than the normal control group (p<.005). in the control group, the mean plasma lpb level that we obtained was 395.689.3 (mg / dl) and there was no significant difference in the levels between the male and the female control groups. observed that the mean plasma lpb levels were 230.943.5 and 424148.9 (mg / dl), which represented no significant difference between the male and the female control groups. our data showed that the mean plasma lpb level was 468.3135.1 (mg / dl) in the hypertensive group, which was significantly higher than that in the control group (p<.005). sohn., observed that the hypertensive group also has a significantly higher lpb level than the control group, but according to ryoo. mean plasma lpb level was not significantly different between the control and the hypertensive groups. our data showed that all of the hypertensive subgroups had significantly higher lpb levels than the control group (p<.005). in the normal control group of our study the ratios of apob / a and total cholesterol / hld cholesterol were 0.7150.236 and 3,6670.394 and there was no significant difference between those for men and those for women. in the hypertensive group the apob / a ratio was 0.7320.212 which was higher than that in the control group. the total cholesterol / hld cholesterol ratio was 3.9361.003, which was significantly higher than that in the control group (p<.05). the total cholesterol / hld cholesterol ratios in hypertensive subgroup with complications and the uncontrolled hypertensive subgroup were significantly higher than those in the control group but the other hypertensive subgroups provided no significant difference when compared with the control group. lipid metabolism in the hypertensive patient is not well understood at present, but several reports shown that patients treated with beta - adrenergic blockers and/or diuretics may develop hyperlipidemia. the current publications in circulation dealing with the effects of beta - adrenergic receptor blockers on specific elements of lipid metabolism sometimes provide conflicting information. it is most often reported that treatment with beta - blockers results in an elevation of the triglyceride level, while total cholesterol remains unchanged. current discussion centers around the possibility of augmented resynthesis of triglycerides in the liver from the increased supply of free fatty acids. the increased supply of fatty acids is due to a mechanism of pronounced lipolysis, independent of catecholamines, which become active in response to the inhibition of catecholamine - induced lipolysis. the finding that diuretics induce changes in the levels of plasma lipids and lipoproteins an element of contradiction as well. diuretic - induced elevation of ldl cholesterol as well as vldl triglyceride has been reported, while hdl cholesterol, on the other hand, appears to be essentially unaffected by diuretics. inas much as in the studies we are referring the reader to, there were no indications that hepatic lipoprotein synthesis increased or that vldl underwent degradation, the possibility of hemoconcentration being caused by diuretics may be excluded with some certainty, in that the rise in ldl is attributed to a reduction of ldl catabolism. in our study the mean plasma hdl cholesterol was not / significantly different between the hypertensive group and the control group. the mean total cholesterol level was elevated significantly in the hypertensive patients with the exception of those in the subgroup without complications and controlled hypertensive subgroup. apob, lpb, and triglyceride were significantly higher in the hypertensive patients in general, even those in the subgroup without complications and those whose hypertension was controlled, than in the normal subjects. however the apob / a and total cholesterol / hdl cholesterol ratios were variable. | we measured apolipoprotein a (apoa), apolipoprotein b (apob), and lipid levels in 115 hypertensive patients and 100 normal subjects in order to evaluate the risk of atherosclerotic heart disease in hypertensive patients. the hypertensive patients were divided into the following subgroups : blood pressure controlled (a) and uncontrolled hypertensive subgroup (b), and without complication (c) and with complication (d).in the hypertensive group, the mean plasma apoa, apob, total cholesterol, triglyceride and beta - lipoprotein (lpb) levels were significantly higher than in the normal control group. the plasma high density lipoprotein cholesterol (hdl - cholesterol) level was not significantly different between the hypertensive group and the normal control group. apob / a ratio in the hypertensive group was higher than in the normal control group, but the difference was not significant statistically.in the hypertensive subgroups, plasma apo a was higher in all the hypertensive subgroups compared to normal control group, but these differences were not statistically significant. in subgroup b, c and d, the plasma apob level increased significantly as compared to the normal control group, but not so in the subgroup a. all of the subgroups had significantly higher levels of triglyceride and lpb level than the normal control group. hld cholesteol level of all of the subgroups did not show any significant difference as compared to the normal control group. in subgroup b, c and d, the apob / a ratio was not significantly different from the normal control group. in the subgroup a, apob / a ratio was lower than in the normal control group, this was not significant statistically. between subgroup a and b, and subgroup c and d, all of the plasma lipids and apolipoproteins did not show significant differences.thus our results showed that apob, lpb and triglyceride, which are closely related to atherosclerotic heart disease, were significantly increased in any of hypertensive subgroups compared to the normal control group. |
prostate cancer constitutes a major health burden, being the most common non - cutaneous malignancy among men in developed countries. in 2007, almost 800,000 new cases of prostate cancer and 250,000 deaths from this disease were estimated to have occurred worldwide. the highest incidence of prostate cancer is observed in the usa, with 192,280 new cases and 27,360 deaths expected in 2009, thereby being the second most common cause of cancer - related death. early autopsy studies have shown a high prevalence of clinically undetected prostate cancer at time of death. in the usa, more than one in three men over 50 years of age had histologic evidence of prostate cancer at autopsy and this prevalence was observed to increase with age, with more than 67% of men aged over 80 years having prostate cancer at time of death. these findings indicate that a high proportion of prostate tumors are clinically insignificant and will never lead to a lethal outcome. furthermore, the introduction and widespread application of prostate - specific antigen (psa) testing has led to increased detection of early - stage, low - volume, non - palpable tumors. this has in turn raised concerns of increased overdiagnosis and unnecessary treatment of indolent disease. to this end, new strategies to help clinicians distinguish between lethal and indolent prostate cancer are urgently needed. prostate cancer is one of the most heritable cancers in men and recent studies have revealed numerous genetic variants associated with this disease. this review will give an overview of the current knowledge of prostate cancer genetics, with a special focus on the ability of genetic variants to predict more aggressive forms. a family history of prostate cancer is one of the strongest risk factors, and twin studies suggest that as much as 42% of the disease risk is explained by heritable factors. attempts to decipher the heritable component of prostate cancer based on candidate gene association studies and genome - wide linkage studies in multiple case families have suggested numerous prostate cancer susceptibility genes and loci. however, an inability to replicate reported linkage and association findings suggest that prostate cancer is genetically complex with multiple common low - penetrance genes involved in prostate cancer predisposition. recently, genome - wide association studies (gwas) have emerged as a powerful method to identify genomic low - risk susceptibility regions for complex diseases, including cancer. through genotyping platforms that explore hundreds of thousands of single nucleotide polymorphisms (snps) simultaneously, it is possible to screen the complete genome for common genetic variation associated with the disease of interest. in 2006 this region was initially identified through linkage analysis in icelandic families with prostate cancer, followed up by association analysis in three independent case - control populations, and separately through admixture mapping in african americans. subsequent gwas and region - focused studies have revealed five distinct linkage disequilibrium blocks harboring prostate cancer susceptibility alleles at 8q24 [11 - 17 ]. the 8q24 region has also been shown to harbor susceptibility alleles for breast cancer, colorectal cancer, bladder cancer, and ovarian cancer. the 1.2 mb sequence at 8q24 containing all observed risk alleles does not code for any known genes, and the biologic mechanisms underlying these associations are unknown. the oncogene c - myc is the closest distal gene to this region and it has been suggested that the observed associations reflect long - range control of myc expression ; however, further functional studies are needed to reveal the role that these variants play in cancer susceptibility. to date, 29 distinct genetic loci harboring prostate cancer risk alleles have been identified and consistently replicated (table 1). in general, the effect of variants in these regions on prostate cancer risk is modest, with odds ratios typically ranging between 1.1 and 1.3. it has been estimated that hitherto identified variants together explain approximately 22% of the familial risk of prostate cancer, and it is anticipated that many more prostate cancer susceptibility variants will be identified in the future. established prostate cancer susceptibility alleles genes within the linkage - disequilibrium block defined by the associated variant : bik, bcl2-interacting killer ; ctbp2, c - terminal binding protein 2 isoform 2 ; eefsec, elongation factor for selenoprotein translation ; ehbp1, eh domain binding protein 1 ; flj20032, hypothetical protein loc54790 ; hnf1b, hepatocyte nuclear factor 1 homeobox b ; itga6, integrin alpha chain 6 ; jazf1, juxtaposed with another zinc finger gene 1 ; klk3, kallikrein 3 ; lmtk2, lemur tyrosine kinase 2 ; msmb, -microseminoprotein isoform a precursor ; nkx3 - 1, nk3 transcription factor related locus 1 ; nudt11, nudix - type motif 11 ; pdlim5, pdz and lim domain 5 isoform d ; ppp1r14a, protein phosphatase 1 regulatory inhibitor ; slc22a3, solute carrier family 22 member 3 ; slc25a37, mitochondrial solute carrier protein ; thada, thyroid adenoma associated isoform 1 ; tnrc6b, trinucleotide repeat containing 6b isoform 2 to date there is no reliable way of predicting whether prostate cancer will be an aggressive, fast - growing disease or a non - aggressive, slow - growing type of cancer. in general, a combination of tumor staging (using the tumor, node, metastasis staging system), tumor grading (using the gleason scoring system) and diagnostic psa serum levels are used to classify patients into different prognostic risk groups to guide clinicians in treatment decisions. in genetic association studies, patients with prostate cancer are commonly classified as having a more aggressive form of the disease if they fulfill any of the following criteria : (1) disease spread outside of the prostate gland, or presence of cancer in the lymph nodes or other metastatic sites ; (2) presence of poorly differentiated cancer as indicated by a high gleason score (that is, 4 + 3 = 7 or higher) ; or (3) a serum psa level associated with a high likelihood of extensive disease (that is, > 20 ng / ml). several studies have explored the capacity of established prostate cancer risk variants to distinguish between less aggressive and more aggressive disease [9 - 13,24 - 46 ]. overall, results are inconclusive, with some studies reporting stronger associations for some of these variants among patients with more aggressive prostate cancer, while others did not. in a large replication study from the practical (prostate cancer association group to investigate cancer associated alterations in the genome) consortium, which evaluated genetic variants at chromosome 3p12, 6q25, 7q21, 10q11, 11q13, 19q13 and xp11 among 7,370 prostate cancer cases and 5,742 controls, fitzgerald and coworkers assessed the same seven variants and an additional six variants at chromosome 7p15, 8q24, 10q26, and 17q12 in a population - based study comprising 1,308 cases and 1,267 controls for association with family history and clinical features of more aggressive disease. no association was observed between any of the evaluated risk variants and a composite measure of disease aggressiveness ; however, two variants, rs10993994 at 10q11 (p = 0.02) and rs5945619 at xp11 (p = 0.03), were nominally significantly associated with gleason score. most of the published studies exploring established risk variants with respect to prostate cancer aggressiveness have had several limitations, including small sample size, heterogeneous definition of aggressive disease across multiple study populations, and reliance on clinical grading and staging of tumors. to address these limitations, kader and coworkers evaluated 20 established risk variants in 17 distinct genomic regions among 5,895 patients with prostate cancer who were of european descent and who underwent radical prostatectomy for treatment of prostate cancer. based on the entire prostate gland, each tumor was uniformly graded and staged using the same protocol. tumors with pathologic gleason scores of 4 + 3 or higher, or pathologic stage of t3b or higher, or non - organ confined disease, were defined as more aggressive disease (n = 1,253) ; tumors with organ confined disease, pathologic gleason score of 3 + 4 or lower, and pathologic stage of t2 were classified as less aggressive disease (n = 4,233). for 18 of the 20 variants explored, no significant difference was observed in risk allele frequencies between patients with more aggressive and less aggressive disease. two variants were significantly associated with disease aggressiveness : snp rs2735839 downstream of the kallikrein 3 gene (klk3 ; p = 8.4 10), which is the gene coding for psa ; and snp rs10993994 in the microseminoprotein gene (msmb ; p = 0.046). to reduce the possible impact of heterogeneity in the definition of aggressive disease, risk variants were also tested for association with gleason score and pathological stage separately. snp rs2735839 in the klk3 gene (p = 7.7 10) and snp rs10993994 in the msmb gene (p = 0.02) were the only variants associated with gleason score. for tumor stage, only snp rs2735839 in the klk3 gene was significant (p = 1.9 10). of note, for both of these variants, the alleles that are associated with increased risk for prostate cancer were more frequent in patients with less aggressive disease. since these risk alleles have been shown to strongly associate with higher psa levels among population controls, it is possible that the observed association with aggressive disease may partly reflect a psa detection bias. it should be noted that the lack of association between established prostate cancer risk variants and disease aggressiveness does not imply non - existence of such genetic variants in the genome. all susceptibility variants identified to date were discovered using case - control designs comparing unaffected individuals with all types of patients with prostate cancer. it has been argued that a more effective design to identify genetic variants associated with aggressive disease should involve a case - case design contrasting patients with more and less aggressive disease. support for this idea was recently provided in a study including 4,829 patients with more aggressive disease and 12,205 patients with less aggressive disease from seven study populations. initially, publicly available genotype data for approximately 27,000 genetic variants across the genome were explored for association with disease severity among patients with prostate cancer from four populations examined in the cancer genetic markers of susceptibility study using a case - case design. a subset of variants (n = 74), showing association within each cancer genetic markers of susceptibility study, and where the direction of association was consistent among all four studies, was selected for further evaluation in an additional three study populations from sweden and the usa. this revealed one genetic variant (rs4054823 at 17p12) for which the tt genotype was consistently higher among patients with more aggressive compared with less aggressive disease in each of the seven populations studied (overall p = 2.1 10under a recessive genetic model). if confirmed in independent study populations, this finding is of great importance, not because of immediate clinical utility, but as a proof of principle that genetic variants predisposing to more aggressive prostate cancer exist. in contrast to exploring inherited genetic variants associated with aggressiveness of disease at time of diagnosis, only a few studies have assessed the importance of established risk variants on prostate cancer progression and prognosis. only one study has explored confirmed risk variants in relation to prostate cancer progression. among 320 patients who were recruited from three hospitals in taiwan where they were treated with radical prostatectomy, huang and co - workers explored association between 20 prostate cancer risk variants and biochemical failure defined by recurrence of psa. during a mean follow - up of 38.5 months, biochemical failure occurred in 113 (35%) of the patients. in univariate analysis, three risk variants (rs1447295 at 8q24, and rs7920517 and rs10993994 at 10q11) were associated with psa recurrence. interestingly, these associations remained significant after adjusting for established prognostic factors such as age, preoperative psa level, tumor stage, gleason score, and surgical margin, suggesting that these variants may improve prediction of psa recurrence among patients treated with radical prostatectomy. penney and co - workers explored eight genetic variants at chromosome 8q24, 17q12, and 17q24.3 for association with prostate cancer mortality in three us prostate cancer study populations comprising a total of 6,460 patients of which 493 died as a result of prostate cancer during follow - up. none of the explored variants was associated with prostate cancer mortality, neither in analysis contrasting lethal cases with long - time survivors (alive over 10 years after diagnosis), nor in survival analysis among all patients. a prospective population - based cohort study of swedish patients with prostate cancer explored the association between 16 established risk variants and prostate cancer mortality. in total, 2,875 patients diagnosed between 2001 and 2003 were followed up for prostate cancer mortality through january 2008. overall, 626 (21%) of the patients died during follow - up and of those 440 (15%) had prostate cancer classified as their underlying cause of death. no association between any of the explored variants and prostate cancer mortality was observed, either in exploring individual variants or in assessing the cumulative effect of all variants. additional studies in large populations are needed to comprehensively explore possible associations, although current evidence suggests that established risk variants are not risk factors for prostate cancer outcome. recent gwas studies have been successful in identifying many low - penetrant susceptibility alleles for prostate cancer, and it is anticipated that many more variants will be detected through combined analysis across existing studies, new generations of larger studies, and increasing size of replication studies. individually, each risk variant has a modest effect on disease risk and they will clearly not be useful for individualized risk prediction. however, risk profiles based on a combination of risk variants lead to an appreciable increased risk of disease and there is potential for the predictive power to increase considerably as more risk variants are detected. this may have important implications for targeted prevention and screening programs for prostate cancer through identification of high - risk groups. since there is considerable co - morbidity associated with curative treatment of prostate cancer (surgery or radiotherapy), there is clear clinical utility in detecting genetic markers that can improve discrimination between those patients that will follow a benign course from those with tumors that carry a poor prognosis and for whom curative therapy is indicated. in addition, inherited genetic markers, in contrast to measurement of a tumor - derived product, can be informative at an earlier stage when the disease is potentially curable. however, it is evident that hitherto identified prostate cancer risk variants provide little or no discriminative capacity between indolent and aggressive forms of prostate cancer. large gwas among affected men contrasting more and less aggressive cases, and exploring association with disease progression and prostate cancer mortality, are clearly needed to detect inherited genetic variants associated with aggressive forms of prostate cancer. initial findings indicate that genetic variants predisposing to more aggressive disease exist and this is also supported by recent epidemiological studies proposing a genetic component in cancer prognosis. the detection of inherited genetic markers capable of discriminating between indolent and fatal forms of prostate cancer holds promise to improve detection and clinical management of this disease in several ways. a genetic - based, targeted psa screening strategy may reduce both overdiagnosis and mortality by identifying those men at risk for fatal prostate cancer at a curable stage. in addition, extended tools to guide clinicians in treatment decisions are critical to improve disease prognosis and decrease treatment - induced morbidity. gwas : genome - wide association study ; psa : prostate - specific antigen ; snp : single - nucleotide polymorphism. | prostate cancer is one of the most heritable cancers in men, and recent genome - wide association studies have revealed numerous genetic variants associated with disease. the risk variants identified using case - control designs that compared unaffected individuals with all types of patients with prostate cancer show little or no ability to discriminate between indolent and fatal forms of this disease. this suggests different genetic components are involved in the initiation as compared with the prognosis of prostate cancer. future studies contrasting patients with more and less aggressive disease, and exploring association with disease progression and prognosis, should be more effective in detecting genetic risk factors for prostate cancer outcome. |
the year 2008 marks the 27th anniversary of the first case report of a new disease today known as acquired immunodeficiency syndrome (aids), whose etiological agent is human immunodeficiency virus type 1 (hiv-1) (1). an estimated 38.6 million people are now living with hiv or aids worldwide, and nearly 11 000 people are infected by hiv daily (joint united nations programme on hiv / aids / world health organization). since the documentation of the first aids case, numerous efforts have focused on vaccine and antiviral drug discovery and development, on identifying measures to prevent hiv transmission, on understanding hiv pathogenesis and the associated host immune responses, and on defining the interactions of hiv-1 proteins with human host cell proteins. the latter is crucial to understanding the individual steps of hiv-1 replication and pathogenesis, and provides an essential foundation for the development of safe and effective therapeutic and prevention strategies to combat aids. as a result of these efforts, thousands of published articles have addressed the interaction of hiv-1 proteins with human host proteins. however, each individual publication addresses only one or a few hiv protein host protein interactions making it cumbersome to collect information on all interactions for one particular hiv or cellular protein. the division of acquired immunodeficiency syndrome (daids) of the national institute of allergy and infectious diseases (niaid) recognized the need for a searchable platform to catalog the interactions of individual hiv proteins with host cell proteins. therefore, the development of an hiv-1, human protein interaction database was initiated in collaboration with southern research institute and the national center for biotechnology information (ncbi). development of the hiv-1, human protein interaction database from the peer - reviewed scientific literature available in pubmed was a 7-year effort starting in 2000. a short communication detailing the development of the database and including a visualization of the hiv-1, human protein interaction network has been published recently (2). briefly, more than 100 000 journal abstracts and publications were identified and screened for original research describing interactions between hiv-1 and human host proteins. in addition, new literature is routinely reviewed to identify interactions described in current publications. review of publications by scientific curator staff is organized by individual hiv-1 proteins and catalogued into an access database by extracting the interaction information from the continuous text. as review of individual interactions is completed, data are provided to ncbi incrementally as a set of comprehensive tab - delimited text files and loaded to a ms sql server 2005 database. validated interaction data are integrated into appropriate records in entrez gene and provided as custom reports and downloads per hiv-1 protein through the reports and downloads tools at http://www.ncbi.nlm.nih.gov / projects / refseq / hivinteractions/. the complete dataset is also available for ftp (ftp://ftp.ncbi.nih.gov/gene/generif/hiv_interactions.gz). an update to the database released on 13 november 2007, which included the interaction data set for the hiv-1 env proteins, marked the milestone of completion of the comprehensive hiv-1, human protein interaction database based on original research articles published since 1984. updates to the database based on interactions described in new scientific reports will be released on a recurring basis. the goal in developing this database was to provide scientists in the field of hiv / aids research a concise, yet detailed, summary of all known interactions between hiv-1 and host cell proteins and it has therefore been designed to track the following information for each protein protein interaction identified in the literature : ncbi reference sequence (refseq) protein accession numbers;ncbi entrez gene i d numbers;brief description of the protein protein interaction;keywords to support searching for interactions;national library of medicine (nlm) pubmed identification numbers (pmids) for all journal articles describing the interaction. ncbi reference sequence (refseq) protein accession numbers ; ncbi entrez gene i d numbers ; brief description of the protein protein interaction ; keywords to support searching for interactions ; national library of medicine (nlm) pubmed identification numbers (pmids) for all journal articles describing the interaction. the purpose of the database is to serve as a central interactive interface for viewing an ensemble of the known interactions between individual hiv-1 proteins and human proteins. the hiv-1, human protein interaction database home page (http://www.ncbi.nlm.nih.gov/refseq/hivinteractions/) enables users to simultaneously view and download a variety of reports detailing interactions for each hiv-1 protein. the database is structured by initial searches for the nine hiv proteins (e.g. gag, pol, env, tat, rev, nef, vif, vpr and vpu), listed in the top right panel of the home page. an alphabetical report of all interacting human proteins is accessed by following the link for any of the hiv-1 proteins. the hiv-1 proteins can also be searched based on their components, for example hiv-1 envelope can be searched either for the entire protein gp160, or separately for the gp120 surface glycoprotein or the gp41 transmembrane protein, which result from proteolytic cleavage of gp160. the hiv - to - human protein interactions are categorized by 43 interaction keywords (e.g. activates, associates with, binds, cleaves, complexes with, deglycosylates, inhibits, modulates, upregulates, etc.). a query interface allows for searching of the database to identify cellular proteins that have a specific type of interaction with a viral protein based on these keywords. the report can be customized to categories of interest by selecting a specific hiv protein and interaction keywords from the drop down menus. reports can be viewed as a web page, or downloaded as a text file for later use. in addition, to help facilitate the retrieval of related data, links to other database resources, such as the database of interacting proteins (dip ; 3), the molecular interaction database (mint ; 4), the binding database (5) and the los alamos national laboratories (lanl) hiv databases (6), are provided on the home page. figure 1 depicts the report and search interface page for the hiv-1 gag polyprotein and its cleavage products. as mentioned earlier, the drop down menus (figure 1a) allow for the selection of data related to the individual gag cleavage products (e.g. matrix, capsid, nucleocapsid, p1 and p6) and also facilitate searching by specific keywords (e.g. associates with, binds and inhibits) that represent the relationship between the viral proteins and the interacting human proteins (figure 1b). reports can either be viewed online or downloaded in ascii format and contain the hiv-1 tax i d, hiv-1 gene i d, hiv-1 protein accession number, hiv-1 protein name, the interaction keyword, the human tax i d, human gene i d, human protein accession number, human protein name, the pmid(s), the modification date and the interaction description. (a) all or part of the interaction data available for an hiv-1 protein can be accessed using the drop down menus. (b) the interacting relationship between hiv-1 and human proteins is reported below the menus. the figure illustrates a query section to display all interactions catalogued for the hiv-1 pr55 (gag) protein. for example, the first two interactions shown are : (i) pr55 (gag) protein associates with atp - binding cassette, sub - family e, member 1 ; and (ii) pr55 (gag) protein binds to adaptor - related protein complex 2, alpha 1 subunit isoform 1. (c) further down, the display shows the association of hiv-1 matrix and p6 with the mitogen - activated protein kinase 1 (mapk1). (d) the arrow points to the link for the entrez gene reports (the green g icon). (a) all or part of the interaction data available for an hiv-1 protein can be accessed using the drop down menus. (b) the interacting relationship between hiv-1 and human proteins is reported below the menus. the figure illustrates a query section to display all interactions catalogued for the hiv-1 pr55 (gag) protein. for example, the first two interactions shown are : (i) pr55 (gag) protein associates with atp - binding cassette, sub - family e, member 1 ; and (ii) pr55 (gag) protein binds to adaptor - related protein complex 2, alpha 1 subunit isoform 1. (c) further down, the display shows the association of hiv-1 matrix and p6 with the mitogen - activated protein kinase 1 (mapk1). (d) the arrow points to the link for the entrez gene reports (the green g icon). currently, the database is composed of 1434 human genes encoding 1448 proteins that directly (e.g. bind, inhibit) or indirectly (e.g. upregulate, modify) interact with hiv-1 proteins. it was found that the majority of the interactions reported are indirect (68%), whereas the rest are direct (2). in addition, the database comprises 2589 unique hiv-1 to human protein interactions and 5135 brief descriptions of the interactions, with a total of 14 312 pmid references to the original articles that reported the interactions. a network of links to supporting literature and cross - references allows users to navigate concomitantly between this database and other resources at ncbi (7), such as entrez gene (8), refseq (9) and pubmed. reports in entrez gene that contain hiv-1 interaction data can be retrieved with the query navigation to a target human protein interaction can be accomplished via one of two primary routes : an hiv-1, human protein interaction database search or an entrez gene text query. for illustration purposes, two search scenarios for the signaling protein mitogen - activated protein kinase 1 (mapk1), which displays a high magnitude of interactions with ten different hiv-1 proteins, are provided subsequently. hiv-1, human protein interaction database search. to view interactions between mapk1 and gag or its cleavage products, users may select gag in the horizontal selection bar on the top right panel of the database home page (http://www.ncbi.nlm.nih.gov/refseq/hivinteractions/), which makes a direct link to the illustration as shown in figure 1. using the scroll down mouse menu, mapk1 can be identified since interacting proteins reported in each interaction session (e.g. associates with and binds in figure 1b) are alphabetic. as a searching result, mapk1 is involved in the process of matrix and p6 phosphorylation (figure 1c). users may click on links to entrez gene (the green g icon ; figure 1d) to view the mapk1 full report. users may begin with the following query : mitogen - activated protein kinase 1[title ] and homo sapiens [organism ]. the entries (e.g. mapk1 and mapk1ip1l) identified with the query are displayed on the entrez gene results page. adding and hiv1interactions[prop] to the query restricts the results to only those entries that have hiv-1 interaction data, and in this example returns a single match to the mapk1 gene report shown in figure 2. protein interactions associated with mapk1 are listed on the entrez gene report page in the hiv-1 protein interactions section (figure 2) ; a link to this section is included in the right column table of contents provided on the full report display (figure 2a). individual hiv-1 proteins (e.g. envelope surface glycoprotein gp120) that interact with mapk1 are listed (figure 2b) along with brief descriptions of the interactions (figure 2c) and links to the supporting literature in pubmed (figure 2d). (d) the interactions and descriptions are linked to the supporting literature in pubmed. (d) the interactions and descriptions are linked to the supporting literature in pubmed. by integrating the hiv-1 interaction data into the entrez gene database, researchers benefit from the additional computation ncbi hiv-1, human protein interaction database home page, there are automatic queries provided to pubmed and the ncbi sequence databases for recent records of interest. via entrez gene, information can be easily obtained about genomic context, pathway membership and protein domain structure. the representative entrez gene search strategies summarized in the following table demonstrate the strength of the data integration and provide examples of how specific subsets of data can be retrieved : query to enter in entrez geneexplanationhiv1interactions[prop ] and human[organism ] and 5[chr ] and 1000000:12000000[base position]genes for which products interact with hiv-1 proteins, based on chromosome location. the value before [chr ] gives the chromosome, and the range separated by : gives the location in base pairs on that chromosome.hiv1interactions[prop ] and human[organism ] and cytoplasm[go]genes for which products interact with hiv-1 proteins, and are coded by the go consortium with at least one term starting with cytoplasm.hiv1interactions[prop ] and human[organism ] and immunoglobulin[domain name]genes for which products interact with hiv-1 proteins, and are calculated by ncbi 's conserved domain database group as having an immunoglobulin domain.hiv1interactions[prop ] and human[organism ] and (kegg or reactome)genes for which products interact with hiv-1 proteins and for which pathways data are available from the kegg or reactome groups. data visualization can be accomplished in multiple ways utilizing the information stored in this database. figure 3 shows an example of data visualization using biological process gene ontology (go) terms (10, http://www.geneontology.org) and individual hiv-1 proteins. this bar chart also demonstrates that a large portion of interactions catalogued in the database are associated with the hiv envelope surface (gp120) and tat proteins. the human cellular proteins interacting with hiv span a wide variety of functional categories, (e.g. signal transduction, protein metabolism, development, etc.) with an overrepresentation of interactions between tat and cellular proteins involved in transcription. in addition, envelope and tat proteins also have a high number of interactions with proteins representing multiple biological processes. figure 3.distribution of interactions based on biological process gene ontology (go) terms and individual hiv-1 proteins. the x - axis shows the individual hiv-1 structural proteins gag, pol and env and their cleavage products, and the regulatory and accessory hiv-1 proteins, tat, rev, nef, vpu, vpr and vif. distribution of interactions based on biological process gene ontology (go) terms and individual hiv-1 proteins. the x - axis shows the individual hiv-1 structural proteins gag, pol and env and their cleavage products, and the regulatory and accessory hiv-1 proteins, tat, rev, nef, vpu, vpr and vif. the hiv-1, human protein interaction database represents an important step towards a more detailed understanding of hiv-1 replication and pathogenesis. a recent example of the value of the database includes the work of brass. (11,12), who used the database as a tool to help analyze and categorize human proteins required for hiv-1 replication. similarly, in order to support their analysis of human pathogen protein protein interactions, dyer. (13) were able to use a subset of the hiv-1 interaction data that has been incorporated into the biomolecular interaction network database (bind ; 14). pathogen protein protein interactions has recently been studied in detail and such maps have revealed global and local networks that relate to known biological properties. studies have indicated that both viral and bacterial proteins tend to interact with hubs (proteins with many interacting partners) and bottlenecks (proteins that are central to many pathways in the network) in human pathogen protein protein interaction networks (13,1517). development of such global and local pathway networks by utilizing the information provided in the hiv-1, human protein interaction database will provide additional insights into hiv-1 replication and disease mechanisms at a systems biology level. these networks may reconfirm and extend known pathways, as well as uncover previously unknown pathway components. in addition, these networks may serve as a starting point for a systems biology modeling of the development of effective therapeutic and prophylactic interventions. hiv-1, human protein interaction database will be continuously updated to keep the database populated with interactions newly reported in the literature. current efforts are also focused on incorporating these data into canada 's biomolecular object network database (bond) (http://bond.unleashedinformatics.com ; successor to bind ; 14), a database cataloguing the interactions between all known cellular proteins. hiv-1, human protein interaction database, or any data contained therein can be provided by using the write to the help desk link at the bottom of the database and entrez gene web pages. national institutes of health, national institute of allergy and infectious diseases, division of aids (n01-ai-05415 and n01-ai-70042 to w.f., b.e.s.- b. and r.g.p.) ; intramural research program of the national institutes of health, national library of medicine (to k.s.k. | the human immunodeficiency virus type 1 (hiv-1), human protein interaction database, available through the national library of medicine at www.ncbi.nlm.nih.gov/refseq/hivinteractions, was created to catalog all interactions between hiv-1 and human proteins published in the peer - reviewed literature. the database serves the scientific community exploring the discovery of novel hiv vaccine candidates and therapeutic targets. to facilitate this discovery approach, the following information for each hiv-1 human protein interaction is provided and can be retrieved without restriction by web - based downloads and ftp protocols : reference sequence (refseq) protein accession numbers, entrez gene identification numbers, brief descriptions of the interactions, searchable keywords for interactions and pubmed identification numbers (pmids) of journal articles describing the interactions. currently, 2589 unique hiv-1 to human protein interactions and 5135 brief descriptions of the interactions, with a total of 14 312 pmid references to the original articles reporting the interactions, are stored in this growing database. in addition, all protein protein interactions documented in the database are integrated into entrez gene records and listed in the hiv-1 protein interactions section of entrez gene reports. the database is also tightly linked to other databases through entrez gene, enabling users to search for an abundance of information related to hiv pathogenesis and replication. |
the prevalence of type 2 diabetes mellitus (t2 dm) in korea is estimated to be 7.3% (in those over 20 years of age), and it has increased about 5-fold over the past 30 years according to a report of the korea national health and nutrition examination survey (knhnes iii, 2005). the number of patients with t2 dm is expected to increase dramatically from about 3.2 million in 2011 (8.8% of the national population) to about 4.25 million (11.1%) by 2030. this enormous increase in the number of t2 dm patients will inevitably be accompanied by chronic diabetic microvascular or macrovascular complications. among the diabetic complications, diabetic peripheral neuropathy (dpn) is the most prevalent and troublesome complication in patients with diabetes mellitus (dm). diabetic neuropathy (dn), which may be focal or diffuse, is diagnosed when diabetic patients complain of symptoms and/or show signs of peripheral nerve dysfunction after the exclusion of other etiologies. peripheral neuropathic pain in diabetic patients is defined as pain arising as a direct consequence of abnormalities in the peripheral somatosensory system in people with diabetes. the symptoms can be present as severe numbness, paresthesia, or hyperesthesia, however, dpn may be asymptomatic in about 50% of patients. as the dpn progresses, the painful symptoms usually disappear. in addition, dpn is also associated with substantial morbidity, which includes not only a susceptibility to foot or ankle fractures and ischemic ulceration leading to lower - limb amputations, but also depression [11 - 13 ]. according to the seattle diabetic foot study, which included 749 diabetic patients, there were a number of findings that independently increase the risk for dm foot ulcer, including certain foot deformities, reduced foot arterial perfusion, and both sensory and autonomic neuropathy. diabetic patients with critical limb ischemia have high risks of lower limb amputation and death. in addition to its influence on morbidity and mortality, painful symptoms in dpn have a significant detrimental impact on quality of life as the condition limits daily activities and interferes with sleep [16 - 18 ]. considering the health - related economic viewpoint, diabetic foot disease significantly increases the health care costs in patients with dm. according to a retrospective study that analyzed insurance costs in the united states, the cost of care for patients with a foot ulcer is 5.4 times higher in the year after the first ulcer episode, but 2.8 times higher in the second year compared with that of diabetic patients without foot ulcers. therefore, early detection and prompt intervention for dpn must be performed for patients with t2 dm. the prevalence of dpn is generally estimated to be 10% to 50% in patients with t2 dm, and the incidence increases with age and duration of dm. the reported prevalence of dn in korea is variable, from 14.1% to 54.5% depending on the study population and the diagnostic method (table 1) [21 - 24 ]. in the diabcare - asia 1998 study, which included 230 dm centers from 12 countries (n=24,317), the frequencies of retinopathy, microalbuminuria, and neuropathy were 21%, 39%, and 34%, respectively. the prevalence of those complications was significantly higher in those patients with higher hemoglobin a1c (hba1c) levels. a nationwide survey performed in 2006 by the committee of the korean diabetes association on the epidemiology of diabetes mellitus (n=5,652) showed that the prevalence of dpn defined by neurologic symptoms or nerve conduction velocity abnormalities was 44.7%. this prevalence was higher than the prevalence of microalbuminuria or retinopathy. in a prospective observational study among 508 korean t2 dm patients, diabetic foot disease occurred in 32 patients (6.3%), and the incidence of diabetic foot disease increased when peripheral neuropathy was present (odds ratio [or ], 2.949 ; 95% confidence interval [ci ], 1.075 to 8.090). while chronic neuropathic pain is present in 13% to 26% of dm patients, it can be found not only in diabetic subjects, but also in impaired glucose tolerance (igt) or impaired fasting glucose individuals. according to a community - based cross - sectional study from the united kingdom, chronic dpn is common and often severe but frequently unreported and interestingly, they showed that 12.5% of patients had never reported their symptoms to their doctors, and 39.3% never received treatment. there are some studies about the relationships between dpn and other diabetic complications in korean t2 dm patients. reported that the prevalence of cardiovascular disease (cvd) was higher in patients with dpn. in their multivariate analysis, dpn was independently associated with cvd (or, 1.801 ; 95% ci, 1.009 to 3.214) in t2 dm patients (n=1,041), with a 52.8% prevalence determined by neurophysiologically diagnosing peripheral polyneuropathy based on electroneuromyographic findings. a close relationship between peripheral sensory neuropathy and peripheral vascular disease was also reported independent of glucose level and other microvascular complications, in particular, retinopathy in t2 dm. the association between cardiovascular risk factors and development of large - fiber nerve dysfunction, which was measured by vibration perception threshold, was reported in type 1 dm patients (n=1,407) in the eurodiab prospective complication study. hyperglycemia not only activates the sorbitol accumulation with a subsequent increase in cellular osmolarity, but it also shunts to the hexose pathway, producing oxidative stress and the formation of advanced glycation end products (table 2). this damage leads to hyperexcitability in central neurons and the generation of spontaneous impulses within the axons as well as the dorsal root ganglion of these peripheral nerves. most of the clinical practice guidelines, including those from the korean diabetes association, recommend that dpn screening should begin at the initial diagnosis of t2 dm and should be performed at least annually thereafter [34 - 36 ]. the exclusion of non - diabetic causes of neuropathy, including alcoholism, vitamin b12 deficiency, endocrinopathies, vasculitides, heavy metal exposure, drug use, and malignancy, is important because these may account for 10% of the cases of neuropathy in people with dm. though the symptoms can exist without signs, the severity of painful symptoms can be reliably assessed by the visual analogue scale or the numerical rating scale (0, no pain ; 10, worst possible pain). in addition, validated scales and questionnaires such as the neuropathic pain symptoms inventory, the brief pain inventory, and the neuropathic pain questionnaires are widely used (table 3). however, the nerve conduction study remains the most reliable, accurate, and sensitive method to evaluate peripheral nerve function, and it has been adopted as the gold standard. this approach is not only time - consuming, expensive, and insensitive for the detection of small - fiber neuropathy, but also it is impractical to perform in an outpatient clinic setting. however, the semmes - weinstein monofilament (swmf) test is simple to use as a screening tool to identify patients at risk for diabetic foot complication in the primary care setting. the inability to sense the 10 g force pressure is considered as insensate and an independent predictor for higher risk of foot ulceration. considered the swmf test as a useful screening tool for dpn. the nerve conduction study (ncs) was used as a gold standard to compare the sensitivity and the specificity of the swmf test. the results were considered as abnormal if the patient could not perceive the 5.07/10 g swmf at more than four of ten sites (37 t2 dm outpatients). the sensitivity and the specificity at two sites (the third and fifth metatarsal head sites) were 93.1% and 100%, the same as at the ten sites. it is likely that the two - site swmf test is a useful screening test for dpn as is the ten - site test. in a study of 126 diabetic patients, 41% complained of dpn symptoms, and swmf and vibration perception were more impaired in patients with subjective sensory symptoms. in 82 diabetic patients, the medial plantar sensory ncs provided a more sensitive diagnosis of dpn, even in patients with normal range measurements in the sural nerve. the medial plantar sensory nerve action potential was abnormal in 46.7% of the symptomatic and 14.3% of the asymptomatic diabetic patients with normal routine ncs in this study. the medial plantar sensory ncs may be helpful in the diagnosis of subclinical dpn in the asymptomatic diabetic patient. compared to the single test, the combinations of tests have a greater than 87% sensitivity in detecting dpn. the aims of dpn treatment are to decrease the painful symptoms, to treat the specific pathogenic mechanism, and to prevent progression or subsequent complications. most of all, strict diabetic control and correction of metabolic risk factors should be initiated. the lifestyle intervention that improves glycemic control and decreases blood pressure with lipid profiles improves both the painful neuropathic symptoms and the intra - epidermal nerve fiber density. these findings suggest that early diagnosis and prompt intervention may be of significant clinical benefit. for pharmacological treatment, first line therapy consists of tricyclic antidepressants (tca), duloxetine, pregabalin or oxycodone (table 4). if the pain is not controlled with first - line therapy, second - line therapy or combination therapy can be used, for which the potential side effects and possible drug interactions must be considered. topical treatment with a 5% lidocaine plaster or capsaicin is also considered. randomized controlled trials have been performed for the treatment of dpn in korean patients with t2 dm using alpha - lipoic acid, pregabalin, or tramadol / acetaminophen combination treatment. the neuropathic symptom score, assessed by the total symptom score, was improved after intravenous alpha - lipoic acid treatment at a dose of 600 mg / day for 14 days in 19 t2 dm patients compared to 13 control subjects (p<0.05). open - label study with oral thioctic acid 600 mg once daily for 8 weeks (n=61) also improved dpn symptoms without serious adverse effects in korean diabetic patients. in an open, randomized, comparative study conducted in 163 t2 dm subjects with dpn, tramadol / acetaminophen (ultracet) treatment (n=79) for 6 weeks was as effective as gabapentin (n=84) for the decrease of pain intensity, an increase in the quality of life, an increase in mood, and a decrease in sleep disturbance in the treatment of painful dn. ten weeks of pregabalin treatment for patients with neuropathic pain (dpn [type 1 or 2 diabetes, n=18 ], postherpetic neuralgia [n=146 ], or posttraumatic neuropathic pain [n=76 ] ; n=162 pregabalin, n=78 placebo) showed a significant reduction in the daily pain rating scale score with improvement in anxiety and a decrease in sleep disturbance. despite the improvement in treatment modalities for chronic pain in recent years, patients with dpn still continue to be inadequately treated. the dramatic increase in the prevalence of t2 dm with its acute or chronic complications are a major health concern in korea. screening of high risk individuals, early detection, and proper management of dpn in patients with t2 dm is urgently needed. careful foot examination, active application of outpatient screening tools including the assessment of pedal pulses, and an organized diabetic foot - care program are needed. despite the improvement in treatment modalities for chronic pain in recent years, therefore, active pharmacologic treatment should be considered to relieve neuropathic pain and improve the quality of life in patients with t2 dm. | diabetic peripheral neuropathy (dpn), a common and troublesome complication in patients with type 2 diabetes mellitus (t2 dm), contributes to a higher risk of diabetic foot ulcer and lower limb amputation. these situations can negatively impact the quality of life of affected individuals. despite its high prevalence and clinical importance, most diabetes mellitus patients not only do not recognize the presence of diabetic neuropathy, but also do not report their symptoms to physicians or other health care providers. therefore, dpn is usually under diagnosed and undertreated. for early detection and appropriate intervention for dpn, a careful history, physical with neurologic examination, and prompt treatment are needed in t2 dm patients. |
syntheses of quinoxalines have attracted a great deal of attention in view of their potent biological and pharmacological activities including anticonvulsant [14 ], antibacterial, antifungal, antiviral, antitubercular, antileishmanial, antiamoebic, analgesic, antihistaminic, antineoplastic, hypoglycemic, mao - a inhibitor, antiarrhythmic, antiatherosclerotic, antiobese, and other diverse pharmacological activities. earlier studies revealed that most of compounds derived from 1,2,4-triazoles have been found to be significant anticonvulsant and tranquillizing agents. furthermore, compounds 1a e in figure 1 which contain triazolo[4,3-a]quinoxaline moiety showed promising anticonvulsant activity. we describe the synthesis and biological evaluation of novel triazolo[4,3-a]quinoxaline derivatives expected to have anticonvulsant activity starting from 1,2-diaminobenzene and oxalic acid via quinoxaline ring build - up. 2,3-dichloroquinoxaline (3) was prepared by chlorination of 2,3-dihydroxyquinoxaline (2), which in turn was prepared by the condensation of the commercially available 1,2-diaminobenzene with oxalic acid in aqueous hydrochloric acid. treatment of 3 with hydrazine hydrate yielded the corresponding 3-hydrazino compound, 4, which was subjected to ring closure to 5 by treatment with triethyl orthoformate. reaction of 5 with thiourea in absolute ethanol afforded the isothiouronium intermediate which upon basic hydrolysis yielded triazolo[4,3-a]quinoxaline-4-thiol (6). the potassium salt 7 was obtained after treatment of 6 with alcoholic koh in a quantitative yield. reaction of the potassium salt 7 with substituted aniline in dmf gave the corresponding anilide 8a d, and its reaction with alkyl chloroacetate in the same solvent gave the corresponding ester 9a e. the hydrazide derivative 10 was synthesized from the reaction of n - butyl ester with hydrazine hydrate. thus, that hydrazide was allowed to react with ethyl acetoacetate and acetylacetone to give the corresponding methylpyrazolone derivative 11 and dimethylpyrazole derivative 12, respectively. when compound 10 was treated with carbon disulphide and potassium hydroxide, allowing the same intermediate 10 to react with isocyanate or with isothiocyanates resulted in the formation of semicarbazide 14 and thiosemicarbazides 15, respectively. acetylation of compound 10 with acetic anhydride and condensation with variety of aromatic aldehydes gave the corresponding n - acetyl derivative 16 and n - arylidene derivatives 17a f, respectively. the structural assignments to the new compounds were based on their elemental analysis and spectral (ir, h nmr, and mass) data. eleven compounds of the newly synthesized derivatives were selected to be screened for their anticonvulsant activity on different groups of mice. swiss albino adult male mice, weighing 2025 g, were used as experimental animals. they were obtained from an animal facility (animal house, department of pharmacology and toxicology, faculty of pharmacy, al - azhar university). animals were kept under well - ventilated conditions at room temperature (2530c). they were fed on an adequate standard laboratory chow (el - nasr co., abou - zabal, egypt) and allowed to acclimatize with free access to food and water for 24-hour period before testing except during the short time the tested compounds were dissolved in dmso and orally administered in a dose regimen ranging from 200 to 800 mg / kg animal weight, using the dosing volume of 0.2 ml per 20 g. pentylenetetrazole was dissolved in normal saline in 2% concentration and was given intraperitoneally in a dose of 60 mg / kg body weight (dose that could induce convulsions in at least 80% of the animals without death during the following 24 hours). phenobarbitone sodium (alex co., egypt) was dissolved in normal saline in 2% concentration and it was intraperitoneally given in doses of 6.25, 12.5, and 25 mg / kg using the same dosing volume. groups of six mice were administered the graded doses of the test compounds and phenobarbitone sodium orally. control animals received an equal volume of saline (10 ml / kg). after one hour, the animals were subcutaneously injected with the convulsive dose of pentylenetetrazole (60 mg / kg). doses that gave full protection against the induced convulsions and those which exhibited 50% protection in addition to the relative potencies of the test compounds to phenobarbitone sodium were recorded in table 1. the ir spectra (in kbr discs) were recorded on potassium bromide discs on a pye unicam sp 3300 spectrophotometer. h nmr spectra were recorded either on gemini 300 mhz (varian usa) or on jeol eca 500 mhz nmr spectrometer (500 mhz) using tms as an internal standard. mass spectra were recorded on a shimadzu gcms - qp 1000 ex mass spectrometer operating at 70 ev. elemental analysis was carried out at the regional center for mycology and biotechnology, al - azhar university, cairo, egypt. progress of the reactions was monitored by tlc sheets precoated with uv fluorescent silica gel merck 60 f254 plates and was visualized using uv lamp and n - hexane : ethyl acetate 9 : 1 as mobile phase. compounds 26 were prepared following the procedures reported in the literature [2224 ]. the used intermediates (chloroacetanilides and alkyl chloroacetates) compound 6 (1.5 g, 0.01 mol) was treated with alcoholic solution of potassium hydroxide (0.4 g, 0.01 mol) ; the reaction mixture was stirred for 1 h ; the solid obtained was filtered, washed with absolute ethanol, and air - dried to give the potassium salt in quantitative yield. ir (kbr) cm : 3391, 3127, 2833, 1629, 1479, 1412, 1309, 1209, 1065, 972. ms (m / z, %): 240 (m, 0.9), 233 (4), 202 (6), 160 (7), 143 (42), 118 (34), 102 (11), 91 (22), 77 (24), 63 (100), 51 (27). h nmr (dmso - d6, 300 mhz) : 7.13 (t, 1h, j = 7.50 hz), 7.27 (t, 1h, j = 7.50 hz), 7.56 (d, 1h, j = 7.20 hz), 8.07 (d, 1h, j = 7.20 hz), 9.77 (s, 1h, triazolo proton). calcd. for c10h8kn4s : c, 44.98 ; h, 2.10 ; n, 23.31. general procedure : a mixture of compound 7 (1.0 g, 0.01 mol) and the appropriate chloroacetanilide (0.01 mol) in dmf (20 ml) was heated over a water bath for 3 h. the reaction mixture was then cooled, poured into ice - cooled water (200 ml), and stirred well for 30 min. the solid thus separated was filtered, washed with water, dried, and crystallized from methanol / toluene mixture (1 : 1) to obtain compounds 8a d. ir (kbr) cm : 3216, 3071, 1666, 1599, 1537, 1463, 1318, 1242, 1066, 955. ms (m / z, %): 335 (m, 0.1), 243 (1), 215 (2), 203 (11), 143 (14), 132 (25), 119 (22), 104 (14), 91 (100), 77 (44), 64 (82), 50 (26). h nmr (dmso - d6, 500 mhz) : 4.34 (s, 2h), 7.02 (t, 1h, j = 7.56 hz), 7.26 (t, 2h, j = 7.56 hz), 7.58 (m, 4h), 7.85 (d, 1h, j = 8.40 hz), 8.32 (d, 1h, j = 7.65 hz), 10.09 (s, 1h, triazolo proton). anal. calcd. for c17h13n5os : c, 60.88 ; h, 3.91 ; n, 20.88. found : c, 61.08 ; h, 3.90 ; n, 20.94%. ir (kbr) cm : 3522, 3205, 3122, 1681, 1591, 1459, 1364, 1247, 1057, 957. h nmr (dmso - d6, 500 mhz) : 4.32 (s, 2h), 7.10 (t, 1h, j = 7.65 hz), 7.43 (m, 4h), 7.89 (d, 2h, j = 7.65 hz), 8.17 (t, 1h, j = 7.65 hz), 10.09 (s, 1h, triazolo proton). calcd. for c18h13n5o3s : c, 56.98 ; h, 3.45 ; n, 18.46. found : c, 57.19 ; h, 3.44 ; n, 18.51%. ir (kbr) cm : 3675, 3566, 3021, 2855, 1678, 1602, 1521, 1451, 1388, 1285, 1056, 959. ms (m / z, %): 351 (m, 0.2), 243 (8), 215 (7), 202 (100), 143 (13), 131 (5), 109 (15), 102 (5), 90 (13), 77 (5), 63 (13), 51 (21). : c, 58.11 ; h, 3.73 ; n, 19.93. found : c, 58.10 ; h, 3.72 ; n, 19.86%. ir (kbr) cm : 3677, 3075, 1695, 1588, 1503, 1464, 1341, 1274, 1066, 952. ms (m / z, %): 380 (m, 0.5), 243 (24), 216 (80), 202 (21), 188 (32), 143 (28), 134 (18), 102 (22), 89 (100), 76 (75), 63 (88), 51 (97). : c, 53.68 ; h, 3.18 ; n, 22.09. found : c, 53.87 ; h, 3.17 ; n, 22.16%. general procedure : equimolar quantities of the potassium salt 7 (1.2 g, 0.01 mol) and alkyl chloroacetate (0.01 mol) in dmf (20 ml) were heated on water bath for 3 h. the reaction mixture was poured onto ice water (200 ml), and stirred for 30 min. ir (kbr) cm : 3454, 1722, 1464, 1316, 1200, 1060, 982, 952. (m / z, %): 274 (m, 10), 243 (4), 215 (100), 188 (55), 170 (15), 161 (26), 143 (36), 122 (16), 116 (13), 102 (11), 76 (8), 58 (46). h nmr (dmso - d6, 500 mhz) : 3.35 (s, 3h), 4.27 (s, 2h), 7.62 (m, 2h), 7.83 (d, 1h, j = 7.65 hz), 8.32 (d, 1h, j = 8.6 hz), 10.09 (s, 1h, triazolo proton). calcd. for c18h13n5o3s : c, 52.54 ; h, 3.67 ; n, 20.43. ir (kbr) cm : 3099, 2966, 2346, 1727, 1641, 1486, 1388, 1171, 1035, 951. (m / z, %): 288 (m, 16), 243 (19), 215 (100), 188 (61), 170 (25), 161 (39), 143 (42), 121 (18), 115 (18), 102 (14), 75 (21), 50 (10). h nmr (cdcl3, 300 mhz) : 1.29 (t, 3h, j = 4.5 hz), 4.18 (q, 2h, j = 3 hz), 4.30 (s, 2h), 7.59 (m, 2h), 7.88 (d, 1h, j = 3.6 hz), 7.94 (d, 1h, j = 3.6 hz), 9.28 (s, 1h, triazolo proton). for c13h12n4o2s : c, 54.15 ; h, 4.20 ; n, 19.43. found : c, 54.34 ; h, 4.21 ; n, 19.37%. ir (kbr) cm : 3423, 3069, 2966, 1743, 1641, 1390, 1299, 1167, 1063, 960. (m / z, %): 303 (m + 1, 40), 302 (m, 16), 243 (14), 215 (100), 188 (26), 170 (13), 161 (9), 143 (16), 122 (12), 116 (8), 102 (7), 90 (13), 63 (4). anal. calcd. for c14h14n4o2s : c, 55.61 ; h, 4.67 ; n, 18.53. ir (kbr) cm : 3446, 2934, 2725, 1728, 1626, 1552, 1468, 1368, 1171, 1063, 953. h nmr (cdcl3, 300 mhz) : 0.77 (t, 3h, j = 7.2 hz), 1.21 (sextet, 2h, j = 7.2 hz), 1.51 (p, 2h, j = 6.3 hz), 4.09 (t, 2h, j = 6.3 hz), 4.29 (s, 2h), 7.30 (m, 2h), 8.18 (d, 1h, j = 6 hz), 8.35 (d, 1h, j = 5.4 hz), 9.96 (s, 1h). anal. calcd. for c15h16n4o2s : c, 56.94 ; h, 5.10 ; n, 17.71. found : c, 56.92 ; h, 5.07 ; n, 17.69%. ir (kbr) cm : 3052, 2976, 2926, 1738, 1466, 1306, 1146, 1068, 956. (m / z, %): 316 (m, 3), 260 (11), 243 (10), 216 (46), 188 (23), 143 (7), 121 (4), 102 (3), 90 (7), 56 (100). h nmr (dmso - d6, 500 mhz) : 1.33 (s, 9h), 4.13 (s, 2h), 7.64 (m, 2h), 7.85 (d, 1h, j = 7.65 hz), 8.33 (d, 1h, j = 7.65 hz). anal. calcd. for c15h16n4o2s : c, 56.94 ; h, 5.10 ; n, 17.71. found : c, 56.72 ; h, 5.09 ; n, 17.63%. compound 9b (3.3 g, 0.01 mol) was dissolved in absolute ethanol (50 ml) and treated with hydrazine hydrate (95%, 20 ml). the reaction mixture was stirred well and heated to 50c for two hours, then cooled and treated with water (200 ml). the solid thus obtained was filtered, washed with water, dried, and then crystallized from glacial acetic acid to give 2.85 g (91%), m.p. > 360c, ir (kbr) cm : 3412, 3196, 3072, 1630, 1488, 1388, 1250, 1164, 1058, 954. ms (m / z, %): 274 (m, 4), 215 (7), 202 (22), 169 (26.86), 143 (16), 128 (47), 102 (22), 76 (100), 51 (68). h nmr (dmso - d6, 500 mhz) : 4.08 (s, 2h), 7.62 (m, 2h), 7.92 (d, 1h, j = 7.6 hz), 8.31 (d, 1h, j = 7.6 hz), 10.05 (s, 1h). anal. calcd. for c11h10n6os found : c, 48.03 ; h, 3.68 ; n, 30.74%. a mixture of 10 (1 g, 0.0028 mol) and acetylacetone (0.36 g, 0.0028 mol) in absolute ethanol (20 ml) was heated at 80c on a water bath for 7 h. the reaction mixture was cooled and poured onto water, and the formed precipitate was filtered and crystallized from ethanol to give 0.78 g (64%), m.p. 236238c, ir (kbr) cm : 3078, 1721, 1572, 1488, 1321, 1230, 1133, 1031, 956. ms (m / z, %): 338 (m, 0.06), 265 (35), 249 (4), 237 (39), 222 (24), 195 (11), 169 (10), 143 (14), 115 (11), 102 (18), 76 (34), 51 (100). h nmr (dmso - d6, 500 mhz) : 2.21 (s, 3h), 3.26 (s, 3h), 4.26 (s, 2h), 6.23 (s, 1h, pyrazole proton), 7.67 (t, 1h, j = 7.65 hz), 7.77 (t, 1h, j = 7.65 hz), 7.98 (d, 1h, j = 8.4 hz), 8.43 (d, 1h, j = 7.65 hz), 10.17 (s, 1h, triazolo proton). anal. calcd. for c16h14n6os found : c, 56.99 ; h, 4.15 ; n, 24.91%. a mixture of 10 (1 g, 0.0028 mol) and ethyl acetoacetate (0.47 g, 0.0028 mol) in dioxane (20 ml) was heated under reflux for 5 h. the reaction mixture was cooled and poured onto water, and the formed precipitate was filtered and then crystallized from dioxane to give 0.37 g (30%), m.p. 242244c, ir (kbr) cm : 3412, 3192, 3069, 1635, 1491, 1388, 1248, 1180, 1062, 970. ms (m / z, %): 340 (m, 2), 221 (3), 201 (2), 135 (6), 121 (3), 69 (48), 55 (100). h nmr (dmso - d6, 300 mhz) : 3.43 (s, 3h), 4.38 (s, 2h, s ch2), 5.61 (s, 2h, ch2 of pyrazole ring), 7.418.39 (m, 4h), 10.13 (s, 1h, triazolo proton). anal. calcd. for c15h12n6o2s found : c, 53.14 ; h, 3.56 ; n, 24.72%. a mixture of 10 (1 g, 0.01 mol), koh (0.01 mol), and cs2 (20 ml) in dmf (20 ml) was heated under reflux until the h2s ceased to evolve (about 5 h). the excess solvent was removed by distillation, the residue was stirred with water and filtered, and the filtrate was acidified with 10% hcl. the precipitated solid was filtered, washed with water, and crystallized from ethanol to give 0.9 g (86%), m.p. > 360c, ir (kbr) cm : 3428, 3111, 2896, 1642, 1467, 1396, 1257, 1098, 952. ms (m / z, %): 316 (m, 6), 242 (10), 215 (6), 210 (21), 202 (81), 143 (11), 121 (7), 105 (17), 77 (18), 52 (100). h nmr (dmso - d6, 500 mhz) : 4.79 (s, 2h), 7.59 (m, 2h), 8.28 (d, 1h, j = 7.65 hz), 9.91 (d, 1h, j = 7.56 hz), 10.10 (s, 1h, triazolo proton). anal. calcd. for c12h8n6os2 : c, 45.56 ; h, 2.55 ; n, 26.56. compound 10 (1 g, 0.006 mol) and cyclohexyl isocyanate (0.45 g, 0.006 mol) in benzene were heated under reflux for 24 h. after cooling, the precipitate was collected and crystallized from ethanol to give 1.1 g (80%), m.p. > 360c, ir (kbr) cm : 3390, 3091, 2929, 1669, 1534, 1497, 1334, 1239, 1175, 954. h nmr (dmso - d6, 500 mhz) : 0.971.78 (m, 11h), 4.15 (s, 2h), 7.087.29 (m, 4h), 9.57 (s, 1h, triazolo proton). : c, 54.12 ; h, 5.30 ; n, 24.54. found : c, 54.31 ; h, 5.30 ; n, 24.61%. a mixture of compound 10 (1 g, 0.006 mol) and the appropriate thiocyanate (0.006 mol) in 20 ml absolute ethanol was heated under reflux for 3 h. after cooling, the precipitate was collected and crystallized from dioxane to obtain compounds (15a - b). 238240c, ir (kbr) cm : 3495, 3066, 1701, 1639, 1522, 1316, 1227, 1051, 955. ms (m / z, %): 361 (m+1, 8), 241 (9), 211 (9), 201 (8.36), 184 (31), 157 (25), 122 (9), 115 (14), 102 (15), 77 (13), 63 (100). h nmr (dmso - d6, 300 mhz) : 0.99 (t, 3h, j = 6.9 hz), 1.06 (q, 2h, j = 6.9 hz), 4.29 (s, 2h), 7.67 (m, 2h), 7.96 (d, 1h, j = 7.2 hz), 8.35 (d, 1h, j = 7.5 hz), 10.11 (s, 1h, triazolo proton). calcd. for c14h15n7os2 : c, 46.52 ; h, 4.18 ; n, 27.13. found : c, 46.68 ; h, 4.19 ; n, 27.21%. 300302c, ir (kbr) cm : 3499, 3173, 2988, 1701, 1688, 1526, 1329, 1246, 1061, 960. h nmr (dmso - d6, 500 mhz) : 4.21 (s, 2h), 7.627.31 (m, 4h), 7.337.65 (m, 4h), 10.02 (s, 1h, triazolo proton). : c, 52.80 ; h, 3.69 ; n, 23.94. found : c, 52.99 ; h, 3.70 ; n, 24.01%. the hydrazide 10 (1 g, 2 mmol) was warmed with acetic anhydride (5 ml) for 1 h, and then the mixture was allowed to attain room temperature. the deposited red solid was filtered, washed with petroleum ether (6080c), and crystallized from ethanol to afford 0.5 g (45%), m.p. 200202c, ir (kbr) cm : 3433, 3130, 3011, 1727, 1670, 1499, 1370, 1238, 1022, 976. ms (m / z, %): 316 (m, 0.1), 242 (36), 200 (86), 170 (100), 143 (89), 116 (50), 75 (48), 50 (34). h nmr (dmso - d6, 300 mhz) : 2.40 (s, 3h), 4.20 (s, 2h), 7.69 (m, 2h), 8.04 (d, 1h, j = 7.8 hz), 8.43 (d, 1h, j = 7.8 hz), 10.19 (s, 1h, triazolo proton). anal. calcd. for c13h12sn6o2s : c, 49.36 ; h, 3.82 ; n, 26.57. found : c, 49.53 ; h, 3.83 ; n, 26.65%. general procedure : a mixture of 1.0 g (0.028 mol) of compound 10 and the appropriate aromatic aldehyde (0.029 mol) in absolute ethanol (20 ml) and catalytic amount of glacial acetic acid (2 ml) was refluxed for 3 - 4 h, and the reaction mixture was then poured onto water. the solid so obtained was filtered and crystallized from acetic acid to obtain compounds 17a f. 350352c, ir (kbr) cm : 3438, 3116, 2925, 1716, 1645, 1480, 1388, 1262, 1169. h nmr (dmso - d6, 500 mhz) : 4.65 (s, 2h), 7.577.63 (m, 5h), 8.07 (s, 1h), 8.098.36 (m, 4h), 9.92 (s, 1h, triazolo proton). anal. calcd. for c18h14n6os 282284c, ir (kbr) cm : 3450, 3097, 1708, 1626, 1478, 1389, 1240, 1045, 955. h nmr (dmso - d6, 300 mhz) : 1.91 (s, 3h), 5.59 (s, 2h), 7.137.34 (m, 4h), 7.687.89 (m, 4h), 8.54 (s, 1h), 10.13 (s, 1h, triazolo proton). calcd. for c19h16n6os : c, 60.62 ; h, 4.28 ; n, 22.33. found : c, 60.88 ; h, 4.29 ; n, 22.40%. 274276c, ir (kbr) cm : 3340, 3192, 3054, 1607, 1471, 1378, 1237, 1160, 1062, 962. ms (m / z, %): 378 (m, 7), 302 (7), 243 (7), 215 (10.32), 201 (16), 143 (19), 120 (30), 102 (16), 76 (28), 51 (100). h nmr (dmso - d6, 500 mhz) : 4.65 (s, 2h), 6.756.85 (m, 4h), 7.13 (t, 1h, j = 7.65 hz), 7.33 (t, 1h, j = 7.65 hz), 8.01 (d, 1h, j = 8.4 hz), 8.32 (d, 1h, j = 8.4 hz), 8.47 (s, 1h), 10.04 (s, 1h, triazolo proton). anal. calcd. for c18h14n6o2s : c, 57.13 ; h, 3.73 ; n, 22.21. found : c, 57.35 ; h, 3.72 ; n 288290c, ir (kbr) cm : 3435, 3101, 3054, 1622, 1457, 1396, 1266, 1193, 1058, 958. h nmr (dmso - d6, 500 mhz) : 4.29 (s, 2h), 6.836.91 (m, 4h), 7.227.32 (m, 4h), 9.00 (s, 1h), 10.03 (s, 1h, triazolo proton). calcd. for c18h14n6o2s : c, 57.13 ; h, 3.73 ; n, 22.21. found : c, 57.10 ; h, 3.69 ; n, 22.15%. 290292c, ir (kbr) cm : 3450, 3093, 1629, 1482, 1349, 1226, 1084, 955, ms (m / z, %): 396 (m, 0.04), 398 (0.03), 361 (0.05), 322 (5), 285 (0.04), 243 (4), 215 (6), 201 (6.02), 185 (100), 143 (7), 123 (34), 102 (57), 76 (45), 50 (89). h nmr (dmso - d6, 300 mhz) : 4.73 (s, 2h), 7.207.39 (m, 4h), 7.487.69 (m, 4h), 8.49 (s, 1h), 9.95 (s, 1h, triazolo proton). anal. calcd. for c18h13cln6os : c, 54.48 ; h, 3.30 ; n, 21.18. found : c, 54.62 ; h, 3.30 ; n, 21.24% 290292c, ir (kbr) cm : 3420, 3328, 3106, 1620, 1476, 1371, 1244, 1158, 1025, 952. ms (m / z, %): 431 (m, 0.03), 395 (0.04), 321 (5), 243 (0.6), 215 (1), 211 (5), 185 (100), 143 (8), 123 (14), 76 (3), 51 (6). anal. calcd. for c18h12c12n6os : c, 50.13 ; h, 2.80 ; n, 19.49. found : c, 50.31 ; h, 2.79 ; n, 19.43%. in the present investigation, 16 new 4-substituted triazolo[4,3-a]quinoxaline derivatives were synthesized and characterized by spectral analysis. the activity may be explained by the presence of substituents on position 4 of the condensed heterocyclic system containing quinoxaline, fused to triazole at 1,2 positions in the backbone structure of title compounds. the electronic factors exerted by the substituents and the hydrophobic nature of phenyl nucleus in the title compounds influenced the activity. | 2-([1,2,4]triazolo[4,3-a]quinoxalin-4-ylthio)acetic acid hydrazide (10) was used as a precursor for the syntheses of novel quinoxaline derivatives with potential anticonvulsant properties. the newly synthesized compounds have been characterized by ir, 1h nmr, and mass spectral data followed by elemental analysis. the anticonvulsant evaluation was carried out for eleven of the synthesized compounds using metrazol induced convulsions model and phenobarbitone sodium as a standard. among this set of tested compounds, two of them (14, and 15b) showed the best anticonvulsant activities. |
the world wide web has become an excellent resource for distribution of educational materials in an electronic format for independent study with computer - aided instruction. students in the health sciences can utilize their own electronic devices with web browsers to access a growing number of web - based educational content. though web - based educational materials may be popular with students as indicated on evaluations, are online resources used in the manner for which they were intended, and do they yield results that suggest students are achieving mastery of subject matter they have studied ? students evaluations and feedback sessions in the preclinical years at the author 's medical school have called for greater availability of practice examinations to prepare for real examinations, including both those in the pathology discipline taught at this school as well as the united states medical licensing examination (usmle) step i examination. in particular, students have shown the greatest interest in access to practice examinations that incorporate imaging. this study was performed to analyze the usage and results of web - based pathology practice examinations that were developed to assist students using formative assessments of their knowledge. these practice examinations were developed to provide a freely available resource for review and self - assessment in major subject areas of pathology, but not to be so extensive or comprehensive that a large amount of student time would be required to complete them. a web site with public internet access to a site entitled web path was developed by the author, starting in 1994, with no direct funding throughout the site 's history. this site provides content for pathology education, primarily using images with text descriptions arranged by subject, along with examination questions. the site began receiving an average million web page hits per month at its inception, and access continued to increase in the 1990 's until average monthly usage ranged from 6.5 to 8.5 million hits / month, and that has remained stable over the past 15 years. usage paralleled the academic calendar of northern hemisphere medical schools, with more hits in fall and spring. web site analysis revealed that over 95% of accesses originated from dynamic internet protocol (ip) addresses of commercial internet service providers, so it is impossible to assess the location of users. on average, over half of users gained access to web path via referral from internet search engines. the menu [figure 2 ] for the examinations is accessed at the following url : http://library.med.utah.edu/webpath/exam/examidx.html the login window for the timed examinations is shown in figure 3. web path home page, with the link to the examinations highlighted by the red arrow examination web page main menu, with links to the nontimed general pathology and organ system pathology banks, and links to the timed review quizzes login popup window showing the data fields collected anonymously, before the start of each timed examination the author developed examination questions for the web site that were representative of content for chapters in the robbins and cotran pathologic basis of disease textbook that followed the usmle content outline. all examination questions were developed in usmle style, and had 5 foils / question. these patient vignette style questions integrated anatomical pathology with laboratory and physical examination findings [figures 4 and 5 ]. the examination templates were developed using javascript for client - side html documents accessible via a web browser. a bank of 431 questions in 9 topic areas for general pathology emphasizing mechanisms of disease, and a bank of 509 questions in 11 topic areas covering organ system pathology, was placed onto the web path site for user - chosen access in a nontimed format, as shown in table 1. these questions provided feedback for each answer chosen by the user, along with a continuously compiled examination score using javascript with cookies. however, the scores were generated client side on the user 's computer and not stored on the web server. sample nontimed examination format from the hematopathology examination, with the question stem in the lower frame, the feedback for the answer in the upper frame, and the cumulative score in the right frame sample timed examination format from the pulmonary subject examination. the stem of this question provides clues to the diagnosis of restrictive lung disease, and the image shows the gross pathology of honeycomb lung, so foil e is the best choice the author developed a smaller bank of timed examinations to mimic a real testing environment. each of the four multi subject review examinations had 30 questions, including 6 questions with images. the topic areas for the timed examinations in order of their display on the web pages are shown in table 1. after clicking on a menu item to open a timed examination, a popup window appeared on the user 's screen, shown in figure 3, with fields for login that recorded via radio buttons choices for the health science field of study for the user, along with free text field for voluntary, self - reported typed entry of the higher educational affiliation by the user. no e - mail address, ip address, or other identifier was collected, so the data remained anonymous and institutional review board exempt. after submitting this form, a sample timed format question is shown in figure 5. following user submission and exit of an examination, scoring occurred using a server - side common gateway interface program using a perl 5 program script. upon completion of the exam, the web server received the information using the html form postmethod and then passed the data to the perl script. the captured data elements written in comma - delimited sequence to an ascii text file included : user 's field of study, user 's academic affiliation, total score, and chosen answer for each question. after a user submitted an examination for scoring, or the exam timed out, only those questions marked were scored. the user was then automatically directed to a web page that provided the overall percentage score for correct answers for the examination taken, along with a key of correct answers for that examination. as in a real examination, the perl script generated a cumulative log file that recorded the individual logins to each examination for each line of the text file. the log files for each examination accumulated following migration of the web site to a new web server and updating with revision of questions in june 2006, until the log files were downloaded in january 2014. during that period, the ascii text formatted log files were downloaded to the author 's computer and loaded into microsoft excel (2011) spreadsheet software (microsoft corporation, redmond, washington) for each examination. the log files of anonymous user data were analyzed for total number of logins, number of logins with all questions completed, scores by question and health science field of study for each of the 21 examinations. the 4 general review examinations [table 2 ] had 31,639 accesses with completion of all questions, for an overall completion rate of 53.7%, and average score of 75.2% with standard deviation of 3.2. review examination 3 with the average farthest from the mean was not a significant outlier (p > 0.05) by grubbs test to detect an outlier. in top to bottom web page menu order, review examination usage was 43.9%, 23.7%, 17.5%, and 14.9% of all accessions [table 2 ]. general review examination usage the 17 subject examinations [table 3 ] had 103,028 completions, with overall completion rate 72.6%, and average score 74.0% with standard deviation of 4.2. the pulmonary subject examination with the average farthest from the mean was not a significant outlier (p > 0.05) by grubbs test to detect an outlier. the first three menu items on the subject examination web page accounted for 12.6%, 10.0%, and 8.2% of all completions, and the bottom three menu items accounted for no more than 2% each [table 3 ]. subject specific examination usage analysis of scores by user 's field of study [table 4 ] showed the highest overall scores were obtained by md and do users, 75.3% for the four general review examinations and users in other health sciences had overall lower averages, ranging from 54.6% to 75.4% for the general review examinations, and from 50.9% to 73.7% for the subject examinations. none of the field of study categories was a significant outlier (p > 0.05) by grubbs test to detect an outlier. examination scores by user 's field of study for the four general review examinations, a score of 100% was achieved by 7% of all users, 21% of users achieved a score 90%, and 95% of users achieved 50% [table 4 ]. for the 17 subject examinations, a score of 100% was achieved by 20% of users overall, 37% of users achieved a score 90%, and 90% achieved a score 50% [table 5 ]. web - based curricular tools have become popular for teaching students since the early 1990 's. the value of web - based formative examinations (giving a score) has been shown to positively influence student performance in curricular assessments that count for a grade. web - based instruction can also promote approaches to deep learning (seeking to understand underlying concepts) that are more effective for students than surface learning (focused on passing the next examination). web - based examinations can provide resources to support distance learning, education beyond a single institution, and share expertise on a national scale. the web path site was designed to provide a variety of pathologic material for review by health science students, but it can not replace a standard curriculum. it serves as an example of what can be done to supplement pathology education with no direct funding for development and no direct cost to users. the total time required for completion of the web path web site question banks, estimating 11.5 min / question, is up to 23 h for untimed questions and up to 6 h for the timed questions. there are now numerous question banks available on the internet, both free and through subscription. novice students are challenged to select material that may be of value to their education. usefulness of educational web sites can be evaluated with a published rating method. the web page menu for this study that leads to the login screen for the examinations is located four clicks away, including a popup login window [figure 3 ], from the main menu [figure 1 ] of the web path web site, tending to discourage casual surfing. there is no way from log analysis to determine how many persons with minimal or no prior education in pathology logged in and took the exams, but of total users, only 4.3% for the review examinations, and only 6.1% for the subject examinations, picked other as a field of study category, and their scores were well within the range of other users who indicated a specific field of study. however, the login did not distinguish students from persons at higher levels of training, such as residents, or practice professionals. the value of anonymous data for research, such as the findings presented here, comes from the large amount of aggregate data that can be collected from users anywhere on earth and over a long period of time and at no cost to users. the major limitation is that the data can not be verified for individual users by location, course of study, scores obtained, or conditions of usage. educational research studies recruiting student volunteers for more rigorous conditions of assessment and analysis generally obtain smaller subject numbers. national organizations providing qualifying examinations for certification can generate extensive statistical analyses, but generally do not publish their findings, results are released only to users and their educational institutions, and the examinations are costly to users. in this study, limiting the data analysis to users completing the examinations served to increase validity of results. reliability was increased through aggregation of data over 9 years with an unchanging set of 290 questions. does posting web - based educational tools insure usage to justify the time needed to develop them ? in this study, the completion rates indicated that the shorter the exercise, the more likely the user would complete it. the general review exams were closer to mimicking a real timed exam, but completion rates were even lower, 53.7% overall, or little more than half the accessions. users may have been accessing the examinations only to assess their potential content and possible educational value, planning to return later. though completion rates were below 100% in this study, the scores achieved on these web - based examinations suggest that some users completing the examinations had sufficient preparation to make use of the examinations to support their pathology education. a high score 90% was achieved by 21% of users of review examinations and 37% of users for the subject examinations [table 5 ]. < 50% suggests that few users were just randomly marking answers [table 5 ]. given that a correct answer key was returned to users following submission of the examination for scoring, it is possible that some users may have retaken an examination one or more times to achieve a higher score, and that skewed the average higher. however, these examinations are not high - stakes for a grade, so there is no tangible reward to the user for obtaining a higher score. if users are striving for a higher score, then that contributes to their education and fulfills a goal of the web site. the variation in scores obtained per examination is primarily due to the nature of the mix of questions on these relatively short examinations. one or two more difficult questions on a 10 questions examination will reduce the average scores achieved [table 3 ]. however, none of the examinations was a statistically significant outlier. the most difficult of the 290 total questions, scoring at 38.8% overall, in the cardiovascular subject examination had an image showing a dense collagenous scar with remote myocardial infarction, and prompted for the most likely current correlate, which was congestive heart failure, but 37.4% of users picked the foil for troponin elevation, reflective of an acute coronary syndrome. however, the foils were nonhomogeneous. the author has used the information from log file analysis to revise the most difficult questions. in this study, to some extent this was a self - fulfilling prophecy, since the order of examinations in the menu, at least for the subject examinations, was arranged by the author according to past experience of subject popularity in a prior study of this web site. similar internet user behavior has been observed even for the web site of the college of american pathologists (cap), where a web page for complex cancer protocols, presumably of interest only to pathologists, recorded the highest number of hits for the adrenal protocol, even though this is an obscure site for malignancies (personal communication). the protocols were arranged alphabetically on that cap web page, with adrenal first. based upon user behavior observed in this study, developers of web pages should place items of greatest interest or impact at the top of a menu. designers of computer - aided instruction should bear in mind that, the longer the exercise, fewer users will complete it in the manner intended. web page authors can evaluate whether developing more content is worth the additional effort, given a declining rate of usage for more items at the bottom of a menu. providing more exercises of a similar nature does not guarantee equivalent usage at the web site. would whole - slide imaging (wsi) lend itself to this kind of examination format ? high - stakes timed examinations for students generally provide additional time, such as 1.5 min / question with imaging, and less for text - only questions. however, use of wsi would require even more time, and reduce the total number of questions for examinations limited to a specific time period. students in health science educational programs have a heavy curricular load, and pathology is just one of many subject areas that require testing. guided instruction with a small number of labeled pathologic images fits within the time available for group learning exercises, such as laboratory sessions, in most curricula. novice students are used to viewing video screens, and are more likely to tolerate such a medium than the multi headed microscope driven by a pathologist moving the slide at so rapid a rate that motion sickness develops in some students. however, there is no practical way to test on wsi with large class sizes, and so students can safely ignore it. though wsi has been applied at the medical student level, it is more effectively suited to training programs in pathology, such as residencies and fellowships. wsi has been evaluated for testing pathology residents taking specialty board examinations administered by the american board of pathology. this study showed that web - based examination completion rates were higher for shorter examinations in a single subject area. usage was higher for shorter 10questions examinations, and for examinations listed closer to the top of the web page menu. users identifying themselves as md / do scored higher than other users, averaging 75%, but this was only slightly higher than the overall average for all users, regardless of their voluntarily stated health science field of study. scores 90% were achieved by 21% of users for review examinations, and by 37% of users for subject examinations and suggest that there were serious users completing the examinations who had sufficient preparation to make use of the examinations to support their pathology education. examinations of shorter length have higher completion ratesusage is highest for items nearer the top of a web page menudeveloping more examinations does not guarantee their equivalent usagea cohort of users had scores 90%, validating the effort in web site development. examinations of shorter length have higher completion rates usage is highest for items nearer the top of a web page menu developing more examinations does not guarantee their equivalent usage a cohort of users had scores 90%, validating the effort in web site development. | context : general and subject specific practice examinations for students in health sciences studying pathology were placed onto a free public internet web site entitled web path and were accessed four clicks from the home web site menu.subjects and methods : multiple choice questions were coded into. html files with javascript functions for web browser viewing in a timed format. a perl programming language script with common gateway interface for web page forms scored examinations and placed results into a log file on an internet computer server. the four general review examinations of 30 questions each could be completed in up to 30 min. the 17 subject specific examinations of 10 questions each with accompanying images could be completed in up to 15 min each. the results of scores and user educational field of study from log files were compiled from june 2006 to january 2014.results:the four general review examinations had 31,639 accesses with completion of all questions, for a completion rate of 54% and average score of 75%. a score of 100% was achieved by 7% of users, 90% by 21%, and 50% score by 95% of users. in top to bottom web page menu order, review examination usage was 44%, 24%, 17%, and 15% of all accessions. the 17 subject specific examinations had 103,028 completions, with completion rate 73% and average score 74%. scoring at 100% was 20% overall, 90% by 37%, and 50% score by 90% of users. the first three menu items on the web page accounted for 12.6%, 10.0%, and 8.2% of all completions, and the bottom three accounted for no more than 2.2% each.conclusions:completion rates were higher for shorter 10 questions subject examinations. users identifying themselves as md / do scored higher than other users, averaging 75%. usage was higher for examinations at the top of the web page menu. scores achieved suggest that a cohort of serious users fully completing the examinations had sufficient preparation to use them to support their pathology education. |
having strong coping skills to reduce stress and satisfaction on the decision - making process to be creative and also having problem - solving skill is necessary in life. in this way, learning should be defined meaningful and problem - solving skill is one of these ways. unfortunately, traditional teaching method in universities transfers a mixture of information and concepts to individuals but leaves them alone in analyzing, prioritizing, and reorganizing emerging knowledge which requires critical thinking and will lead to effective and meaningful learning. critical thinking is considered is an essential part of clinical decision - making and professional competence of nursing staff and medical emergencies. emphasize the need for this skill in these jobs is due to rapid change in the healthcare field and complexities of this current system, which face staff working in health care services with the special situation to render safe, decent, and high quality services. in general, health care staff has to use critical thinking in taking important and vital decisions inevitable. education experts agree that critical thinking should be an integral part of any education in every level, because, it is thinking that will lead to make a decision the best possible solution with analyzing, assessing, selecting, and application and this is what is needed in the contemporary world of today. interest in the abilities of critical thinking in educational circles is not a new phenomenon, and its origin dates back to plato 's academy. accordingly, this skill is considered as a priority in training medical specialties. however, using and expanding it by staff of medical emergencies is a necessity, which causes delineating comprehensive and purposeful care plan and also increasing the probability of success in the management of victims and the scene. furthermore, increase the number and scale of natural disasters over the past decade has caused that staff of medical emergencies, working in the field of healthcare and treatment cares in current situation, face with very complicated problems comprehensively as a result of advanced technology, ethical and cultural factors. in this regard, it is necessary that traditional methods should be replaced with decentralized emergency management systems to meet demands and attain considerable success in severe events and catastrophic disasters. some of these techniques use decision - making skills, creative thinking, and problem - solving skill in today 's world. critical thinking skills in medical education are considered as the ultimate goal of learning and staff and personnel, who think creatively and critically, less commit false judgment and conclusion, rather, they try to concentrate on topics that are relevant to the clinical area and adopt proper decision in this respect, the issue of which can reduce gap between theoretical and clinical education to a great extent. however, making a decision is considered the most important and risky part of health care professions. therefore, knowing the decision making and applying useful strategies for creating this skill is essential for health care personnel, particularly, the staff working in medical emergencies. triage is just one of complicated decision - making examples which include considering patient and other factors of the treatment system. therefore, knowing decision - making and applying fruitful strategies for the creation of this skill is essential for healthcare workers, especially those working in medical emergencies. considering the job sensitivity of medical emergencies staff and significance of decision - making power and ability of solving problem in them, as well as selecting and adopting accurate decision at the time of triage of patients, they (health - care workers) are in dire need of skill more than anything else to adopt proper decision. on the other hand, moreover all these problems, staff working in medical emergencies will face unique problems which are specific to their job environment such as working with multiple staff and people in treatment team of patients and crisis hit families, happy and sad moments, life and death, accidents and disaster. therefore, with due observance to the said issues, the effect of training problem - solving skills on decision - making power and critical thinking of staff working in medical emergencies is the main aim of this study. this is an experimental study with two pre- and post test stages, in which, effect of teaching problem - solving skill (independent variable) has been studied on decision - making skill and critical thinking of 95 staff working in medical emergencies (dependent variable). this 95 staff was divided into two groups : experimental (n = 47) and control (n = 48). a sample of this study includes all staff working in medical emergencies as many as 95 persons who showed their interest to participate in this study. purposive sampling method and size in this study were similar to the total population. in iran, prehospital emergency medical handled by the graduate of an emergency medical technician and a bachelor and only men is graduates in this field. moreover, there are a number of general physicians in the central dispatch who provide medical consultation for people who call emergency medical services (ems) and also give medical advice to the technicians who treat victims at the crash scene or on the way to the hospital., it was tried to control factors affecting decision - making power and critical thinking (such as age, marital status, working experience, education, number of children, mental illness and consumption of psychotropic drugs and experience of participating in classes (emotional intelligence, stress management, yoga, problem - solving and decision - making within 6 months) in each two groups. in this regard, any significant difference was not observed between research units in both groups (p > 0.05). decision - making and critical thinking skill of personnel was evaluated before and after the intervention. decision - making skill was evaluated in twenty questions using decision - making questionnaire. according to likert scale, each question was scored in four levels from 0.25 to 1. the minimum and maximum score in this questionnaire stood at 5 and 20, respectively. in other words, the lowest score in this questionnaire translation and preparation stages of questionnaire were conducted. in the same direction, face and content validity of the questionnaire the reliability of this test was obtained at 0.87 and approved using retest method in a 2-week time interval for 30 persons. california critical thinking skills test is not a test only, rather, it includes various editions for measuring critical thinking skills in children, young adults, students in various academic levels and also various health, law and business professions, etc. three cognitive critical thinking skills including analyzing, evaluating, and inference are measured while in the second category, two cognitive skills of inductive reasoning and deductive reasoning are evaluated. in fact, all forms and various editions of this test are measured the five mentioned cognitive skills. the questionnaire used in this study consists of 34 four or five option questions with only one option is correct. some questions should be answered with thinking and inferring a series of assumptions while some others should be answered with well grounded assessment of a conclusion. this questionnaire is appropriate for evaluating students critical thinking and also for assessment of those people who are in dire need of solving problem and making a decision. the range of test scores is between 0 and 34. in a study conducted by khalili. on 405 students in nursing at tehran, iran and shahid beheshti universities of medical sciences, persian translation of this form was studied in terms of validity and reliability. the reliability coefficient obtained in the above study, using kr-20 (62%), had a high correlation with the reliability coefficient obtained in the standardization process of this test conducted in the us (68%-70%). furthermore, construct validity, which has been translated as the most important validity type in tests, indicates correlation between the structure of this test and its basic theory. to control data transfer between personnel of test and control groups, personnel of the experimental group were requested not to talk with the control group at the time of research with regard to the conducted interventions. then, training a course of problem - solving skill was held in eight 2-h sessions during 8 weeks in the presence of personnel of experimental group, using group discussion methods, brainstorming and discussion in small four member groups with the guidance of a pertinent professor, taking advantage of social problem - solving model as practiced by d the stages of this model include : the ability to identify problemacknowledging the problem as a changeable potentially natural phenomenonbelieved to be effective in dealing with the problem - solving frameworkhigh self efficiency expectations to implement stages of modelaccustomed to stop, think, and then making effort to solve a problem. the ability to identify problem acknowledging the problem as a changeable potentially natural phenomenon believed to be effective in dealing with the problem - solving framework high self efficiency expectations to implement stages of model accustomed to stop, think, and then making effort to solve a problem. collection of all information availableseparation of facts is of the assumptions which require investigationanalysis of the problemspecifying the actual objectives. collection of all information available separation of facts is of the assumptions which require investigation analysis of the problem specifying the actual objectives. to determine wide range of possible solutionsability to choose the most effective response to replies. to determine wide range of possible solutions ability to choose the most effective response to replies. predict probable consequences of each actionpaying due attention to the usefulness of these consequences. predict probable consequences of each action paying due attention to the usefulness of these consequences. observing the results of execution all sessions of the training course were designed in tandem with this pattern, and one stage of this pattern was executed in each session. in each session, so the presence of all members of the experimental group, the instructor explained the objectives of the meeting and the participants requested that their experiences in dealing with different problems on the job fit the theme of the meeting, express. then, discuss a case study, and participation in meeting goals, training was provided to them. chicago : spss inc.) in addition, statistical t - test, chi square test, and paired t - test were used. the ability to identify problemacknowledging the problem as a changeable potentially natural phenomenonbelieved to be effective in dealing with the problem - solving frameworkhigh self efficiency expectations to implement stages of modelaccustomed to stop, think, and then making effort to solve a problem. the ability to identify problem acknowledging the problem as a changeable potentially natural phenomenon believed to be effective in dealing with the problem - solving framework high self efficiency expectations to implement stages of model accustomed to stop, think, and then making effort to solve a problem. collection of all information availableseparation of facts is of the assumptions which require investigationanalysis of the problemspecifying the actual objectives. collection of all information available separation of facts is of the assumptions which require investigation analysis of the problem specifying the actual objectives. to determine wide range of possible solutionsability to choose the most effective response to replies. to determine wide range of possible solutions ability to choose the most effective response to replies predict probable consequences of each actionpaying due attention to the usefulness of these consequences. predict probable consequences of each action paying due attention to the usefulness of these consequences. observing the results of executionevaluation. observing the results of execution all sessions of the training course were designed in tandem with this pattern, and one stage of this pattern was executed in each session. in each session, so the presence of all members of the experimental group, the instructor explained the objectives of the meeting and the participants requested that their experiences in dealing with different problems on the job fit the theme of the meeting, express. then, discuss a case study, and participation in meeting goals, training was provided to them. chicago : spss inc.) in addition, statistical t - test, chi square test, and paired t - test were used. 100% of participants in this study were men, and all were employed in prehospital emergency centers in isfahan province. the age range of participants was between 23 and 51 years, and their mean age stood at 32.15 5.21. of total participants, 29 (30.52%) and 66 (69.47%) persons were single and married, respectively. the minimum and maximum job experience of participants in this study stood at 1 and 29 years, respectively. of total participants in this study, 10 participants (10.52%) held a diploma degree, 32 participants (33.68%) with associate 's degree, 50 participants (52.63%) with bachelor 's degree and three participants (3.15%) with master 's degree. of total participants, 48 persons (50.52%) graduated in nursing, 25 persons (26.31%) in medical emergencies, nine persons (9.47%) in anesthesiology, five persons (5.26%) in operating room, and eight persons (8.42%) graduated in other fields of study. in terms of employment status, 20 of participants (2.10%) were official crew members, whereas 54 participants (56.84%) had been employed in contracting basis, 11 of participants (11.57%) employed in contractual basis, whereas 10 participants (10.52%) had been employed as corporate manpower. it should be noted that 60 participants (63.15%) were employed in urban emergency bases while 35 of them (36.84%) had been employed in road emergency centers. before intervention, mean and standard deviation of decision - making scores in experimental and control groups stood at (12.85 2.57) and (11.79 2.12), respectively, while total score of critical thinking in test and control groups stood at (10.42 1.85) and (10.61 2.12), respectively. given the above issue, independent t - test did not show a significant statistical difference between these means. furthermore, chi square statistical test showed that there is not any significant difference between two groups of experimental and control statistically in terms of demographic variables. none of the groups, that is, experimental and control showed experience of participating in the following classes such as yoga, problem solving, emotional intelligence, and stress control and management. the average decision - making score in whole samples before intervention stood at 11.84 1.38 while critical thinking score stood at 11.03 1.09 the mean decision - making score before intervention showed no a significant difference between the two groups statistically (p > 0.05), but the mean between two groups showed the significant difference after intervention (p 0.05), but this mean showed a significant difference between the two groups after intervention (p 0.05). comparing mean difference of decision - making skill scores before and after intervention comparing mean difference of critical thinking scores before and after intervention any significant relationship was not observed between demographic variables with critical thinking and decision - making power (p > 0.05). the results of this study show the weakness of decision - making and critical thinking skill in personnel of medical emergencies and an increase of this skill with training problem - solving skill. unfortunately, a study has not thus far been conducted with regard to the determination of decision - making skill among personnel of medical emergencies. in a study which was conducted by gunnarsson and stomberg in sweden, he examined factors affecting decision making among ems personnel in emergency centers. in his study, he reported that various factors affect decision - making power of these personnel which includes as follows : factors related to patient, factors related to the environment, factors related to colleagues, factors related to patients privacy issues, performance of team leader, staff technical know how and knowledge and moral contradictions, etc. it should be noted that these issues make decision making very difficult for these people and sometimes, would lead to unsuccessful decisions. in a study conducted by franklin., they examined the way of decision - making among staff of medical emergency and reported that method of staff 's decision - making has high relationship with the mental processes, cognitive abilities, degree of sensitiveness of decision, power of cognition (judgment), solving problem, and organizational situation of their workplace. therefore, quality decision - making training courses should be organized at higher levels by adopting better decisions. hence, to make better decision, staff must make an educated decision at higher levels, and education in healthcare environment merely should go step further furthermore, results of study conducted by dy and purnell showed that a variety of factors effect on the complexity of decision - making process among personnel in healthcare and treatment system, the most important of which include as follows : ability and talent of individuals, level of culture, ability of patient, level of knowledge and information, method of establishing relationship, ability of solving problem, etc. to adopt the best decision pitt. also reported that promoting critical thinking skills can increase professional competency and qualifications of nurses to a great extent, the issue of which as of paramount importance for personnel working in special emergency wards. thaiposri and wannapiroon also studied the impact of conceptual methods in promoting critical thinking skill and enumerated problem - solving method as one of methods of strengthening and improving critical thinking skill which is a solid evidence of the said claim. popil also showed that the critical thinking skill can be promoted using challenge methods such as case study. the results a study conducted by roberts. and heidari and ebrahimi emphasizing on decision - making skill, confirm these results. with due observance to the results of this study and given the significance which is considered for empowering associate 's degree personnel of medical emergencies in terms of problem - solving skills, it can be concluded that these skills are weak among them. hence, to attain the best decisions, providers should receive on - the - job training to foster strong problem - solving and decision - making skills that can be utilized in the field and on the front lines of iranian emergency medicine. finally, despite the significance of decision making and its influence on the way of managing and caring victims, the results of studies conducted in iran show the insufficient skill of personnel in decision - making and critical thinking process. considering the job sensitivity of staff working in medical emergencies and significance of decision - making power and ability of critical thinking in them, it can be said that giant stride can be taken in line with promoting scientific and job level of associate 's degree personnel working in medical emergencies. in general, status of this scientific course considering that this study has been conducted among students in medical emergencies, generalization of results of this study to other students is impossible, so that this study is recommended to be conducted in other academic courses with high number of students. in other words, results of this study can not be generalized to other students. for this reason, this study is recommended to be conducted for students of other academic courses. | background : the aim of this study was to determine the effect of problem - solving training on decision - making skill and critical thinking in emergency medical personnel.materials and methods : this study is an experimental study that performed in 95 emergency medical personnel in two groups of control (48) and experimental (47). then, a short problem - solving course based on 8 sessions of 2 h during the term, was performed for the experimental group. of data gathering was used demographic and researcher made decision - making and california critical thinking skills questionnaires. data were analyzed using spss software.results:the finding revealed that decision - making and critical thinking score in emergency medical personnel are low and problem - solving course, positively affected the personnel decision - making skill and critical thinking after the educational program (p < 0.05).conclusions : therefore, this kind of education on problem - solving in various emergency medicine domains such as education, research, and management, is recommended. |
these infectious diseases have spread worldwide and have become a serious public health problem (1). e. histolytica possesses mitosomes, a type of mitochondrion - related organelle (mro) (25). this organelle was originally called crypton when its discovery was reported, and such organelles are currently known as mitosomes, the widely accepted name (6, 7). mros are derived from canonical mitochondria and are ubiquitously found in anaerobic / microaerophilic eukaryotes (2, 4). mros display a variety of unique characteristics which are conferred by essentially reduced and/or modified mitochondrial functions and that occasionally result from the addition of new functions acquired through lateral gene transfer (5, 8). it has been postulated that the uniqueness of mros helps organisms to adapt to various niches for their survival (25, 8). entamoeba mitosomes have largely lost typical mitochondrial functions, such as those involved in the tricarboxylic acid cycle, electron transport, oxidative phosphorylation, and -oxidation of fatty acids (4, 5). what are their biological roles in this organism ? despite being recognized, these important issues have not been satisfactorily addressed. we previously showed that sulfate activation, which is not generally compartmentalized to mitochondria, is a major function in e. histolytica mitosomes (3, 5, 9). furthermore, we demonstrated that 3-phosphoadenosine 5-phosphosulfate (paps), which is synthesized through mitosomal metabolism, acts as an activated sulfur donor mainly to produce sulfolipids by the catalytic actions of putative cytosolic sulfotransferases (sults) (5). cholesteryl sulfate (cs) is one such sulfolipid that plays an important role in encystation, a cell differentiation process necessary for maintaining the entamoeba life cycle (9). these findings provide not only an explanation for the biological role of entamoeba mitosomes but also evidence to support the uniqueness of mros. importantly, our findings indicate that in entamoeba, paps, a metabolite synthesized through the mitosomal pathway, needs to be translocated to the cytosol for the production of sulfolipids. therefore, a molecule that can translocate paps from mitosomes to the cytosol must be present. three candidates which could be responsible for this important function are encoded in the e. histolytica genome. one is a paps transporter, and the others are mitochondrial carrier (mc) proteins (ehi_068590, ehi_095150, and ehi_153760, respectively) (amoebadb ; http://amoebadb.org/amoeba/). however, the paps transporter can be ruled out because its nonmitosomal localization has already been demonstrated (5). mc proteins belong to a large family of mitochondrial inner membrane carriers that transport various metabolites between the cytosol and mitochondrial matrix (10, 11). most mc proteins are localized in mitochondria, but atypical localizations in chloroplasts and peroxisomes have recently been reported (11). the structural features conserved in mc proteins include a size of 30 to 35 kda, three tandemly repeated homologous domains, each of which has 100 amino acid residues, and six transmembrane helices forming an aqueous cavity. substances transported by mc proteins include nucleotides, amino acids, keto acids, and inorganic phosphate (pi) (10, 11). in this study, to address the issue of how the mitosomal sulfate activation pathway and putative cytosolic sults cooperate in e. histolytica, we performed biochemical and cell biological characterizations of an e. histolytica mc protein, e. histolytica mitochondrial carrier family (ehmcf), and related sulfate metabolism enzymes, the e. histolytica sults (ehsults) and e. histolytica 3(2),5-bisphosphate nucleotidases (ehpapases). [c]leucine (stock radioactivity, 100 ci / ml), [p]atp (stock radioactivity, 10 mci / ml), and [s]paps (stock radioactivity, 1 mci / ml) were purchased from perkinelmer japan (yokohama, japan). the e. histolytica hm-1:imss cl6 strain was routinely cultured in vitro in diamond 's bi - s-33 medium at 37c as described previously (3, 5). expression plasmids for hemagglutinin (ha)-tagged ehsults and ehpapases were constructed by pcr amplification of the corresponding open reading frames (orfs) using appropriate primers sets (table 1). amplicons, except for the one containing ehsult7, were then digested with bglii and inserted into the corresponding sites of the pehex - ha plasmid (5). the pcr - amplified ehsult7 fragment was directly cloned into bglii - digested plasmid pehex - ha using an in - fusion hd cloning kit from takara bio (otsu, japan) according to the manufacturer 's instructions. lipofection transfection of e. histolytica trophozoites with the constructed plasmids, drug selection, maintenance of selected transformants, and indirect immunofluorescence analysis of independent established transformants were performed as described previously (5). the recombinant ehmcf (rehmcf) protein that is encoded in the e. histolytica genome (ehi_095150 in amoebadb ; http://amoebadb.org/amoeba/) was produced using a wheat germ cell - free translation system in the presence of asolectin liposomes, which were freshly prepared just prior to use, as described previously (12). two plasmids, pyt08-ehmcf and pyt08-codon - optimized ehmcf, were constructed as the templates for in vitro mrna synthesis. for pyt08-ehmcf, a dna fragment encoding the ehmcf orf was amplified from e. histolytica cdna by pcr with an appropriate primer set (table 1), digested with spei and sali, and inserted into the corresponding sites of the pyt08 vector (12). for the pyt08-codon - optimized ehmcf, a synthetic dna encoding an ehmcf orf in which the codon usage is optimized to that of the wheat germ translation system (genbank accession number lc036596) was custom synthesized by genscript japan (tokyo, japan) and cloned into spei and sali sites of the pyt08 vector (12). the mrnas synthesized from the constructed plasmids were then used in the cell - free translation system in the presence of asolectin liposomes and, when needed, with [c]leucine (final radioactivity, 2 ci / ml) as described previously (12). subsequently, the reaction mixtures were centrifuged at 20,000 g for 20 min at 4c, and the supernatant and the precipitate were separately collected. finally, the precipitate was suspended in 150 l 10 mm pipes - naoh (ph 6.5) and subjected to ultrasonication (digital sonifier model 250 d ; branson, danbury, ct, usa) for 18 s (50% duty cycle). the suspension obtained was used for vesicle preparations for the transporter activity assay as described in the following section. as a blank suspension, the reaction mixture operated in the absence of mrna was treated in the same way as the rehmcf protein suspension. to verify the production of rehmcf and its purity, 1/100 volumes from each fraction were analyzed by sds - page, followed by visualization through either autoradiography or coomassie brilliant blue staining. two types of the vesicles, namely, substrate - loaded vesicles and empty vesicles, were prepared and used for the assays. the substrate - loaded vesicles were prepared essentially as described previously (12). in detail, the rehmcf protein suspension (see the section above) was mixed with an equal volume of solution containing liposomes into which one of the countersubstrates examined was preloaded. to prepare the liposome solution, 200 mm pipes naoh (ph 6.5) containing 40 mm potassium gluconate and 50 or 20 mm countersubstrate was added on acetone - washed asolectin (80 mg lipid / ml), followed by ultrasonication (digital sonifier model 250 d ; branson, danbury, ct, usa) for 5 min on ice. subsequently, the obtained mixture was frozen in liquid nitrogen, thawed at room temperature, and subjected to ultrasonication (digital sonifier model 250 d ; branson, danbury, ct, usa) for 18 s (50% duty cycle). finally, the countersubstrate that was not loaded into vesicles was removed by gel filtration with a dowex ag-1x8 column (bio - rad, tokyo, japan) using 10 mm pipes naoh (ph 6.5) containing 40 mm potassium gluconate and 100 mm sodium gluconate. the empty vesicles were prepared in a same way as described for the substrate - loaded vesicles except that no countersubstrates were preloaded. blank controls for each type of vesicle were likewise prepared using the blank (see the section above) suspension in place of the rehmcf protein suspension. the transport activity of the rehmcf in vesicles was measured as described previously (12). briefly, a quantitative evaluation of the uptake of either [p]atp or [s]paps into substrate - loaded or empty vesicles was performed. the final concentration of preloaded countersubstrate in the transport assay of the uptake of [p]atp or [s]paps was 25 mm or 10 mm, respectively, and that of added radiolabeled substrate (either [p]atp or [s]paps) was 0.5 mm. the reaction was initiated at 25c upon addition of 5 l radiolabeled substrate into 100 l 10 mm pipes naoh (ph 6.5) containing either type of vesicle, [p]atp (final radioactivity, 2.7 ci / ml) or [s]paps (final radioactivity, 1.5 ci / ml). reactions were terminated by the addition of 15 l stop solution (360 mm pyridoxal 5-phosphate, 64 mm mersalyl acid). after removal of extravesicle radiolabeled substrate by gel filtration performed with a dowex ag-1x8 column (bio - rad, tokyo, japan) using 200 mm sodium acetate, the radioactivity associated with each type of vesicle was measured as units of disintegrations per minute using an lsc-6100 liquid - scintillation counter from aloka (tokyo, japan). in parallel, finally, the amount of substrate transported into the vesicles was calculated after subtracting the radioactivity of a blank control from that of the corresponding sample. the amount of protein which was estimated prior to vesicle preparation was used to determine the specific transporter activity. its estimation was accomplished by measuring the intensity of the corresponding bands in sds - page gels stained by coomassie brilliant blue. bovine serum albumin was used as a standard. for a time course analysis, a quantitative evaluation of the uptake of [p]atp into substrate - loaded vesicles into which nonradiolabeled atp was preloaded or into empty vesicles was performed at 0, 5, 10, or 20 min after starting the incubation. for a countersubstrate specificity analysis, a quantitative evaluation of the uptake of either [p]atp or [s]paps into the substrate - loaded vesicles into which one of the various substances tested was preloaded or into empty vesicles was performed after 10 min of incubation. the proteins described in this study (genbank accession numbers, e. histolytica genome identification numbers) and the accession number of the ehmcf synthetic gene are as follows : mcf (xp_649800, genome identification number ehi_095150), pi transporter (xp_656350, ehi_153760), paps transporter (xp_654175, ehi_068590), sult1 (xp_654200, ehi_140740), sult2 (xp_654101, ehi_166030), sult3 (xp_651675, ehi_197340), sult4 (xp_655605, ehi_092490), sult5 (xp_650013, ehi_090430), sult6 (xp_649714, ehi_146990), sult7 (xp_651768, ehi_114190), sult8 (xp_652989, ehi_181190), sult9 (xp_653539, ehi_031640), sult10 (xp_650544, ehi_062680), papase1 (xp_651950, ehi_193350), papase2 (xp_655585, ehi_179820), papase3 (xp_650613, ehi_175410), and the synthetic ehmcf cdna (lc036596). the e. histolytica genome contains 10 genes that encode putative sults, which can catalyze the production of sulfated molecules, e.g., sulfolipids, from paps and sulfur acceptors (amoebadb ; http://amoebadb.org/amoeba/ [5, 9 ]). we previously demonstrated the cytosolic localization of ehsult1 and -2 (5). to fully determine the localization of all ehsults, we established independent transformants expressing ha - tagged ehsult3 to -10 in e. histolytica and analyzed the transformants obtained. the fluorescent signals detected in these transformants were distributed throughout cells, except in organelles such as the nucleus, vacuoles, and small vesicles (fig. this result, combined with the data corresponding to the cytosolic localization of ehsult1 and -2 (5), indicates that all ehsults are localized in the cytosol (fig. importantly, it also confirms the cytosolic localization of ehsult6, which catalyzes the production of cs, a sulfolipid important for entamoeba encystation (9). these findings are consistent with the requirement for paps, a mitosomal sulfate - activated metabolite, to be transported into the cytosol for it to be a substrate for ehsults in e. histolytica. two e. histolytica mc proteins (ehi_095150 and ehi_153760, respectively) have been characterized as an adp / atp carrier (aac) and a pi carrier (amoebadb ; http://amoebadb.org/amoeba/ [6, 9, 13 ]). in addition, phylogenetic analysis showed that only the e. histolytica aac (ehaac) and not the pi carrier is a member of a subfamily cluster of mc proteins, which includes carriers transporting adenine nucleotides and coenzyme a (coa) (13). recently, a carrier that was originally characterized as an aac of the thylakoid membrane (14) was shown to possess a capacity for countertransporting paps ; therefore, it is now known as a paps transporter (15). these findings narrow down the mitosomal membrane paps transporter candidates and are consistent with our hypothesis that ehaac acts as an antiporter that can transport paps across mitosomal membranes in e. histolytica. ehaac is more commonly termed e. histolytica mitochondrial carrier family (ehmcf) (3, 5, 6, 16) ; therefore, unless otherwise stated, we use the name ehmcf here. obtaining functional rehmcf protein is necessary to address how the mitosomal sulfate activation pathway cooperates with cytosolic sults in e. histolytica. to achieve this, we exploited a wheat germ cell - free translation system in the presence of asolectin liposomes because this system circumvents several problems encountered in expressing recombinant membrane proteins in surrogate organisms or cells (17, 18). in this system, we can trace only the target recombinant protein as a nascent protein using a radiolabeled leucine. the molecular size of the synthesized radiolabeled protein was 30 kda, which is close to the deduced molecular mass of ehmcf (30,443 da), and it was predominantly recovered from the fraction precipitated at 20,000 g (fig. the yield of rehmcf could be improved by using a synthetic ehmcf cdna in which the codon usage is optimized to that of the wheat germ translation system (fig. most importantly, rehmcf was highly enriched and became a major protein in the precipitated fraction, indicating that centrifugal fractionation is sufficient to purify rehmcf nearly to homogeneity (fig. production (a) and purification (b) of rehmcf and the assay system for measurement of its transporter activity (c). autoradiograph (a) and coomassie brilliant blue - stained (b) sds - page gels are shown. the loaded samples, which were prepared by centrifugal fractionation of a reaction mixture from the cell - free translation system, were as follows : i, initial material ; s, supernatant ; p, precipitate. the different dna fragments carrying an ehmcf orf in the pyt-08 vector are as follows : native, a pcr - amplified dna from e. histolytica cdna ; codon - optimized, a synthetic dna in which the codon usage of the ehmcf orf is optimized to that of the wheat germ translation system. the uptake of external [p]atp into the substrate - loaded vesicles preloaded with nonradiolabeled atp (open) or into empty vesicles (filled) was measured. the data are shown as means standard deviations (sds) (indicated with error bars) of the results from more than three independent experiments. asterisks indicate significant differences from empty vesicles (, p < 0.05 ; p < 0.01). we then examined whether the rehmcf purified as the precipitated fraction was functional by measuring its ability to transport atp, a standard substrate for mc proteins. uptake of p - labeled atp into substrate - loaded vesicles proceeded in a time - dependent manner, while uptake of p - labeled atp into empty vesicles could not be detected (fig. 2c). furthermore, they indicate that rehmcf is properly reconstituted into the lipid bilayer of asolectin liposomes, indicating that rehmcf can function as an antiporter. our primary question does ehmcf, a mitosomal protein, participate in the translocation of paps across mitosomal membranes in e. histolytica ? can be now addressed. to address this question, we measured the countertransport activity of the reconstituted rehmcf for atp with paps, atp, adp, or amp. the uptake of p - labeled atp into the substrate - loaded vesicles was significantly higher when paps was used as a countersubstrate (11.7 0.4 nmol / mg protein) than when adenosine mono-, di-, or triphosphates were used (3.2 0.2, 0.0 0.5, or 1.8 0.7 nmol / mg protein, respectively). the uptake of p - labeled atp into empty vesicles could not be detected (fig. these data are partly inconsistent with those from a previous study owing to differences in the atp / adp exchange activity (6). in this study, no uptake of p - labeled atp into the adp - loaded vesicles or the empty vesicles could be detected, while in the previous study, a significant uptake of c - labeled atp into adp - loaded vesicles was observed. assessment of this difference is hampered by the lack of demonstration of uptake of c - labeled atp into the empty vesicles in the former work. they also showed a significant uptake of c - labeled adp into atp - loaded vesicles, a result which was well supported by showing competitive inhibition by excess cold adp as well as the absence of uptake into the empty vesicles. this inconsistency in the reported atp / adp exchange activity may be due to differences in proteoliposome preparations, in components in the reactions (e.g., the use of radiolabeled materials as a substrate or of preloaded molecules as a countersubstrate), and/or in assay conditions (e.g., incubation time or concentrations of substrate added and of countersubstrate preloaded). another explanation is possible differences in the lipid compositions because of different membrane sources having been used for protein reconstitutions (this study and reference 6). 3a) clearly indicate that ehmcf indeed has the capacity for countertransport of atp using paps as a preferred countersubstrate. the substrate - loaded vesicles (preloaded substances are indicated by names) and the empty vesicles (indicated by a minus sign) were assayed for the uptake of either [p]atp (a) or [s]paps (b). the specific transport activity (a) and the transport activity relative to that of paps as the control (set as 100%) (b) are shown. the data are presented as the means sds calculated from the results of three independent experiments. the raw data used to calculate the relative activity levels are as follows : paps, 32.8 7.2 ; pap, 39.1 13.9 ; atp, 14.1 5.0 ; adp, 13.6 3.2 ; amp, 2.4 0.7 ; aps, 7.3 1.6 ; coa, 4.2 0.7 ; sulfate, 0.1 0.1 ; gtp, 0.1 0.2 ;, 0.0 0.1 (nmol / mg protein / min). asterisks indicate significant differences between paps and other countersubstrates (, p < 0.05 ; p < 0.01). to investigate further the countersubstrate specificity, we measured the activity of paps countertransport with various substances. among potential countersubstrates examined, adenosine 3,5-bisphosphate (pap) gave the highest activity for the uptake of s - labeled paps into the substrate - loaded vesicles (119.2 42.4% relative to that of paps as 100% control). adenosine mono-, di-, and triphosphates all gave moderate activities, but a preference for atp and adp over amp (43.0 15.2% and 41.5 9.6% over 7.3 2.2%, respectively) was observed. similarly, adenosine 5-phosphosulfate (aps) and coenzyme a (coa) also gave moderate activities (22.3 4.8% and 12.8 2.2%, respectively). sulfate and gtp gave nearly untraceable activities (0.3 0.2% and 0.3 0.5%, respectively). consistent with the assays using p - labeled atp, uptake of s - labeled paps into empty vesicles could not be detected at all (fig. 3) indicate that ehmcf is different from archetypal aacs regarding countersubstrate specificity and are in agreement with the previous finding that carboxyatractyloside and bongkrekic acid, specific inhibitors of most aacs, are not effective against ehaac (6). more importantly, these data indicate that ehmcf functions mainly as a paps / pap and paps / paps antiporter in vitro. generally, pap is produced together with a sulfated molecule through the catalytic action of sult with respect to paps and a sulfur acceptor. pap is then degraded to adenosine 5-phosphate and pi by 3(2),5-bisphosphate nucleotidase [19, 20 ; 3(2),5-bisphosphate nucleotidase is described here as papase for ease of reading ]. in e. histolytica 1) (5), and ehmcf has a high capacity for countertransport of paps with pap or paps in vitro (fig. 3). therefore, we inferred that knowing the localization of ehpapase is important for predicting the availability of the substrates for ehmcf in vivo ; this will help unravel its precise role. in the e. histolytica genome, three genes encoding putative papase1 to -3 (ehi_193350, ehi_179820, and ehi_175410, respectively) (amoebadb ; http://amoebadb.org/amoeba/) are present. to determine the localization of all the papases in e. histolytica, we established independent transformants expressing each ha - tagged ehpapase. in all the transformants analyzed, except in organelles such as the nucleus, vacuoles, and small vesicles, the fluorescent signals were evenly distributed throughout cells, indicating that ehpapase1 to -3 are localized in the cytosol (fig. consistent with these observations, the cytosolic localizations of ehpapase1 and -2 have been recently reported (21, 22). moreover, ehpapase1 has been biochemically characterized (21) and active transcription of ehpapase1 to -3 has been reported (23). collectively, these findings indicate that all the ehpapases are functional in the e. histolytica cytosol. 1) (5), suggests that pap produced by the catalytic action of ehsults is sequentially degraded by ehpapases ; therefore, the pap concentration in the cytosol is maintained at a low level in e. histolytica. this is consistent with the general idea that pap is toxic to cells (24) but contradicts another interpretation of the results (fig. 3), i.e., that ehmcf functions mainly as a paps / pap and paps / paps antiporter in vitro. however, the results (fig. 3) also showed that the reconstituted rehmcf had high activity for the exchange of atp with paps. atp, which is mainly synthesized through cytosolic pathways, is a crucial molecule for the sulfate activation pathway to produce paps in e. histolytica mitosomes, whereas paps is a necessary substrate for ehsults to produce sulfolipids in the cytosol. hence, maintaining a molecule such as ehmcf that has a high capacity for exchanging atp with paps across the mitosomal membrane could be beneficial for e. histolytica. in agreement with this interpretation, we previously demonstrated that mcfgs, an e. histolytica g3 strain in which ehmcf was knocked down by gene silencing, showed a significant reduction in sulfate activation activity and a marked growth defect (3). this finding can be now explained by that the shortage of atp in the mitosomes as well by as a shortage of paps in the cytosol. this would impair the synthesis of sulfolipids, which plays important roles in e. histolytica growth. however, a study to confirm the importance of sulfolipids in e. histolytica is needed. mechanistically, the ehmcf knockdown causes an evident reduction in the exchange activity of paps with atp across the mitosomal membrane in e. histolytica ; this reduction lowers the flow of cytosolic atp into mitosomes, leading to a mitosomal atp shortage. this shortage severely impairs mitosomal sulfate activation activity ; therefore, paps production significantly decreases. in addition, even the paps produced can not be efficiently translocated into the cytosol because of the defect in ehmcf activity. consequently, paps levels in the cytosol become remarkably low, resulting in the halting of almost all ehsult sulfolipid synthesis. in conclusion, we suggest that ehmcf functions mainly as a paps / atp antiporter and links the mitosomal sulfate activation pathway to the cytosolic chain reaction that is composed of ehsults and ehpapases in e. histolytica (fig. 5). a scheme for sulfate metabolism in e. histolytica. the flow of metabolites and the enzymes involved are depicted, based on evidence from previous studies (3, 5, 13, 21, 22) as well as from the present study. aps, adenosine 5-phosphosulfate ; apsk, aps kinase ; as, atp sulfurylase ; ipp, inorganic pyrophosphatase ; mcf, mitochondrial carrier family ; nas, sodium / sulfate symporter ; pap, adenosine 3,5-bisphosphate ; papase, 3(2),5-bisphosphate nucleotidase ; paps, 3-phosphoadenosine 5-phosphosulfate ; pi, inorganic phosphate ; pic, pi carrier ; ppi, pyrophosphate ; sult, sulfotransferase. | entamoeba histolytica, a microaerophilic protozoan parasite, possesses mitosomes. mitosomes are mitochondrion - related organelles that have largely lost typical mitochondrial functions, such as those involved in the tricarboxylic acid cycle and oxidative phosphorylation. the biological roles of entamoeba mitosomes have been a long - standing enigma. we previously demonstrated that sulfate activation, which is not generally compartmentalized to mitochondria, is a major function of e. histolytica mitosomes. sulfate activation cooperates with cytosolic enzymes, i.e., sulfotransferases (sults), for the synthesis of sulfolipids, one of which is cholesteryl sulfate. notably, cholesteryl sulfate plays an important role in encystation, an essential process in the entamoeba life cycle. these findings identified a biological role for entamoeba mitosomes ; however, they simultaneously raised a new issue concerning how the reactions of the pathway, separated by the mitosomal membranes, cooperate. here, we demonstrated that the e. histolytica mitochondrial carrier family (ehmcf) has the capacity to exchange 3-phosphoadenosine 5-phosphosulfate (paps) with atp. we also confirmed the cytosolic localization of all the e. histolytica sults, suggesting that in entamoeba, paps, which is produced through mitosomal sulfate activation, is translocated to the cytosol and becomes a substrate for sults. in contrast, atp, which is produced through cytosolic pathways, is translocated into the mitosomes and is a necessary substrate for sulfate activation. taking our findings collectively, we suggest that ehmcf functions as a paps / atp antiporter and plays a crucial role in linking the mitosomal sulfate activation pathway to cytosolic sults for the production of sulfolipids. |
wild - type phi29 dnap (833,000 u ml ; specific activity 83,000 u mg) was supplied by enzymatics corporation. phosphoramidites (including abasic residue (dspacer), 3-spacer c3 cpg, and acridine) were from glen research. dna oligonucleotides were synthesized at the stanford university protein and nucleic acid (pan) facility. lyophilized oligonucleotides received from pan were re - suspended in 7 m urea, 0.1 tbe, purified by denaturing polyacrylamide gel electrophoresis, and quantified using a nanodrop 1000 (thermo scientific). 2 m each of a 70mer dna template (supplementary fig. 1) and a 23-nt dna primer bearing a 5 fluorescein (6-fam) label, were combined in the presence or absence of blocking oligomer (fig 1b(ii)) at 2.4 m final concentration. this mixture was incubated at 90 c for three minutes in 1x te, 100 mm kcl buffer, followed by slow - cooling to room temperature. 1 m of preannealed dna substrate was then incubated for 5 hours at room temperature (approximately 23c) in nanopore buffer (22.5 l final volume) supplemented with 10 mm mgcl2, 0.75 m phi29 dnap, and 100 m dntps as indicated (fig. reactions were terminated by the addition of buffer - saturated phenol. following phenol / chloroform extraction and ethanol precipitation, dried dna pellets were dissolved in 7 m urea, 0.1x tbe and resolved by denaturing polyacrylamide gel electrophoresis. gels were 17% acrylamide : bisacrylamide (19:1), 7 m urea, 1 tbe, and run at 18 w. 6-fam labeled dna primer products were visualized on a uvp gel documentation device using a sybr gold filter. setup of the nanopore device and insertion of an -hl nanopore into a lipid bilayer have been described. briefly, a single -hl nanopore was inserted into a lipid bilayer that separates two wells that each contained 100 l of nanopore buffer (ph 8). a 180 mv potential was applied across the bilayer and ionic current was measure through the nanopore between agcl electrodes in series with an integrating patch clamp amplifier (axopatch 200b, molecular devices) in voltage clamp mode. data were recorded using an analog - to - digital converter (digidata 1440a, molecular devices) at 100 khz bandwidth in whole - cell configuration then filtered at 5 khz using a analogue low - pass bessel filter. experiments were conducted at 23c with 1 m dna substrate preannealed to blocking oligomer in nanopore buffer with 0.75 m phi29 dnap, 10 mm mg, and 100 m of each dntp added to the nanopore cis well unless otherwise noted. a single nanopore experiment is defined as the time during which ionic current data were acquired from one hl nanopore in an intact bilayer before termination by bilayer rupture, loss of channel conductance, or completion of a preset number of translocation events. successful forward and reverse dna template translocations ranged for 50 to 500 per experiment.the ratio of fast (< 200 ms) dna alone events to polymerase - bound dna events was approximately 10 to 1 unless otherwise noted. ionic current output from the nanopore device was analyzed using clampfit 10.2 software (axon instruments) after additional smoothing of data at 2 khz using a low - pass gaussian filter unless otherwise noted. the standardized ionic current map in figure 3b was compiled from ten translocation events from the experiment in figure 2. these ten events each started at 23 pa, traversed two peaks at 35 pa, and ended by stalling at 23 - 24 pa. ionic current steps within these ten translocation events were counted as discrete states if their durations were 3 ms or greater. for the data summarized in supplementary figure 4, sequence - dependent pauses were scored if the duration of a discrete ionic current state between 0 and 16 was 200 ms or greater, or if fluctuations between any two discrete states between 0 and 16 was 200 ms or greater. wild - type phi29 dnap (833,000 u ml ; specific activity 83,000 u mg) was supplied by enzymatics corporation. phosphoramidites (including abasic residue (dspacer), 3-spacer c3 cpg, and acridine) were from glen research. dna oligonucleotides were synthesized at the stanford university protein and nucleic acid (pan) facility. lyophilized oligonucleotides received from pan were re - suspended in 7 m urea, 0.1 tbe, purified by denaturing polyacrylamide gel electrophoresis, and quantified using a nanodrop 1000 (thermo scientific). 2 m each of a 70mer dna template (supplementary fig. 1) and a 23-nt dna primer bearing a 5 fluorescein (6-fam) label, were combined in the presence or absence of blocking oligomer (fig 1b(ii)) at 2.4 m final concentration. this mixture was incubated at 90 c for three minutes in 1x te, 100 mm kcl buffer, followed by slow - cooling to room temperature. 1 m of preannealed dna substrate was then incubated for 5 hours at room temperature (approximately 23c) in nanopore buffer (22.5 l final volume) supplemented with 10 mm mgcl2, 0.75 m phi29 dnap, and 100 m dntps as indicated (fig. following phenol / chloroform extraction and ethanol precipitation, dried dna pellets were dissolved in 7 m urea, 0.1x tbe and resolved by denaturing polyacrylamide gel electrophoresis. gels were 17% acrylamide : bisacrylamide (19:1), 7 m urea, 1 tbe, and run at 18 w. 6-fam labeled dna primer products were visualized on a uvp gel documentation device using a sybr gold filter. setup of the nanopore device and insertion of an -hl nanopore into a lipid bilayer have been described. briefly, a single -hl nanopore was inserted into a lipid bilayer that separates two wells that each contained 100 l of nanopore buffer (ph 8). a 180 mv potential was applied across the bilayer and ionic current was measure through the nanopore between agcl electrodes in series with an integrating patch clamp amplifier (axopatch 200b, molecular devices) in voltage clamp mode. data were recorded using an analog - to - digital converter (digidata 1440a, molecular devices) at 100 khz bandwidth in whole - cell configuration then filtered at 5 khz using a analogue low - pass bessel filter. experiments were conducted at 23c with 1 m dna substrate preannealed to blocking oligomer in nanopore buffer with 0.75 m phi29 dnap, 10 mm mg, and 100 m of each dntp added to the nanopore cis well unless otherwise noted. a single nanopore experiment is defined as the time during which ionic current data were acquired from one hl nanopore in an intact bilayer before termination by bilayer rupture, loss of channel conductance, or completion of a preset number of translocation events. successful forward and reverse dna template translocations ranged for 50 to 500 per experiment.the ratio of fast (< 200 ms) dna alone events to polymerase - bound dna events was approximately 10 to 1 unless otherwise noted. ionic current output from the nanopore device was analyzed using clampfit 10.2 software (axon instruments) after additional smoothing of data at 2 khz using a low - pass gaussian filter unless otherwise noted. the standardized ionic current map in figure 3b was compiled from ten translocation events from the experiment in figure 2. these ten events each started at 23 pa, traversed two peaks at 35 pa, and ended by stalling at 23 - 24 pa. ionic current steps within these ten translocation events were counted as discrete states if their durations were 3 ms or greater. for the data summarized in supplementary figure 4, sequence - dependent pauses were scored if the duration of a discrete ionic current state between 0 and 16 was 200 ms or greater, or if fluctuations between any two discrete states between 0 and 16 was 200 ms or greater. | single - molecule techniques have been developed for commercial dna sequencing1,2. one emerging strategy uses a nanopore to analyze dna molecules as they are driven electrophoretically in single file order past a sensor3 - 5. however, uncontrolled dna strand electrophoresis through nanopores is too fast for accurate base reads6. a proposed solution would employ processive enzymes to deliver dna through the pore at a slower average rate7. here, we describe forward and reverse ratcheting of dna templates through the hemolysin (-hl) nanopore controlled by wild - type phi29 dna polymerase (phi29 dnap). dna strands were examined in single file order at one nucleotide spatial precision in real time. the registry error probability (either an insertion or deletion during one pass along a template strand) ranged from 10% to 24.5% absent optimization. this general strategy facilitates multiple reads of individual template strands and is transferrable to other nanopore devices for implementation of dna sequence analysis. |
the treatment of periodontal diseases has now moved toward an antimicrobial model of disease management. with the threat of widespread antibiotic resistance rendering many antibiotics useless against important diseases, there is an increased necessity not only to minimize antibiotic use and develop novel nonantibiotic - based treatments, but also to raise the profile of disease prevention. probiotics are defined as live microorganisms that when administered in adequate amounts confer health benefits on the host. they repopulate the beneficial bacteria, which can help kill pathogenic bacteria and fight against infection. probiotics administered orally may benefit oral health by preventing the growth of harmful microbiota or by modulating mucosal immunity in the oral cavity. mechanical removal of supragingival plaque is the most effective tool to prevent gingivitis (loe. 1965) but most individuals do not adequately control plaque accumulation and gingivitis continues to be prevalent. to overcome this hindrance, antimicrobial products in the form of dentifrices or mouthwashes have been tested for their adjunctive efficacy in reducing plaque and gingivitis. among them, chlorhexidine is regarded as a gold standard in dentistry for the prevention of dental plaque. though very effective, it has certain side effects such as brown discoloration of teeth, oral mucosal erosion, and bitter taste. as an alternative preventive tool however, only a few clinical studies have been so far conducted on the use of probiotics in the prevention of oral diseases. thus taking into consideration, all these above facts and claims, this study was carried out to test the potential antiplaque and anti - inflammatory properties of probiotic in the form of a mouthwash. a total of 45 systemically healthy subjects (29 males and 16 females) visiting the department of periodontology, at the new horizon dental college and research institute bilaspur, chhattisgarh, were recruited for the study. the inclusion criteria for the study included : subjects between the age group 20 and 30 years of agesubjects with chronic gingivitissubjects are having a dentition with 20 evaluable teeth (minimum of five teeth per quadrant). subjects between the age group 20 and 30 years of age subjects with chronic gingivitis subjects are having a dentition with 20 evaluable teeth (minimum of five teeth per quadrant). the exclusion criteria of the study included : history of systemic diseasespregnant, lactating femaleshistory of antibiotic therapy in the past 3 monthshistory of oral prophylaxis within 6 months previous to the studysubjects with mouth breathing habitsubjects with orthodontic and prosthodontic appliancessubjects with deleterious habit such as smokinghistory of undergoing nonsurgical and surgical periodontal therapy in the last 6 months. history of systemic diseases pregnant, lactating females history of antibiotic therapy in the past 3 months history of oral prophylaxis within 6 months previous to the study subjects with mouth breathing habit subjects with orthodontic and prosthodontic appliances subjects with deleterious habit such as smoking history of undergoing nonsurgical and surgical periodontal therapy in the last 6 months. a randomized, parallel group clinical study was conducted on 45 systemically healthy patients reporting to the outpatient department of periodontics and oral implantology, at the new horizon dental college and research institute, sakri, bilaspur (chhattisgarh) with chronic gingivitis. the subjects were assessed for plaque and gingival inflammation by recording the plaque index (pi) (loe and silness 1964), gingival index (gi) (loe and silness), and oral hygiene index - simplified (ohi - s) (green and vermillion) by a single investigator experienced with index system recording at baseline. thorough scaling and polishing were performed and the patients were randomly divided into three groups consisting of 15 patients each as under : group a - probiotic mouthrinse (sporlac plus [sanzyme ltd. india ] + distilled water)group b - chlorhexidine mouthwash 0.02% (hexidine [icpa])group c - control (saline) group a - probiotic mouthrinse (sporlac plus [sanzyme ltd. india ] + distilled water) group b - chlorhexidine mouthwash 0.02% (hexidine [icpa ]) group c - control (saline) an informed written consent was obtained from each patient included in the study. the patients in group a were given sporlac plus satchets (a probiotic formulation containing lactobacillus acidophilus, lactobacillus rhamnosus, lactobacillus sporogenes, bifidobacterium longum, and saccharomyces boulardii) and 10 ml ampules of distilled water which are commercially available at the chemist as shown in figure 1. the patients were demonstrated and instructed to prepare the experimental probiotic mouthwash by mixing together the contents of the sachet and 10 ml of distilled water. emphasis was made to explain to the patient that the solution had to be stirred thoroughly until all the contents were completely dissolved in the distilled water. the formulation had to be prepared and rinsed immediately once prepared and could not be stored. photograph showing the sporlac plus probiotic sachet, distilled water ampules, and chlorhexidine mouthwash all the three groups were advised to rinse their mouths with the respective mouthwashes prescribed to them for 15 days without any dilution for 1 min twice daily half an hour after brushing. they were advised not to eat anything for half an hour after using the mouthwash. the clinical parameters of pi, gi, and ohi - s recorded at baseline were repeated on 14 and 28 day. descriptive analysis was done and independent sample t - test was employed between 2 groups. the study protocol was in accordance with the local ethical guidelines and approved by the local ethics committee. a randomized, parallel group clinical study was conducted on 45 systemically healthy patients reporting to the outpatient department of periodontics and oral implantology, at the new horizon dental college and research institute, sakri, bilaspur (chhattisgarh) with chronic gingivitis. the subjects were assessed for plaque and gingival inflammation by recording the plaque index (pi) (loe and silness 1964), gingival index (gi) (loe and silness), and oral hygiene index - simplified (ohi - s) (green and vermillion) by a single investigator experienced with index system recording at baseline. thorough scaling and polishing were performed and the patients were randomly divided into three groups consisting of 15 patients each as under : group a - probiotic mouthrinse (sporlac plus [sanzyme ltd. india ] + distilled water)group b - chlorhexidine mouthwash 0.02% (hexidine [icpa])group c - control (saline) group a - probiotic mouthrinse (sporlac plus [sanzyme ltd. india ] + distilled water) group b - chlorhexidine mouthwash 0.02% (hexidine [icpa ]) group c - control (saline) an informed written consent was obtained from each patient included in the study. the patients in group a were given sporlac plus satchets (a probiotic formulation containing lactobacillus acidophilus, lactobacillus rhamnosus, lactobacillus sporogenes, bifidobacterium longum, and saccharomyces boulardii) and 10 ml ampules of distilled water which are commercially available at the chemist as shown in figure 1. the patients were demonstrated and instructed to prepare the experimental probiotic mouthwash by mixing together the contents of the sachet and 10 ml of distilled water. emphasis was made to explain to the patient that the solution had to be stirred thoroughly until all the contents were completely dissolved in the distilled water. the formulation had to be prepared and rinsed immediately once prepared and could not be stored. photograph showing the sporlac plus probiotic sachet, distilled water ampules, and chlorhexidine mouthwash all the three groups were advised to rinse their mouths with the respective mouthwashes prescribed to them for 15 days without any dilution for 1 min twice daily half an hour after brushing. they were advised not to eat anything for half an hour after using the mouthwash. the clinical parameters of pi, gi, and ohi - s recorded at baseline were repeated on 14 and 28 day. descriptive analysis was done and independent sample t - test was employed between 2 groups. the study protocol was in accordance with the local ethical guidelines and approved by the local ethics committee. the mean pi values for all three mouthrinses were 0 at baseline as scaling and polishing were done for all tooth surfaces. a pi score of 0 represented a tooth surface that was entirely free of clinically detectable plaque. mean values of pi scores for probiotics was 0.36 0.14, control 1.10 0.22, and chlorhexidine 0.21 0.15 as cited in table 1. the degree of increment of mean pi scores was more pronounced for control rinse (saline) as compared to probiotic and chlorhexidine rinses. the differences in the increment of the mean pi scores before and after rinse for probiotic and control were significant, whereas that between probiotic and chlorhexidine were statistically insignificant. the mean gi scores were 0.45 0.174 for probiotic, 1.03 0.142 for control, and 0.40 0.124 for chlorhexidine as cited in table 2. in comparison to the baseline data, there was a significant decrease in mean gi scores of probiotic and chlorhexidine rinses as compared to the control rinses, whereas that between probiotic and chlorhexidine it was statistically insignificant. the degree of reduction of mean gi scores was more pronounced for the probiotic and chlorhexidine rinses as compared to control rinse. intergroup comparisons in pi intergroup comparisons in gi the mean ohi - s values were 0.35 0.106 for probiotic, 0.97 0.139 for control, and 0.48 0.341 for chlorhexidine, respectively, as cited in table 3. in comparison to baseline data, there was a significant decrease in the ohi - s score of the probiotic and chlorhexidine group as compared to the control group. intergroup comparisons in ohi - s whereas clinical significance after intergroup comparison between control and chlorhexidine was p = 0.00, probiotic and chlorhexidine was p = 0.16, probiotic and control was p = 0.00 for pi. between control and chlorhexidine, it was p = 0.00, probiotic and chlorhexidine it was p = 0.75, probiotic and control was p = 0.00 when comparison for gi was calculated. between control and chlorhexidine, p value was at 0.00 for probiotic, and chlorhexidine p value was at 0.41 for probiotic and control p value was at 0.00, when ohi - s was calculated. this intergroup comparison shows that there is a statistically significant difference in the control group and probiotic group in the reduction of pi, gi, and ohi - s scores. these side effects are brown discoloration of the teeth and tongue, oral mucosal erosion, and taste perturbation. several side effects associated with its use have stimulated the search for an alternative antiplaque agent. antibacterial mouthrinses act by nonspecifically reducing the levels of both friendly and harmful oral bacteria. in contrast, probiotic has been developed utilizing natural beneficial bacteria to promote a healthy balance of microorganisms in the mouth. probiotic technology represents a breakthrough approach to maintain oral health by utilizing natural beneficial bacteria commonly found in healthy mouths to provide a natural defense against those bacteria thought to be harmful to teeth and gums. probiotic species mostly belong to the genera lactobacillus and bifidobacterium. to be able to exert probiotic properties in the oral cavity, however, it is essential for the microorganism to resist the oral environmental conditions and defense mechanism, to be able to colonize and grow in the mouth, and to inhibit oral pathogens. the potential benefits of probiotics on systemic health and medical disorders, such as gastrointestinal diseases, have been elaborately described. the first species introduced into research were l. acidophilus and bifidobacterium bifidum. among a number of potential benefits that have been proposed are reduced susceptibility to infections, reductions in allergies, and lactose intolerance, as well as lowered blood pressure and serum cholesterol values. within dentistry, the previous studies with lactobacilli strains such as l. rhamnosus, lactobacillus. casei, lactobacillus reuteri, or a lactobacilli mix have revealed mixed results on oral microorganisms. a variety of mechanisms have been proposed for their actions, and some effects have been stated to be systemic rather than only local. it is likely that these mechanisms vary according to the specific strain or combinations of strains used, the presence of prebiotics and the condition that is being treated, as well as the stage of the disease process in which the probiotic is administered. there are common themes emerging in studies of the modes of action of probiotics and numerous mechanisms have been proposed including prevention of adhesion of pathogens to host tissues, stimulation, and modulation of the mucosal immune system, e.g., by reducing production of pro - inflammatory cytokines through actions on nfkb pathways, increasing production of anti - inflammatory cytokines such as interleukin-10 (il-10, and host defense peptides such as beta - defensin 2, enhancing immunoglobulin a defenses, and influencing dendritic cell maturation. killing or inhibition of growth of pathogens through production of bacteriocins or other products, such as acid or peroxide, which are antagonistic toward pathogenic bacteria has also been reported. the current concept concerning the etiology of periodontal disease considers three groups of factors which determine whether active periodontal disease will occur : a susceptible host, the presence of pathogenic species, and the absence of so - called beneficial bacteria. it is generally accepted that the oral biofilm in association with an aerobic bacteria is the main etiological factor in periodontal disease. did a study in patients with moderate to severe gingivitis who were given either freeze - dried lactobacillus salivarius wb21 (wb21)-containing tablets or xylitol containing placebos regularly (3 times daily for 8 weeks). the intake of tablets containing l. salivarius resulted in benefits in terms of pocket probing depth and pi in individuals at high - risk of periodontal disease (smokers) as compared to a placebo control group. did a study in patients with moderate to severe gingivitis who were given one of the two different l. reuteri formulations (lr-1 or lr-2) at a dose of 2 10 (8) cfu / day, or a corresponding placebo. l. reuteri was efficacious in reducing both gingivitis and plaque in patients with moderate to severe gingivitis. other studies have aimed to identify organisms which have the potential for probiotic action that may protect against periodontal diseases. some oral strains of lactobacilli, streptococci, and bifidobacteria have been reported to have in vitro inhibitory activity against periodontal pathogens while others are more active against mutans streptococci. observed that l. gasseri strains isolated from periodontally healthy subjects were more efficient at inhibiting the growth of aggregatibacter actinomycetemcomitans than strains from periodontally diseased subjects, and also inhibited the growth of porphyromonas gingivalis and porphyromonas intermedia. this correlated with an inverse relationship between carriage of homofermentative lactobacilli and subgingival colonization by a. actinomycetemcomitans, p. gingivalis, and p. intermedia. ishikawa. observed in vitro inhibition of p. gingivalis, p. intermedia, and prevotella nigrescens by l. salivarius. daily ingestion of l. salivarius - containing tablets resulted in reduced salivary counts of these black pigmented anaerobes. noordin and kamin conducted a trial among 90 school children and assigned them into placebo, chlorhexidine, and probiotic groups ; and plaque scores were recorded at baseline (0 day), on 15 day (after 14 days of intervention), and 3 weeks (after discontinuation of intervention). probiotic mouthrinse was more effective for inhibition of dental plaque accumulation after 14 days of intervention and also after 3 weeks of discontinuation of intervention. vivekananda. evaluated the effects of lactobacilli reuteri (prodentis) alone and in combination with scaling and root planing (srp) in patients with chronic periodontitis for a period of 42 days. their trial confirmed the plaque inhibition, anti - inflammatory, and antimicrobial effects of l. reuteri (prodentis) and they recommended the use of probiotic during nonsurgical and maintenance phase of periodontal treatment. harini and anegundi evaluated clinically the efficacy of a probiotic and chlorhexidine mouthrinses on plaque and gingival accumulation in children for 14 days and concluded that the probiotic mouthrinse was found effective in reducing plaque accumulation and gingival inflammation. evaluated the effects of l. reuteri - containing probiotic lozenges and placebos as an adjunct to srp in 30 patients with chronic periodontitis, monitored clinically, and microbiologically at baseline, 3, 6, 9, and 12 weeks after therapy. significant improvement in all clinical parameters reduced p. gingivalis levels, more pocket depth reduction and attachment gain in moderate and deep pockets was observed in the srp + probiotic group. maekawa and hajishengallis studied whether lactobacillus brevis cd2 or placebo could inhibit periodontal inflammation and bone loss in experimentally induced periodontitis in mice. mice topically treated with l. brevis cd2 displayed significantly decreased bone loss, lower expression of tumor necrosis factor, il-1, -6 and -17a, lower counts of anaerobic bacteria, but higher counts of aerobic bacteria as compared to placebo - treated mice. hence, l. brevis cd2 could inhibit periodontitis through modulatory effects on the host response and the periodontal microbiota. purunaik. aimed to investigate the efficacy of probiotic (1 g powder of 1.25 billion freeze dried combination, a mixture of l. acidophilus, l. rhamnosus, b. longum, and s. boulardii), 0.2% of chlorhexidine and placebo mouthrinses in reducing plaque and gingivitis among 90 school children aged 1516 years. it was found that both probiotic and chlorhexidine mouthrinses were able to significantly reduce plaque and gingival levels after 14 days. did a study to test and compare in vitro antimicrobial activity of l. reuteri on pathogenic bacteria involved in the formation of dental caries : streptococcus mutans, streptococcus gordonii, and periodontal disease : actinomyces naeslundii and tannerella forsythia chlorhexidine of 0.12% was used as a positive control. l. reuteri was shown to have an inhibitory effect against s. mutans, followed by t. forsythia and s. gordonii, and a less significant effect against a. naeslundii. our results indicate that probiotics could be useful in the improvement / maintenance of oral health in subjects at a high - risk of periodontal disease and add to the body of data supporting the effectiveness of both chlorhexidine and probiotic as antiplaque / antigingivitis agents. the advantages of using a probiotic mouthrinse are that as it contains friendly commensals, there is no issue of antibiotic resistance, and there are no known / proven toxicities caused due to their use. sporlac plus sachet powder for oral solution is a commercially available powder containing probiotics lactobacillus and bifidobacterium not < 1.25 billion cells (l. acidophilus, l. rhamnosus, lactic acid bacillus, b. bifidum, b. longum, and s. boulardii) popularly used in the treatment of diarrhea of any etiology infantile, weaning, and neonatal diarrhea along with any anti - diarrheal agent. several clinical studies have already demonstrated the effectiveness of this probiotic in the treatment of systemic diseases and infectious diseases such as acute diarrhea and crohn 's disease. very few studies till date have studied the basic / initial treatment for periodontal patients in terms of srp and use of probiotic mouthwash in the reduction of clinical parameters of gingivitis in india. an initial attempt was made in this randomized, parallel clinical trial to evaluate the benefits of scaling and sporlac plus probiotic sachet in the treatment of chronic gingivitis and to compare it with chlorhexidine, which has been regarded as the gold standard in dentistry for the prevention of plaque and gingivitis. in all the three groups namely chlorhexidine, probiotic, and saline, pi, gi, and ohi - s were significantly reduced within each treatment group over the 30 days, and thus, even the control had a significant effect. however, such placebo effects are known to occur as a simple consequence of the hawthorne effect. the results of our study are in accordance with studies done by vivekanand., harini and anegundi, and noordin and kamin showing the effect of the probiotics in the management of periodontal disease. the combination of scaling and probiotics and scaling and chlorhexidine demonstrated a significant reduction of pi, gi, and ohi - s when compared with scaling and saline group. the effect of srp on plaque reduction is similar to that reported by mousques. and proye. thus, the plaque reduction brought about by scaling was enhanced by the use of probiotics and chlorhexidine, which is in accordance with the studies done by krasse. in the present study, using a negative (saline) and a positive control (chlorhexidine), we were able to state that the probiotic mouth wash (sporlac plus) has shown a good potential as an antiplaque agent and its effectiveness in reducing the plaque accumulation and gingival inflammation is comparable to chlorhexidine. considering the local side effects of chlorhexidine including brown staining, taste disturbance, enhanced supra gingival calculus formation, and less commonly desquamation of the oral mucosa. sporlac plus probiotic mouthrinse seems a very effective and economical alternative for patients with periodontal disease. probiotic effects are strain - specific, thus, each individual bacterial strain needs to be tested separately, and the effects described for one strain can not be directly applied to others. most of the studies carried out to evaluate the effects of probiotics on periodontal disease have been associated with an individual bacterial strain. unfortunately, mislabeling of strains in probiotic products seems to be a common problem. on the other hand, multispecies or multi - strain probiotic products can be even more effective than products with only one bacterial strain. in our study, we used a probiotic mouthrinse containing the combination of lactobacillus, bifidobacterium, and sacchromyces strains. it is possible, in the complex environment of the human mouth that probiotic cocktail of multiple strains would be more effective than any single probiotic agent. this combination of probiotic strain was similar to those used by haukioja. and purunaik. the results of our study showed a significant reduction of plaque and gingival status and were in accordance with the above - mentioned studies suggesting that combinations of probiotics strains may have synergistic adhesion effect. though these strains tested maintained the oro - microbiological balance, their action in the oral cavity is dubious as oral mucosa is not their innate habitat. furthermore, there is also a need to evaluate whether these lactobacilli strains are momentary or stable oral colonizers. however, it seems plausible that prolonged administration of probiotic preparations may have a preventive role against the development of plaque and gingivitis. however, we would like to state that the major limitation of our probiotic preparation is that it needs to be used immediately once prepared and can not be stored. thus, we would recommend that a proper vehicle is needed for delivering probiotics so that patient compliance can be improved. apart from the unacceptable taste, no other adverse effects on the oral mucosa such as ulcerations were noted with probiotic mouthrinse. likewise, it would be interesting to evaluate the additional gi benefits of probiotics in studies, where the patient is instructed to ingest rather than expectorate the probiotic mouthrinse. this was not possible to evaluate in this study, since it was a comparative clinical trial assessing the efficacy of three mouthrinses. longitudinal studies involving probiotics and further microbiological evaluation are also essential when prescribing them in place of antiseptics and antimicrobials. in the recent times, when organisms are fast developing resistance to antibiotics, the emergence of probiotics appears to be a boon for the treatment of diseases. researchers have confirmed that diseases of the periodontium are not confined to the oral cavity but have strong systemic effects. hence, probiotics offer a natural and promising option to establish both a good oral and systemic health. in the present study, therefore, probiotic mouth rinse has a potential therapeutic value, and further long - term studies are recommended to determine its efficacy. | background : the aim of our clinical trial was to assess and compare the antiplaque and anti - inflammatory potential of a probiotic mouthwash with 0.2% chlorhexidine and saline.materials and methods : a randomized parallel group study was designed for a period of 4 weeks on 45 systemically healthy subjects between 20 and 30 years having chronic gingivitis. the study population was divided into three groups. group a - 15 subjects were advised experimental (probiotic) mouthwash. group b - 15 subjects were advised positive control (chlorhexidine) mouthwash and group c - 15 subjects into a negative control group (normal saline). oral prophylaxis was done for all groups at baseline. after the proper oral hygiene instructions, all the three groups were instructed to rinse their mouth with 10 ml of their respective mouthrinse, undiluted for 1 min twice daily, 30 min after brushing. clinical parameters such as plaque index (pi), gingival index (gi), and oral hygiene index simplified (ohi - s) were assessed at baseline, 2 weeks and 4 weeks, respectively.results:at day 28, the pi, gi, and ohi - s were significantly reduced by all treatment modalities ranking probiotic and chlorhexidine is greater than saline.conclusion:the probiotic mouthrinses tested was effectively used as an adjunct to mechanical plaque control in the prevention of plaque and gingivitis. thus, the probiotic mouthrinse has a great therapeutic potential. |
today, trauma is considered as a major healthcare problem in every society with any health, social and economic status. in addition to being cause of death of millions of people, it causes temporary or permanent disabilities for millions of others. this affects not only the individual, but also the family, society and other involved systems insofar as this has been one of the priorities identified by the world health organization. although loss of life is considered as a main indicator for expressing the significance of an accident from the perspective of health and society, it is sometimes ignored that for every life lost due to an accident, a large number of injuries including amputation occurs at the same time, which leads to hospitalization, receiving medical and pre - hospital services, and utilizing equipment and human resources for treatment and rehabilitation of the injured people. some causes of amputation include traumas, infections, diabetes, cardiovascular diseases, cancers and other diseases. however, the causes of amputation due to trauma vary from country to country, in countries with a recent war in their history ; about 80 percent of amputations are due to war, which differs in different countries. for example, in cambodia, 94.5% of amputations are caused by the war related traumas, 4.5% by the other traumas and 1% by diseases. these figures are respectively 65%, 25% and 10% in zimbabwe, 3%, 32% and 65% in us and 2%, 30% and 68% in denmark. most of people under 50 with amputations caused by trauma and injuries associated with car accidents, industrial accidents or war live in the developing countries. the most frequent cause of amputation in veterans is traumatic factors (direct contact with mines and fragmentations). amputation of the lower limbs, more common in men than women, includes about 80 - 87% of all cases of amputation and may occur in one or both lower extremities. lower limb amputation occurs frequently in accident and war injuries and its most common form is below - knee amputation. the level of lower limb amputation may be from finger tip to hip joint or even removal of a part of pelvic bone. the impact of amputation on an individual 's psychological condition as well as family and social relationships is undeniable because physical disability affects not only the psycho - social adjustment, but also the mental health. when compared to normal people, such people mostly experience social isolation. due to failure to comply with the new condition, these people may suffer from psycho - social difficulties such as depression, sense of hopelessness, low self - esteem, boredom, anxiety, frustration, feeling guilty, and fear of the family 's future, which sometimes lead them to commit suicide. furthermore, they involve other problems such as abnormal behaviors (addiction to drugs or alcohol) and poor social performance. the economic, social, personal, familial, and environmental problems are other problems faced by the people with amputations, making their life more difficult. however, research has shown that social support has a great role in adaptation and coping with diseases, such as amputation. support refers to protecting and establishing a social interaction that starts with communication and after a while, it leads to an empathic relationship and ultimately an immune system for the patient. in fact, support is a vital and multi - dimensional need and patients should always enjoy from it. social support can be provided in the form of psychological - emotional, information, tangible and sociable support. in other words, it seems that the understanding and attitude of patients towards the received support is more important than the level of presented support. in fact, living with a disease means to overcome feelings and tags of low value that are the usual outcome of a chronic disease ; and those who have more social support, pass the transition phase easier than others. therefore, the role of support in this winding path is clear, because understanding social support can prevent the occurrence of adverse physiological adverse effects in patients, increase the level of self - care, positively influence their physical, mental, and social conditions and clearly increase their performance. family members, relatives, friends, doctors, other health - care workers and also support associations can be such a support source for the patients. although, the importance of friends and family, especially peers in adaptation with chronic diseases has been mentioned in several studies, one of the aspects of social support is the informal family support. the individual 's self - care ability and behavior are affected by underlying causes like family. undoubtedly, family (spouse, children and parents) is the first - line support for the patient to comply with the disorder occurred in his / her life. family members, especially spouse are the first individuals providing care to the patient with chronic disease, meeting the needs for emotional, ethical, and knowledge support, the needs for positive and intimate feedback, which lead to enhanced motivation and adaptation. although, the spouse of a disabled person is one of the closest relatives, who has the largest and the most extensive (emotional and cognitive, and verbal and non - verbal) relation with that individual, he / she deal with numerous afflictions, as shown in different studies. considering the considerable number of stressors in the life of amputees and long - term physical problems and limitations, which suggests the possibility of reduced quality of life more than before, cooperation with other groups and individuals seems to be necessary. if amputees are encouraged to use positive - coping strategies, they will express their feelings more easily (expressing feelings is a way of dealing with amputation more appropriately), which contributes to their adaptation process. given that social support and cohesion are currently considered as the important factors in adapting and coping with acute and chronic diseases and it is important that patients understand the protection provided, the present study aims to explain the understanding of trauma patients and experience of support sources during the process of adaptation with a lower limb amputation. this study is part of a comprehensive study entitled process of adjustment to lower limb amputation in traumatic patients using qualitative content analysis. in this study, using purposive sampling, 20 participants from among the amputees continuously referring to the foundation of veterans and the state welfare organization of ardebil and tabriz for follow - up treatment was selected and interviews were conducted until the point of data saturation. in a qualitative research, sampling is often started purposefully aiming to select people who have experienced the phenomenon in question, and are able to offer their experiences ; i.e. those rich in information to participate actively and help the researcher to understand their lives and their social interactions in a better way. this process continues by theoretical sampling, in which selection of each new participant depends on previous samples or participants and the data obtained from them. selection of the next subjects depends on who were selected first and what information has been obtained from them. in the course of the study, purposive sampling is gradually replaced with theoretical sampling. at the start of sampling, the researcher was looking for people who, as the key knower 's, were rich sources of information about social support for coping with lower limb amputation ; thus, could help to have a better understanding about the phenomenon of coping with amputation. therefore, the first selection of the participants was performed with the help and consult of experts in the foundation of martyrs and veterans affairs and the department of social welfare, which were in close contact with the participants from the beginning of affection and during amputation and hence knew them well. sampling was performed at first through an objective - based method and after formation of initial concepts and their characteristics and spectrum, the next objectives were selected via theoretical sampling to further complete the concepts and the obtained classes, and further discover the relations between classes. from among the 20 participants in this study, 85% and 95% were male and married, respectively. the participants age ranged from 25 to 57 (with an average age of 41.7 7.9) and the history of lower limb amputation ranged from 2 to 31 years (with an average of 20.4 10.5). participants amounting to 43.8%, were amputated following an accident and 56.2% due to war. forty percentof participants were under diploma, 45% had an associate or bachelor degree, and 15% had a master or doctorate degree. forty - five percent were governmental employees, 30% self - employed, and 25% unemployed or housewives. in addition, the participants in this study had different types of trauma (war, accidents and, injuries, burns, etc.). participants were selected with the highest diversity (in terms of duration of amputation, age, sex, marital status, education, occupation, etc.). the goal of the maximum variation sampling is to obtain rich, extensive, and deep data covering a wide range of social behaviors (including social support). confirming this, polit. (1993) stated that in maximum variation sampling, the data obtained are more extensive and diverse. the main method used for data collection was in - depth unstructured interviewing with open - ended questions. this type of interview is appropriate for qualitative research because of its flexibility and depth. interview with open - ended questions allowed the participants to fully explain their experiences about the phenomenon under study. all interviews were conducted by the main researcher and started off with a general question : tell me about your amputation. the following questions were asked based on the information provided by the participant and were focused on clarifying the main question that was the process of adjustment to amputation of lower limbs. the length of personal interviews varied between 30 to 120 minutes and was 72 minutes, on average. all interviews were recorded, and were immediately analyzed word - by - word using maxqd10 software. given that in qualitative research the researcher is needed to be immersed in the information researcher listened to interviews and reviewed the typed texts several times. the alternative way of data collection in this study was observation, which was performed to study the interactions and understand the meaning and practices of the participants during the process of coping with amputation. observation of the behaviors and their documentation help perceiving the difference between what people say and what they really do. therefore, the researcher observed interactions and relationships of the participants with family members, peers, and colleagues before and after the interview at workspace, home, and the rehabilitation clinic ; then prepared field notes from himself. field notes are pieces of writings by the researcher from observations, which were used to document the observations and analyze the data. during documentation, the researcher wrote on a paper whatever that he heard, saw, felt, experienced, or thought. in order to observe the ethics, the researcher as an observer participant, monitored and perceived the subjects overtly and with prior notice. the memo allowed the researcher to write his opinions and views about the data ; it served also to document the researcher 's evolving ideas about codes and their interactions. data was collected and analyzed simultaneously, using the qualitative content analysis and constant comparison techniques. qualitative content analysis is a research method for the subjective interpretation of the content of text data. in this method, through the systematic classification process, coding categories can be directly and inductively extracted from the raw data. content analysis goes beyond merely extracting the objective content from text data, and thereby, key concepts and hidden patterns can be extracted from the content of data collected from the participants. in qualitative content analysis process, data collection and analysis, proceed simultaneously. in this method, in order to collect the new data for answering research questions, the researcher goes back and forth between data collection and analysis. in other words, the systematic analysis process was interactive and rotational, meaning that while analyzing the data, the researcher frequently referred to the previous data and the topics which they included, and compared the new data with the old ones. these units are the analyzable parts of the text data, which help the researcher achieve the research goals. the initial codes will be extracted from the meaning units, which are the important and reliable units of analysis. in this study, the content of each interview was analyzed immediately and the next interview was conducted based on the extracted concepts. during data analysis, the emphasis in data was on conceptualization, which proceeded through interaction between the researcher and the data, and also through constant comparisons. he tried to compare events, incidents, and features of a class with those of other classes and to pay attention to the main differences and similarities. the initial codes may contain the exact content of interview or be an abstraction derived from the content. then, based on their similarities and differences, the initial codes are reduced into sub - categories, which in turn are reduced into abstract categories and key concepts. data analysis was performed through a five - step content analysis as recommended by granheimand lundman. in the first step, the audio - taped interviews were immediately transcribed verbatim and used as the main data of research. in the second step, after listening carefully to the tape - recorded interviews several times and reading the transcribed material, the text was divided into meaning units. in the third step, the meaning units were abstracted and labeled with codes. according to the experiences of the participants, the visible and hidden meanings were identified as sentences or paragraphs, and then they were summarized and labeled with codes. in the fourth step, based on the similarities, differences and proportions, then, the categories, sub - categories and key codes were formed. using the group members comments and suggestions, the vague and obscure points requiring more attention were discovered and revised in the next interview. concepts were specified based on the inner content of the text, which was reviewed and revised according to all data. during the study, some methods were used to ensure the data accuracy and stability. long - term relationship with amputees increases trust and makes them more willing to share the truth, which in turn increases generation of real and authentic data. member checking or respondent validation was used to verify data accuracy and to ensure data validity. sharing with participants some parts of the interview and his interpretations of their words, the researcher discussed his own interpretation and the meaning of participants words with them to achieve identical ideas and concepts. member checking, reading the transcribed interviews, revision of initial codes, categories and concepts as well as receiving the participants feedback were all the techniques used for data validation. data credibility was confirmed by peer review and the interview transcripts, cods and categories were extracted. additionally, the results were examined by three faculty members and there was compatibility between the extracted data. the researcher shared the findings with some amputees who were not participated in the study and data compatibility was confirmed by them as well. interviewing with different participants, using direct quoting and providing examples and data dependability was provided by immediate transcription of interview and re - examination of all data using the external checking. the accuracy of the data collection and analysis process according to the methodology principles, was approved by professors and advisors, while it was reviewed as well in the meetings held every six - month, with experts and skilled people within the field of qualitative research ; the ambiguities or drawbacks were discussed and analyzed, and consensus was finally achieved. this study is a part of a phd dissertation in nursing from tabriz university of medical sciences, which was approved by the ethics committee of that university. firstly, the participants were fully informed about the purpose of research and method of interview and were ensured about the confidentiality of information. they were told about their right to withdraw from the study and their informed consent was obtained. the date and time of interview were set based on the participant 's choice, after extracting initial codes from interviews, codes were reviewed and summarized several times and were classified based on the similarities and congruence. then, by further reviewing and comparing the levels, their inner meaning was identified as initial themes. thus, these themes showed the nature and dimensions of the patient 's understanding of support sources. these themes include : 1-supportive family, 2-gaining the friends support, 3-gaining morale from peers, and 4-assurance and satisfaction with the workplace. among the mentioned support sources, the participants prominently stated the importance of family support in the interviews. they feel they are respected and loved by others, they are valuable components and they belong to a social network of mutual obligations and relationships. the first category of these patients family includes parents, especially for single people, and spouse for married people. they help the disabled person in various ways such as helping them in doing daily activities, joining them when faced with problems, giving them morale, keeping them entertained, especially in dealing with anxiety, presenting financial support, giving hope, having mutual understanding in their lows and ups of life. about confabulating and creating a quiet and intimate environment with his / her mother to gain peace of mind, a participant says : whenever i need to relax mentally, i would say it to my mom and hug her to give me peace of mind so that i can easily handle problems even when i was humiliated. she is fully satisfied with the support of his brothers, and considers them as a protection shield against external damages, especially from her husband : it was hard to cope with these crises (divorce). you know the only people who really supported me in the divorce problem were my brothers. they were really supportive and i was frightened that they may not even allow me come back home and that they may disagree with divorce. (p9) one of the most fundamental concerns of mutilated people is to grapple with financial problems. on the one hand, medical equipment is costly and on the other hand, they are unable or have limitations to do their affairs and find suitable jobs due to defects caused by amputation. most participants expressed that their families did not skimp in financial difficulties and assist them as much as they could. until my older sister was alive, i had no financial problems. if it was empty, without chicken and meat, she would say my brother to fill my fridge. (p15) a participant who received financial support, in addition to mental and emotional support from her / his families, says : after the accident, my family was bothered too much since i was a single child. you will dance in your wedding. they lived up to their words. (p4) however, one of the most important support elements is the prominent role of spouse emphasized by many participants. their spouses were the most important supporter and a close friend in dealing with great odds and difficulties arising from amputation. their spouses were sympathetic and tireless friends in all fields and in different complicated situations. their understanding and dedication were the main tools to overcome the problems and deficiencies caused by amputation. i would go to the terminal at night to transport passengers so that nobody can know me. (p5) another participant stated that his wife has hidden financial problems from her family. in addition, she has retained her and her family 's self - esteem and independence., she would go to his father 's home without jacket in the winter blizzard. often her family would take her home with their car ; otherwise, she would hug the baby, walking 7 - 8 km to get home. but she never broke her pride and did not accept the second hand and old clothes of others even as gifts. (p2) as noted above, although most people believed that the family, especially spouse, is the most important support for disabled individuals in dealing with their daily problems, few of the participants stated that their family did not help them in problems and even in some cases they were disregarded by both the family and spouse. for example, a participant says about his brother 's indifference and lack of his support for moving the store items, which incurred costs, and he was even ridiculed by his brother : today, i wanted to move my store items to my home. i had to spend money to move my items while my brother was over there ! (p18) a divorced participant introduced her husband as a hindarance for her scientific progress and higher education. even when i was working in his office, i registered for participation in the law exam at payam - e- noor university. unfortunately, at the time of the entrance exam, he took me to a trip. i found out later that he did not want me to continue my education. close relationship between two people leads to relaxation of the parties, support for each other in case of need and the sense of security and belonging. by telling good memories, assuming amputation simple, advising in everyday life such as job selection, helping and showing empathy in activities and understanding their situation, close friends improve their morale and positive outlook on life. for example, they advised me how to do a certain thing or in some cases they comforted me in difficult times. he was a teacher for me, a great teacher who told me to forget. he taught me to forget some things.(p12) despite the emphasis of most participants on favorable support of friends, one of the participants believed that after mutilation, his friends abandoned him and did not have good feeling for him. (p12) another set of effective factors in dealing with their situation at this stage is to gain morale from peers. with peers, we mean a group with lower limb amputation or other amputations all participants gave a positive evaluation of this group and felt that the problems of these people are similar to theirs, and they easily understand their problems in everyday life. observing similar defects in these individuals becomes an effective aid in adaptation with the current state. families must gather together, guys must be together, because our situation is somewhat analogous. (p13) sometimes, the peer group and similar patients caused the peace of mind for families, in addition to the individual. i was put on the wheelchair to go down to see my mother. at that moment, of god 's grace, he has lost his both feet. that scene really made us relaxed. (p8) the last group of support sources is workplace. most participants expressed that authorities understand their situation and pave the ground to provide their financial and emotional support. the general manager instructed to give me a villa house because it has no stairs. he explicitly told that my house must have a yard so that i can use my car easier. moreover, he instructed the deputy to give me a comfortable chair they recently bought for the office.. a participant says about the advices and guidance of his colleagues on the importance and encouragement to early marriage : my colleagues in the company really guided me to marry due to my situation. they said that it must no longer be delayed ; you must marry and have a child so that you can solve part of your problems and also your child and wife can provide a special help for you tomorrow. (p3) however, one participant grumbled about the lack of understanding and attention of his boss. he stated that his organization did not follow the rules and justice in giving him his rights and this is the reason of his early retirement. my amputation was 45% legally ; i could come in the workplace with 1.5 hours of delay in the morning. i said to my boss that i come in the morning on time, and instead, considered it overtime for me. he called me one morning and said that, from tomorrow, you can either come late in the morning or leave early at the afternoon. among the mentioned support sources, the participants prominently stated the importance of family support in the interviews. they feel they are respected and loved by others, they are valuable components and they belong to a social network of mutual obligations and relationships. the first category of these patients family includes parents, especially for single people, and spouse for married people. they help the disabled person in various ways such as helping them in doing daily activities, joining them when faced with problems, giving them morale, keeping them entertained, especially in dealing with anxiety, presenting financial support, giving hope, having mutual understanding in their lows and ups of life. about confabulating and creating a quiet and intimate environment with his / her mother to gain peace of mind, a participant says : whenever i need to relax mentally, i would say it to my mom and hug her to give me peace of mind so that i can easily handle problems even when i was humiliated. she is fully satisfied with the support of his brothers, and considers them as a protection shield against external damages, especially from her husband : it was hard to cope with these crises (divorce). you know the only people who really supported me in the divorce problem were my brothers. they were really supportive and i was frightened that they may not even allow me come back home and that they may disagree with divorce. (p9) one of the most fundamental concerns of mutilated people is to grapple with financial problems. on the one hand, medical equipment is costly and on the other hand, they are unable or have limitations to do their affairs and find suitable jobs due to defects caused by amputation. most participants expressed that their families did not skimp in financial difficulties and assist them as much as they could. until my older sister was alive, i had no financial problems. if it was empty, without chicken and meat, she would say my brother to fill my fridge. (p15) a participant who received financial support, in addition to mental and emotional support from her / his families, says : after the accident, my family was bothered too much since i was a single child. you will dance in your wedding. they lived up to their words. (p4) however, one of the most important support elements is the prominent role of spouse emphasized by many participants. their spouses were the most important supporter and a close friend in dealing with great odds and difficulties arising from amputation. their spouses were sympathetic and tireless friends in all fields and in different complicated situations. their understanding and dedication were the main tools to overcome the problems and deficiencies caused by amputation. i would go to the terminal at night to transport passengers so that nobody can know me. (p5) another participant stated that his wife has hidden financial problems from her family. in addition, she has retained her and her family 's self - esteem and independence. she never told her family my problems. once a week, she would go to his father 's home without jacket in the winter blizzard. often her family would take her home with their car ; otherwise, she would hug the baby, walking 7 - 8 km to get home. but she never broke her pride and did not accept the second hand and old clothes of others even as gifts. (p2) as noted above, although most people believed that the family, especially spouse, is the most important support for disabled individuals in dealing with their daily problems, few of the participants stated that their family did not help them in problems and even in some cases they were disregarded by both the family and spouse. for example, a participant says about his brother 's indifference and lack of his support for moving the store items, which incurred costs, and he was even ridiculed by his brother : today, i wanted to move my store items to my home. i had to spend money to move my items while my brother was over there ! (p18) a divorced participant introduced her husband as a hindarance for her scientific progress and higher education. even when i was working in his office, i registered for participation in the law exam at payam - e- noor university. unfortunately, at the time of the entrance exam, he took me to a trip. i found out later that he did not want me to continue my education. close relationship between two people leads to relaxation of the parties, support for each other in case of need and the sense of security and belonging. by telling good memories, assuming amputation simple, advising in everyday life such as job selection, helping and showing empathy in activities and understanding their situation, close friends improve their morale and positive outlook on life. for example, they advised me how to do a certain thing or in some cases they comforted me in difficult times. he was a teacher for me, a great teacher who told me to forget. he taught me to forget some things.(p12) despite the emphasis of most participants on favorable support of friends, one of the participants believed that after mutilation, his friends abandoned him and did not have good feeling for him. another set of effective factors in dealing with their situation at this stage is to gain morale from peers. with peers, we mean a group with lower limb amputation or other amputations. all participants gave a positive evaluation of this group and felt that the problems of these people are similar to theirs, and they easily understand their problems in everyday life. observing similar defects in these individuals becomes an effective aid in adaptation with the current state. families must gather together, guys must be together, because our situation is somewhat analogous. (p13) sometimes, the peer group and similar patients caused the peace of mind for families, in addition to the individual. i was put on the wheelchair to go down to see my mother. at that moment, of god 's grace, i saw another veteran, passing before me. he has lost his both feet. that scene really made us relaxed. (p8) most participants expressed that authorities understand their situation and pave the ground to provide their financial and emotional support. the general manager instructed to give me a villa house because it has no stairs. he explicitly told that my house must have a yard so that i can use my car easier. moreover, he instructed the deputy to give me a comfortable chair they recently bought for the office.. a participant says about the advices and guidance of his colleagues on the importance and encouragement to early marriage : my colleagues in the company really guided me to marry due to my situation. they said that it must no longer be delayed ; you must marry and have a child so that you can solve part of your problems and also your child and wife can provide a special help for you tomorrow. (p3) however, one participant grumbled about the lack of understanding and attention of his boss. he stated that his organization did not follow the rules and justice in giving him his rights and this is the reason of his early retirement. unfortunately, up to now there is no support from my boss. my amputation was 45% legally ; i could come in the workplace with 1.5 hours of delay in the morning. i said to my boss that i come in the morning on time, and instead, considered it overtime for me. he called me one morning and said that, from tomorrow, you can either come late in the morning or leave early at the afternoon. support (protection) is the most decisive determinant of health and social network means a system of social connections that binds individuals to the larger social structure. and without supporting these patients their likelihood of success diminished in disease control and thus returning to the normal life. the results showed that understanding support by sources, trauma patients have four special dimensions. the supportive family means that attention and support from family bring hope and encouragement to the patient and make him / her feel respected, loved by others, and belonged to a social network of relationships and mutual obligations. social factors, particularly family, play an important and effective role in mental health of individuals. many studies have discussed the role of family in preserving health and strengthening social relations. pistulka.,(2002) argued that the social support may serve as a protective shield between stress and depression. in a study on korean immigrants in maryland, they studied stress, social support and depression in 60 - 89-year - old people and found that stress and social support have a significant relationship with depression. the results showed that the presence of veterans at home beside the family had a positive impact on the disease symptoms. moreover, the degree of suicidal ideation and the severity of depression in the veterans hospitalized at home were reported significantly lower than those in hospices. given the extensive relevant literature, there is no doubt about the importance of family sources in the preservation and promotion of hope in patients with amputation and even patients with other chronic diseases. for example, a study by bland and darlington (2002) showed that family support is the important source of hope for patients with mental disorders. studies by vellone (2006) in italy and tan (2005) in turkey showed that there is a positive significant relationship between the level of family support and hope in cancer patients. in this regard, a study by baider., (2003) since people with amputation spend much of their time with their families, family support is important in helping them to cope with amputation. the patients considered spouse, among family members, as the key member in supporting them. in fact, support by the patient 's spouse is the main support source during disease periods. spouse is the first support source for the patient and his / her presence as an individual who increases a sense of solidarity and belonging to others can have impacts on the health and function of the patient. on the role of the patient 's spouse in the patient recovery, thompson and lewin (2000) write : there is ample evidence that successful recovery and rehabilitation of stroke patients, adaptation in the convalescent period and restarting activities are largely dependent on the behavior of family members, particularly spouse. in a cross - cultural study in three female groups (european - american, chinese and japanese), the results showed that japanese women are extremely grateful for the support of their husbands, they have tangible support of their husbands in helping them to face with the consequences of the disease and their husbands take great efforts to protect them practically. chinese women also expressed that their husbands are the most important source of support in the face of disease. while european women expressed that they have had the attention and care from their spouses in the face of disease, but they expected more support from them and what they saw was not what they expected. given the relative cultural similarity of iran and eastern societies, our results are consistent with japanese and chinese women on the support of spouses, because eastern women play roles such as supporter, relaxer and helper in the family. they do not expect a lot of care and attention, so changes in spouse 's behavior and playing more supportive role were manifested very important for patients and in some cases, they were surprised because they did not expect all this attention and love from their husbands and perhaps they have been never faced with this kind of behavior on their part in the past. this theme was one of frequent items in facing the disease diagnosis and at the end of treatment. in iran, the study by taleghani., (2005) showed that the spouse is the most important coping sources for breast cancer patients and the study by stone., (2005) showed that in total, patients in south asia had a nice emotional support. only in two of the participants, spouse could not bear problems and separated. although, the participant experienced a very hard mental trauma, the family including spouse was prone to mental, social and health injuries due to multiple stress intolerance. in fact, spouse of a disabled person can primarily affect the mental health level of his / her spouse. due to the problems caused by the disability of their spouses, they may experience different stresses so that some of them may go for divorce. the participants also mentioned the supportive role of friends in the process of adapting with amputation. the supportive role and behavior of their friends bolstered the sense that they are not alone in facing with problems and they can overcome the problems with the support of friends. in a study by rambod (2010), cited by ghodsi, most patients expressed that there is someone who love them and worry about them, and this increase their assurance and ability to cope with the disease. in a study on patients with rheumatoid arthritis, fyrand (2002) suggests that people with more friends have more social support and this social support promotes consistency behaviors in them.. in his study, decker (2007) concluded that in cancer survivors, the support received from friends and mother has the greatest role in coping with the disease. however, the support received from mother is more important than friends. in studies, conducted by taleghani (2005), hassankhani (2009) and rahmani (2012), patients put emphasis on the importance and supporting role of friends in adapting and coping with their situation. despite the emphasis of the majority of participants on good support of friends, only one of them believed that after amputation, his friends abandoned him. in confirmation of this case, lew (2007) suggests that due to the long duration of treatment and many problems of these patients, friends attention to these people reduces over time. rejection by friends was reported not only in people with amputation, but also in other chronic diseases in some cases. in this regard, in a study on hemodialysis patients, rambod.,(2010) state that when patients refer to the dialysis centers, often none of the family members and friends are present. even patients stated that their relationships with relatives and friends were reduced over time and now they do not receive any support from them. however, the change of life caused by hemodialysis and chronic renal failure increases the need to support from others. subsequent to the support by family and friends, the participants seem to benefit from the support of the peer group and this group plays a prominent role in adaptation with amputation. individuals in a particular social network, especially in the peer groups such as groups consisted of patients with similar disease can help each other in finding a solution to the problem, authenticating, navigating to the information, creating positive emotions and comfort. communication with others is a central part in social support because social support is focused on understanding and recognizing each other 's needs. hildingh and fridlund (2004) studied the impact of social support on the matched patients with similar clinical conditions and re - admission to the hospital. the results revealed that patients who participate in support programs of matched patients have more physical activity. they concluded that matched groups exchange experiences with each other and a collective spirit dominates the group so that they help each other to cope better with their disease. in addition, social processes such as social comparison, social learning and social exchange formed in the groups expedite the patients recovery and adaptation. in the study by hernandez., (1999), the participants believed that non - diabetic people can not understand what they say. moreover, powerful communication and emotional networks in iranian society is another reason for the use of peers and family as learning sources. dan., (1999) evaluated support programs through the matched (homogeneous) group in patients with breast cancer. the patients stated that a meeting with volunteers who have experienced breast cancer caused them to feel less alone in dealing with the disease and be more hopeful about the future. several studies have pointed out the importance and positive role of the peer group in adapting with the disease. the workplace organization of the participants was the last support source, which paves the ground to strengthen a sense of hope to cope with the disease by creating job motivation and encouragement. increasing productivity in the organization requires several conditions such as addressing the mental health of workforce from different aspects. today, social support perceived by the employees of an organization is known as one of the most important psychological factors influencing the labor productivity. in their study, dolan., (2008) noted that the lack of supportive factors in the workplace reduces the health level and quality of life of employees. the lack of social support in the workplace detaches employees from each other and eventually ruptures the bond between them. in such a situation, disharmony and disruption make the staff to see their efforts inconclusive and become dissatisfied with their jobs and the governing relations in the workplace due to the lack of supportive feedback for their professional efforts. subsequently, a kind of disappointment appears in personnel, threatening the survival, quality of life and organizational stability, in general. in examining labor productivity indices, bahadori.,(2010) wyatt and chan (2007) believe that understanding and supporting the staff in the form of appreciation of the work are a strong index for increasing their job satisfaction and efficiency. they also believe that addressing the respect need of personnel as well as material and moral support will help the organization to achieve its lofty goals. throughout the process of recovery, rehabilitation and return to the ordinary life of trauma patients, they enjoyed social support of family members, peers, friends and the workplace organization. in fact, adaptation with the disease occurred in the context of social interactions with others. given the importance of social support in adapting with lower limb amputation, future studies are recommended to identify other support sources and causes. | introduction : the effect of amputation on an individual 's psychological condition as well as family and social relationships is undeniable because physical disability not just affects the psycho - social adjustment, but also the mental health. when compared to normal people, such people are mostly experiencing social isolation. on the other hand, social support is known as the most powerful force to cope with stressful situations and it allows patients to withstand problems. the present study aims to explain understanding the trauma of patients and the experience of support sources during the process of adaptation to a lower limb amputation.materials and methods : the present study was conducted using qualitative content analysis. participants included 20 patients with lower limb amputation due to trauma. sampling was purposive initially and continued until data saturation. unstructured interviews were used as the main method of data collection. collected data were analyzed using qualitative content analysis and constant comparison methods.results:the main theme extracted from the data was support sources. the classes include supportive family, gaining friends support, gaining morale from peers, and assurance and satisfaction with the workplace.conclusion:given the high number of physical, mental and social problems in trauma patients, identifying and strengthening support sources can be effective in their adaptation with the disease and improvement of the quality of their life. |
in 2008, ticks were collected from animals from 2 different biotopes where each babesia species had been known to circulate : a farm on which a herd was infected with b. divergens and a reserve on which wild fauna were infected with babesia sp. eu1. a dairy farm in la verrie (vende, france) was selected as a favorable biotope for b. divergens transmission on the basis not only of the presence of numerous ticks on cows and in pastures in 2007 but also of the parasite circulation in the herd, attested by serologic testing (prevalence of 37.5% by immunofluorescence antibody test [ifat ]) and confirmed by its isolation from cattle erythrocytes (prevalence 25% by culture) (9). of the cows tested by ifat, 56% had positive results, which indicated that new infections from ticks were occurring within the herd. because we assumed that sporozoite differentiation is stimulated by blood ingestion and because of experimental proof that female ticks can transmit b. divergens (10), we collected only adult ticks feeding on cows. the 324 collected ticks were morphologically identified as i. ricinus and weighed to estimate their repletion status (range 3398 mg). of these, 223 ticks (4.7339 mg) were dissected under a stereomicroscope to isolate both salivary glands, which were subsequently crushed in 30 l phosphate - buffered saline in a 1.5-ml microtube with an adapted pestle. a droplet of this suspension was deposited on an 18-well slide, stained with may - grnwald - giemsa, and examined under a light microscope. when parasites were seen, and for 41 additional negative samples within the same weight range, 5 l of the infected suspension was added to the culture medium with bovine (b. divergens selective growth) or sheep (both species growth) erythrocytes, rpmi (roswell park memorial institute medium ; lonza, basel, switzerland), and 20% fetal calf serum (lonza) in 96-well plates (11). to identify the parasites, we directly sequenced the amplified 18s rdna babesia gene. pcr with phusion high - fidelity dna polymerase (finnzymes, espoo, finland) was performed on extracted dna (wizard genomic dna purification kit ; promega, madison, wi, usa) from the remaining crushed salivary gland suspensions (bab primers gf2 and gr2, 540 bases long, variable part of the gene) (4) and from resulting parasitized erythrocytes (primers cryptof and cryptor, 1,727 bases long, complete gene) (12). to confirm the identity of the infected ticks, we directly sequenced a variable part of the 16s rdna mitochondrial gene of ixodes ticks (310 bases long) (primers irup1 5-ttgctgtggtattttgactatac-3 and irdo2 5-aattattacgctgttatccctga-3). microscopic observation of crushed salivary gland suspensions identified small pear - shaped elements in only 3 ticks ; weights were 11.7, 25.3, and 277 mg. these millions of pyriform parasites were considered to be sporozoites (13) : they measured about 2 m in length and 1 m in diameter (figure, panel a). only a few parasites had unusual forms, which suggests binary fission (figure, panel b). microscopic appearance of babesia sp. eu1 sporozoites isolated from tick salivary glands and of subsequent asexual development in erythrocytes. sporozoites were stained with giemsa and observed in the suspension of crushed salivary glands (a, b) and from salivary glands directly crushed between slides (c, d, e). a composite panel of asexual stages cultivated in sheep erythrocytes from these sporozoites is presented (f) ; developmental stages are indicated by letters (d, dividing stages ; m, free merozoites ; s, schizont - like form ; t, trophozoite). development of intraerythrocytic parasites was observed, which proved the parasites capacity to directly infect erythrocytes. of the 3 tick salivary glands containing pear - shaped elements, 3 days after inoculation onto a culture, 1/10,000 erythrocytes was infected. starting wells, 10-ml amplified cultures (10% parasitized erythrocytes) could be established within 1 month (figure, panel f). typical babesiidae developmental forms (trophozoite, dividing stages, and free merozoites) were observed, as were more atypical schizont - like parasites, which seemed to produce numerous merozoites. when sporozoites were not observed, parasites were never observed in the cultures of either bovine or sheep erythrocytes. 18s rdna gene (blast [www.ncbi.nlm.nih.gov/blast/blast.cgi ] search in genbank) showed the sequences to be 100% identical to the babesia sp. eu1 sequence (ay046575) for the 3 infected ticks (sporozoites and culture). the partial (sporozoites) and complete (culture) 18s rdna sequences obtained have been deposited in genbank, accession nos. identity of the ticks was confirmed by sequence analysis and comparison with the 16s rdna i. ricinus gene (u14154). for the wild fauna reserve, we used the same approach. at the reserve of chiz (deux - svres, france), where high prevalence babesia sp. eu1 has been described (4), we captured 18 roe deer, then collected and analyzed blood samples from them. presence of babesia sp. eu1 was attested by culture of samples from 4 of the deer. for 31 female ticks, half of the ticks were processed as previously described, and the salivary glands of the other half were simply crushed between 2 slides so parasites could be better seen and quantified. with the latter method, a huge number of sporozoites, 10 to 10, were observed (figure, panels c, d). the inner structures were well preserved, nuclei were clearly visible, and we could observe apparent dividing forms (figure, panel e). from the ticks collected from roe deer, only 2 tick salivary glands contained parasites ; pcr products using bab primers showed 100% identity with babesia sp. our study shows that i. ricinus ticks are competent vectors for babesia sp. eu1. not only can these ticks carry babesia sp. eu1 dna, but more importantly, they enable these parasites to complete their life cycle up to the production of infectious sporozoites eu1 undoubtedly classifies this species in the genus babesia, a feature generally not proven for most babesia spp. the proportions of babesia sp. eu1infective ticks found in our study (3/223 from cattle farm and 2/31 from wild fauna reserve, not statistically different) are comparable to published prevalence of infected ticks (1%2%) collected either from animals or vegetation (68,14,15). millions of parasites inside salivary glands were observed and could be injected to the vertebrate host, from the early stage of the tick feeding (11.7 mg) until repletion (277 mg), which represents a massive infection. these 2 epidemiologic features, combined with the increasing number of immunocompromised persons, should lead to more awareness of the risk related to this zoonotic pathogen. b. divergens sporozoites were never seen in the salivary glands of adult i. ricinus ticks, even when ticks were collected from cattle. this finding is despite the large number of ticks examined (223), the prevalence of nymphs carrying b. divergens dna collected from the farm pastures (87% in 2007 on 113 nymphs analyzed, data not shown), and the infectious status of the herd (serologic prevalence 56%). we therefore raise questions about the main transmitting stage (larvae, nymph, or adult ?) and about the quantitative transmission of b. divergens by i. ricinus ticks (low number of produced and infectious sporozoites ?). in europe, each of which is transmitted by i. ricinus ticks but probably with different sporozoite - production features. | to determine characteristics of natural transmission of babesia sp. eu1 and b. divergens by adult ixodes ricinus ticks, we examined tick salivary gland contents. we found that i. ricinus is a competent vector for eu1 and that their sporozoites directly invade erythrocytes. we conclude that eu1 is naturally transmitted by i. ricinus. |
in the last two decades, there was a remarkable increase in studies concerning food science performed by means of nuclear magnetic resonance (nmr) spectroscopy [1, 2 ]. the reason for nmr success in food analysis lies essentially in the possibility to study complex matrices, obtaining a large number of information on metabolites within a single experiment, with minimal or no sample preparation. in fact, even if other widely used analytical techniques such as gas chromatography (gc) have a higher sensitivity, they need quite sophisticated extraction procedures [3, 4 ]. furthermore, advanced nmr hardware and user - friendly software have been developed as well as bidimensional techniques that allow easy metabolite identification. the area below each proton nmr signal is directly proportional to the numbers of nuclei so its knowledge allows the determination of the quantitative chemical composition. finally, its use in synergy with multivariate statistical analyses permitted a number of relevant studies on food metabolomics and chemometrics especially after the increasing needs for the control on food quality and safety [6, 7 ]. in order to obtain good quality nmr spectra in fact, nonliquid systems are characterized by strong anisotropic interactions that can not be averaged out and produce unresolved broad peaks. in particular, the nmr spectral sensitivity and resolution are limited by those mechanisms that provoke line - broadening effects also with high magnetic fields. dipolar coupling and susceptibility heterogeneity are examples of these kinds of mechanisms that occur within biological samples. therefore, one technique that can average multiple line - broadening mechanisms was developed to limit and resolve these problems, particularly strong for solid - state nmr. this technique is known as magic angle spinning (mas) nmr spectroscopy (see section 2) and, taking advantage of geometric constrains, can be used to acquire high - resolution (hr) spectra of heterogeneous samples such as tissues and cells. this allows the determination of the metabolic profile of the studied system under the considered conditions. therefore, hr - mas nmr has become more popular in food science and in the biological and biomedical fields [9, 10 ]. as a matter of fact, many studies performed by means of hr - mas on different organs and tissues have been reported, demonstrating for example, the ability of this technique to discriminate between malignant and benign disease [1113 ]. moreover, the possibility to follow the metabolic changes has led to apply the hr - mas technique in different fields ranging from the characterization and authentication of different foods to the study of the cellulose degradation happening over centuries in ancient documents. the experimental results we present in this work concern the application of this interesting and powerful technique to the study of the metabolic profile of some typical foods of the mediterranean diet. in the early 1960s, in greece and southern italy, adult life expectancy was among the highest in the world and rates of coronary heart disease, certain cancers, and other diet - related chronic diseases were among the lowest. this was attributed to the particular diet adopted in those regions and today known as mediterranean diet. the mediterranean diet is principally characterized by the consumption of olive oil and wine together with numerous plant foods (vegetables, breads, other forms of cereals, potatoes, beans, nuts, and seeds), fresh fruit (e.g., citrus), fish, and cheese. poultry is consumed from low to moderate amounts ; zero to four eggs are consumed weekly and red meat is consumed in low amounts. this diet is able to provide all of the known essential micronutrients (i.e., vitamins and minerals), fiber, and other plant food substances believed to promote health. it is noteworthy that the mediterranean diet was inscribed in 2013 on the representative list of the intangible cultural heritage of humanity (unesco). among the different foods of the mediterranean diet, we focused our attention on four important food products characterized by a protected geographical status : the pgi (protected geographical indication) cherry tomato of pachino, the pgi interdonato lemon of messina, several pdo (protected designation of origin) extra virgin olive oils (evoos) from sicily, and the pat (traditional italian food product) red garlic of nubia. the european union (eu) has restrictive laws about the food safety policy aimed at protecting consumer health and interests while guaranteeing the smooth operation of the single market. in particular, the eu ensures that control standards are established and adhered to regarding food and food product hygiene, animal health and welfare, and plant health and preventing the risk of contamination from external substances. it also establishes the bases for an appropriate labelling, in line with the approach from the farm to the fork, thereby guaranteeing a high level of safety for foodstuffs and food products marketed within the eu, at all stages of the production and distribution chains. indeed, in this paper we present hr - mas nmr results on the mentioned four typical food products of the mediterranean diet. the increasing demand of quality control by consumers pushes the development analytical techniques able to characterize the metabolic profile of a particular food. we were able to identify and quantify the main metabolites within the studied systems that can be considered their fingerprint. in fact, the used technique can reveal and quantify a number of metabolites even on few amounts of samples and without any chemical treatment. in spite of its quite low sensitivity, the rapidity and easiness of the hr - mas technique, together with the reduction of chemical consumption and waste production, make the methodology very attractive for industry. h one and two - dimensional nmr experiments were conducted at atmospheric pressure by using a bruker avance spectrometer operating at 700 mhz, h resonance frequency, in the experimental configuration known as magic angle spinning (mas). this technique was developed to reduce the two main line - broadening mechanisms that are important in acquiring spectra of a tissue or cell sample, namely, dipolar coupling and heterogeneous isotropic susceptibility. spinning the sample at the magic angle ~ 54.74 by few thousands of hertz averages, these interactions to zero and our experiments were performed at the temperature of 300 k calibrated against the standard ch3oh reference (4% ch3oh in cd3od) with an accuracy of 0.2 k. temperature calibration is very important for this kind of experiments because of the heat produced by the high rotational speed. in fact, the real sample temperature is higher with respect to that read by the thermocouple. for each experiment we use a 4 mm - diameter zirconia sample holder (rotor) with a spherical insert for a total volume of 50 l and a kel - f rotor cap. we use deuterated solvents (d2o and cdcl3) in order to have a lock signal for a chemical shift reference and for a fine optimization of the static magnetic field homogeneity. furthermore, the use of deuterated solvent is necessary in order to avoid any excessive proton signal from the solvent itself. in aqueous preparation we use a 1 mm solution of d2o with 2,2-dimethyl-2-silapentane-5-sulfonate (dss) as an internal standard for the quantification of the assigned metabolites. high purity reagents were bought from sigma - aldrich co. (saint luis, mo, usa). the acquired spectra were processed (fourier transform, phase correction, and baseline adjustment) by means of the standard routines of the software package xwinnmr version 3.5 (bruker biospin, deutschland). peaks assignment was performed by means of literature data and of a well - established software package : nmr suite professional version 7.1 (chenomx, alberta, canada). this latter software is based on a highly sophisticated targeted profiling technology which allows an easy deconvolution of complex nmr spectra and the corresponding quantification of the identified compounds. for the complete and unambiguous assignment of some compounds we performed standard two - dimensional nmr techniques such as cosy and hsqc. for those metabolites not included in the software database (such as gallic acid) (see section 3.2) and also for a confirmation of the chenomx output, we used the standard bruker program nmrquant for the quantification of metabolites. the nmr technique is less sensitive with respect to other well - established analytical techniques such as gc and icp - ms (inductively coupled plasma mass spectrometry) and can quantify metabolites whose concentration is usually above one part per million. the quantification is obtained by using a reference compound of known concentration within the studied solution (1 mm dss in d2o in our case). the area below each proton signal is proportional to the amount of the corresponding substance, so that by the knowledge of the chemical structure and molecular weight of the assigned metabolites, it is possible to obtain the molar concentration by a simple proportion between the peak areas. by means of the chenomx software, all the spectral contributions belonging to the considered molecule should fit the experimental spectrum whereas by using nmrquant, the most intense and resolved peaks should be used for metabolites quantification. finally, we can state that the advantages of the used method rely on the rapidity of the analysis without the needs for any sample treatment that allows, at least in principle, the reuse of the sample. furthermore, the method is precise and allows observing a great number of compounds simultaneously. on the contrary, the method is not very sensitive for the detection of compounds whose concentration is below one part per million. precise extraction procedures are needed in order to observe particular compounds (or secondary metabolites) at very low concentration. in the following subsections, we describe the sample preparation and experimental procedures for each single food products we have analyzed. the pgi cherry tomato of pachino is produced within an area located in the south east of sicily (italy) that includes the entire municipality of pachino and portopalo di capo passero and part of the territories of noto and ispica. we analyzed 14 cherry tomato samples of pachino and 14 of dubious provenience (non - pachino) including 2 coming directly from beijing (china). for a statistically significant outcome we analyzed at least 5 samples for each kind of tomato. the pgi cherry tomatoes of pachino were provided by istituto zooprofilattico sperimentale della sicilia a. mirri (http://www.izssicilia.it/) which is the official institution recognized by the eu as to able to certificate this food product. all samples were studied in the red stage which is when more than 90% of the surface, in the aggregate, is red. in doing so we reduce the eventual metabolic differences due to different ripening stage [21, 22 ]. we diluted 6 mg of freeze - dried tomato in 100 l of a 1 mm solution of dss in d2o. then, we vortexed for a couple of minutes and put fifty microliters into the rotor that in this case was spun at 6000 hz. the duration of the hard pulse was of 8 s with a relative attenuation of 3 db, the spectral width was 10 khz, the acquisition time was 2.9 s, the points in the time domain were 64 k, the number of transient was 128, and the relaxation time was 2 s for a total time of about 10 min per experiment. for the reduction of the residual signal of water we use the standard bruker presaturation pulse sequence zgpr with a presaturation pulse attenuation of 60 db. when processing the spectra we considered 32 k points in the frequency domain. they are rich in sugars, vitamin c, and flavonoids, which are very important in human metabolism. we have analyzed 10 different interdonato lemon samples cultivated in sicily and 10 cultivated in turkey at the same ripening stage. for each sample we have repeated the measurements on 6 different replicates in order to have a statistically significant outcome. the pgi interdonato lemons were provided by 4 different companies belonging to the consorzio di tutela del limone interdonato di sicilia igp. we extracted the lemon juice by a simple mechanical procedure and diluted 20 l of juice in 30 l of 1 mm dss dissolved in d2o directly into the rotor with a spherical insert and a kel - f rotor cap. the sample was kept at ambient temperature (300 k) by a cold n2 flow and a heating element. we used the following experimental parameters : rotor spinning rate 6000 hz, duration of the hard pulse 8 s, spectral width 10 khz, acquisition time 2.9 s, 64 k points in the time domain, 128 transients, and 2 s of relaxation time. the total time necessary for each experiment was of about 10 min and we used 32 k points in the frequency domain for processing the spectra. 16 samples of different evoos were selected from different geographical areas of sicily and in particular 8 from the province of trapani (tp), 5 from that of messina (me), and 3 from that of agrigento (ag). among these we consider the following pdo cultivars : valle del belice (tp), val di mazara (tp), valli trapanesi (tp), and valdemone (me). all samples, after being carefully kept away from light and possible temperature changes that would alter the nature of the oils, were analyzed by taking 30 l of cdcl3 and 20 l of sample, placed in the rotor. the spectral width used was of 20 ppm (~14 khz), the repetition time was 5 s, and the number of transients was 128. the duration of the hard pulse was of 5 s with an attenuation of 3 db. the rotation speed of the rotor was set to 7000 hz and the total time was of about 15 min per experiment. the main signals of the typical proton spectrum of evoos come from fatty acids [2325 ]. we used two different methods based on peaks integration for the determination of the fatty acids composition. consider that all fatty acyl chains are esterified to a common moiety, glycerol, in order to form triacylglycerols. this means that it is possible to obtain the fatty acid composition by the inherent connection between the areas of the characteristic signals of each fatty acyl chain and one of the glycerol backbone () in the h nmr spectra (figure 1, e.g., signals at 4.27 ppm). in particular, fatty acids can be esterified up to three times to the same glycerol moiety and this fact must be taken into account in order to calculate the correct amount. for example, it is possible to have up to three linolenic acid groups esterified to the same glycerol moiety so a ratio of 22.2 glycerol to 100 linolenic acid hydrogens should be used when integrating the signal e (0.98 ppm) in figure 1. special attention should be given to the integral determination (limits, slope, etc.). for example, in our case (700 mhz) the c satellites of most intense peaks have to be subtracted by the integrated region in order to obtain correct values (e.g., as shown in the expansion of the linolenic (e) signal at 0.98 ppm reported in figure 1). accordingly, the percentage of linoleic (a) acid can be determined by integrating the signal at about 2.74 ppm (signal iv = a + 2e in figure 1), which refers to the methylene hydrogens between two double bonds or olefins. therefore, by setting the integral of the chosen glycerol signal to 33.3, the relative area found for the signal at 2.74 ppm directly gives the percentage of linoleic plus linolenic acids. since linoleic (a) and linolenic (e) acids have, respectively, two and four methylene hydrogens between olefins, and the percentage of linoleic acid is obtained by subtracting twice the content of linolenic acid that was previously determined. the signal (ii in figure 1) at about 2.02 ppm refers to the methylene olefin hydrogens of all unsaturated fatty acids, and being the ratio of 2 glycerol hydrogens to 12 possible olefin hydrogens, if the signal of glycerol hydrogens is set to 16.7, the area of the signal at 2.02 ppm provides the percentage of all unsaturated fatty acids : linolenic (e), linoleic (a), and oleic (c). the percentage of oleic acid can be then obtained by subtracting from the value found, the contributions of the unsaturated acids previously obtained. finally, by setting again the integral of the chosen glycerol signal to 33.3, that of the signal at 2.28 ppm (from six carbonyl hydrogens of all fatty acids esterified to the glycerol moiety) is approximately 100. therefore, the percentage of saturated fatty acids can then be determined by subtracting from the area of the signal at 2.28 ppm, the contributions of the unsaturated oleic, linoleic, and linolenic acids found earlier. the second method that we used was introduced by vigli. by considering that the content of linoleic acid can be determined by referring the intensity of its characteristic methyl signal at 0.95 ppm (signal e in figure 1) to the intensity of the methyl signal at 0.85 ppm (signal labeled i in figure 1) belonging to all acids except linolenic. the relative amount of linoleic acid can be determined by subtracting from the signal of diallylic protons at 2.73 ppm (signal iv in figure 1) the relative amount of linolenic calculated earlier as well as the oleic acid content that can be determined by referring the allylic protons centered at 2.02 ppm (signal ii in figure 1) to all fatty chains as measured from the intensity of the c-2 protons around 2.3 ppm (signal iii in figure 1). one has(2)linoleic=3iv4e3iiioleic = ii2iiilinoleiclinolenicsaturated = ie+ilinoleicoleiclinolenic. the red garlic of nubia is cultivated within the municipality of paceco (trapani) and in particular in the integral natural reserve of saline in trapani. it is a food product registered to the italian ministry of agricultural, food, and forestry policies as a traditional italian food product (pat). we have prepared the garlic samples by accurately cutting a thin strip of about 20 mg, rolling it directly into the rotor together with 30 l of 1 mm dss in d2o. in our one - dimensional experiments we used 32 k points in the time domain and a spectral width of 14 ppm (~10 khz). the repetition time was set to 3 s and the number of transients was 256. the duration of the hard pulse was of 6.4 s with an attenuation of 3 db. also in this case, to reduce the residual water signal, we use the standard bruker presaturation pulse sequence zgpr. the rotation speed of the rotor was set to 7000 hz and the total duration was of about 20 min per experiment. we have performed the experiments on 6 samples and 6 different replicates in order to have a statistically significant outcome. tomato (solanum lycopersicum) is probably the most consumed fresh vegetable all over the world. tomato is low in calories and shows antioxidant, antitumoral, and antidepressive properties due to the relatively high concentration of lycopene, ascorbic acid, vitamin e, flavonoids, and so forth. the first tomato accredited by the pgi certificate (council regulation (eec) number 2081/92) and one of the most counterfeit food product is the sicilian cherry tomato of pachino. its special taste comes from the right combination of sugars, organic acids, free amino acids, and salts [20, 27 ]. we were able to identify and quantify the molar concentration of the main metabolites that can be observable by means of our nmr technique. in such a way we aim to obtain a metabolic fingerprint of this protected foodstuff that allows for its characterization and authentication. figure 2 reports in the main plot the complete typical proton spectrum of a pgi cherry tomato of pachino sample. the expansions are the enlargement of the phenolic region (left side, blue line) and part of the aminoacidic region (right side, red line). one can note the high resolution of the obtained spectra especially in the phenolic region that is usually characterized by high noise level. we used for peaks assignment literature data [21, 22, 28 ] and the above mentioned software package nmr suite professional version 7.1 (chenomx, ab, canada) that allows also the determination of metabolites concentration. we followed the same procedures also for the non - pachino cherry tomato samples in order to execute a multivariate statistical analysis in terms of the principal components analysis (pca). this kind of multivariate statistical analysis, being based on an unsupervised pattern recognition technique, allows the identification of differences and similarities between nmr metabolic fingerprints. in particular, nmr spectra were processed by means of a custom - written prometab 3.3 software in matlab version r2009b (the math works, natick, ma, usa) ; spectra were binned from 0.7 to 10.0 ppm with 0.005 ppm bin size ; the residual water signal (4.654.95 ppm) was excluded ; spectra were normalized to the total area and were generalized by log transformation (with a transformation parameter, = 10) to stabilize the variance across the spectral bins and to increase the weightings of the less intense peaks. finally we use the software package unscrambler x version 10.0.1 (camo software as, oslo, no) for the pca analysis with cross validation ; data were mean - centered and the singular value decomposition (svd) algorithm was used. our aim is to establish if there are any metabolites that can account for sample differentiation. we report in figure 3 the results of the pca analysis in the form of a score plot, where samples that are metabolically similar cluster together. as it can be noticed, the principal component 1 (pc1) is able to separate pachino from non - pachino cherry tomatoes except for one sample. the final identification of the metabolites that can account for sample differentiation can indeed be obtained by analyzing the loadings corresponding to the pc1. the loadings of a principal component represent the weight by which each standardized original variable should be multiplied to get the component score. in particular, positive loadings values represent metabolites that are predominant in non - pachino samples and vice versa. therefore, we were able to identify those metabolites whose concentration can determine the sample clustering. student 's t - test analysis, performed by means of the software package microsoft excel (microsoft co., wa, usa), allows to determine only the statistically significant (p value less than 0.05) changes of metabolites that we report in table 1. sugars, gaba, glutamic acid, trigonelline, tryptophan, and tyrosine concentration is higher in pachino cherry tomatoes whereas that of alanine, guanosine, and methanol is higher in non - pachino ones. we want to stress that our result should be independent of factors such as the annual weather variations that can provoke some metabolic changes. in fact, it was shown that despite the marked variability showed only by antioxidants content, greenhouse - growing conditions in sicily induce the accumulation of relatively high levels of ascorbic acid, phenolic compounds, and carotenoids in cherry tomatoes for most of the year. moreover, very recent studies on the response of tomato to constraining the intensity of solar radiation showed that the tomato plant 's metabolism has a strong adaptation to cope with the limitation in light availability such as increasing the specific leaf area and reducing respiration. this was only of little concern to the fruit quality, because no effect of constraining the intensity of solar radiation on the concentration of total dry matter, sugars, and lycopene in the fruits was observed. lemon (citrus limon (l.) burm.), similarly to tomato, is one of the most consumed fresh fruit. lemon is low in calories and displays antioxidant and antineoplastic properties that depend on the relatively high concentration of potassium, magnesium, calcium, vitamin c, phenolic compounds, and so forth. in particular, lemon is the third most important health - promoting fruit rich in phenolic compounds as well as vitamins, minerals, dietary fiber, essential oils, and carotenoids. furthermore, it is widely used also by the food industry as raw materials or flavoring additives for a wide variety of products. indeed, lemons have a strong commercial value for fresh products market and food industry. it is cultivated in the province of messina (italy) within an area delimited by the ionian sea and the peloritans mountains and it is one of the few citrus accredited by the european pgi certificate (commission regulation (ec) number 1081/2009). in particular, we have studied the one - dimensional proton spectrum of lemon juice for different samples of both pgi interdonato lemon of messina and interdonato lemon from turkey. we have assigned and quantified the main metabolites that are present in these two hybrids by means of literature data and the above mentioned chenomx software package. in fact, other studies performed on the different tissues of lemon with the hr - mas technique are present in literature that agree with our results. however, in this mentioned study no metabolites quantification was performed. figure 4 reports the comparison between the one - dimensional proton spectrum of the pgi interdonato lemon (red line) and that of the turkish one (black line) in the chemical shift region of amino acids. in the figure, all the identified metabolites are numbered and the most evident spectral differences are highlighted by means of rectangular shapes. in detail, in turkish lemon there is a greater amount of both saturated (1) and unsaturated (6) fatty acids, lactic acid (8), arginine (10), and -aminobutyric acid or gaba (13). in contrast, asparagine (21) and malic acid signals (20), even if their peaks are cut in the figure, are more intense in pgi interdonato lemon of messina. in lemon juice the major contribution of unsaturated fatty acids comes from oleic, linoleic, and linolenic acids whereas that of saturated fatty acids comes from palmitic and stearic acids. organic acids such as citric, isocitric, and lactic acids mainly contribute to determine the lemon acidity that plays the major role in the criteria assessing the commercial acceptability of the fruit. citric acid (peaks in figure 4 at 2.85 and 3.00 ppm cut because of their extreme intensity) content in lemon juice is about 5% to 6%. for what concerns the sugars, namely, sucrose, fructose, and glucose, that represent the major component of carbohydrates in citrus fruits and hold the key to sweetness of the juice, the spectral comparison is reported in figure 5. fructose and -glucose have a higher concentration in pgi interdonato lemon of messina whereas sucrose content is essentially identical. another metabolite that is very important for nutritional consideration and that displays the same concentration in the two hybrids is vitamin c. the question mark at about 4.15 ppm in figure 5 represents a metabolite that displays different chemical shift and that we were not able to assign with certainty but should belong to some malonic compounds. some minor but important metabolites that we were able to assign are myoinositol (peaks at about 3.28, 3.53, 3.62, and 4.06 ppm), scyllo - inositol (peak at about 3.34 ppm), and stachydrine (peak at about 3.11 ppm). inositols are present in many vegetable species as minor components and have a positive physiologically activity in human. many important studies have demonstrated the importance of inositols in the treatment of several diseases such as the polycystic ovary syndrome. furthermore, myoinositol content and myoinositol / fructose ratio have been found to provide information on the quality and genuineness of orange juice. stachydrine is an osmoprotectant or osmoprotective compound, which helps organisms to survive extreme osmotic stress. our evaluation of stachydrine content (about 0.6 mm) (see table 2) in both interdonato lemon juices agrees with that of a recent work on the effect of stachydrine on endothelial cell senescence under high glucose stimulation. finally, the methanol peak at about 3.36 ppm is well evident in the spectrum of interdonato turkish lemon but it is not so intense in that of the pgi interdonato lemon of messina. even if we are dealing with low concentrations we want to stress that an excess of methanol is not well tolerated by the human body since it interferes with liver metabolism where it is oxidized. also in the phenolic region (figure 6) we were able to identify and quantify a good number of metabolites. here we observe signals coming from nucleosides compounds (33), trigonelline (31), tryptophan (37), tyrosine (34), phenylalanine (38), gallic acid (38), and so forth. in particular, we want to stress the relatively high concentration, for both hybrids, of gallic acid which is a hydroxybenzoic acid present in food of plant origin and exhibit antioxidative properties. gallic acid is one example of metabolite identified by means of hsqc experiments. in table 2, we report the molar concentration, together with the standard deviation, of those metabolites whose p value is below 0.005 and so that can be taken into account for sample differentiation. in particular, the metabolic differences can be considered due to the different geographical origin of the two hybrids of interdonato lemon. olive oil contains a large proportion of monounsaturated fat, is relatively low in saturated fat, and is another source of the antioxidant vitamin e. these characteristics make olive oil preferable to animal fats just from the standpoint of health [44, 45 ]. in fact, diets high in monounsaturated fat seem to reduce the risk of atherogenesis and coronary heart diseases, because they increase the concentration of high - density lipoproteins (hdls) without increasing that of the low - density lipoproteins (ldls). several cultivars of sicilian evoos have been certified with the protected designation of origin (pdo) certificate by the european commission such as valdemone, valle del belice, valli trapanesi, and val di mazara. we aim to study their peculiar characteristics in terms of fatty acids concentration and of minor compounds such as terpenes and aldehydes. we have studied the fat composition of several evoos produced in sicily by means of proton hr - mas nmr, by means of two different but almost equivalent nmr methodologies (based on peak integration) described in section 2.4. we obtained essentially the same results, for both methods (nmr1 and nmr2) that are reported in table 3 and compared with the corresponding values obtained by means of gas chromatography on some of them. the results are very promising and confirm that h nmr spectroscopy can be considered a very useful tool for assessing virgin olive oil quality and genuineness. furthermore, we investigate the possibility to discriminate between samples coming from different sicilian provinces. to this purpose, we executed the pca analysis on the processed spectra and the corresponding score plot is reported in figure 7. even though there is no clear distinction between different sicilian regions, being the results superimposed to each other, the two samples grown in the province of trapani, highlighted with an orange ellipse, belong to two spanish cultivars : arbosana and arbequina. these two samples show a minor concentration of oleic acid and a major concentration of saturated fatty acids with respect to the original sicilian cultivars. it is noteworthy that oleic acid is considered to be antithrombotic compared with saturated fatty acids. furthermore, arbosana and arbequina evoos possess a minor amount of squalene as pointed out by figure 8 where the comparison between the spectra of arbequina and valle del belice cultivars is reported. indeed in figure 8 the expansion on the left side (green arrow) shows that even though the peak at about 2.83 ppm (representing the total amount of fatty acids) (see methods section 2.4) has the same intensity for the two cultivars, that at about 2.02 ppm is more intense for the valle del belice sample rather than for arbequina. moreover, the expansion on the right side (blue arrow) shows that the peak at about 2.83 ppm, corresponding to squalene, is more intense for valle del belice rather than for arbequina. squalene is a hydrocarbon and a triterpene involved in the synthesis of all plant and animal sterols. it is known that squalene assumption for olive oil consumption reduces the risk of cancer. garlic (allium sativum l.) and garlic supplements are consumed in many cultures for their healthy effects. since the ancient times, garlic was consumed as a remedy for different alimentary disorders and infections. in fact, in literature, there are many studies that investigate garlic preparations and their properties. the most important garlic compounds that have beneficial effects on human health are the organosulphur ones. however, once garlic is cut or crushed, compounds in the intact garlic are converted into hundreds of organosulfur compounds in a short period of time. allicin is an effective antimicrobial agent that can be found in limited amounts only in freshly crushed garlic. another important allylic compound is s - allyl cysteine (sac) that has a strong antifungal action and that seems to be highly present just in the red garlic of nubia (see the region at about 6 ppm of nmr spectra in figure 9). we have determined the molar concentration of the main metabolites of the red garlic of nubia by studying the one - dimensional proton spectrum obtained by means of hr - mas nmr. the peak assignment was particularly difficult in the carbohydrates and allylic regions of the nmr spectra for the superposition of signals belonging to many similar chemical species. our results confirm and extend those obtained by means of the same technique by ritota. on white and red italian garlic. in figure 9 we report the comparison between the hr - mas nmr proton spectra of two different italian red garlics : the red garlic of nubia (measured by us with a 700 mhz spectrometer) and the red garlic of sulmona (measured by ritota. with a 400 mhz spectrometer). in the figure, we have highlighted five different chemical shift regions that are relevant for the comparison. the region centered at about 4 ppm belongs to carbohydrates and is similar for both spectra. the regions centered at about 6 and 8 ppm have instead a different intensity (higher for the nubia sample) and correspond to allylic compounds and riboflavin, respectively. the major differences between the spectra of the two red garlics are showed in the first two regions. in particular, it is noteworthy that only in the spectrum of the red garlic of sulmona (black line in figure 9) there is a sharp triplet at about 1.23 ppm that the authors, together with a signal at about 3.95 ppm, assign to an unknown compound. we believe that these signals belong to diethylthiophosphate, that is, an organophosphorus compound widely used as pesticide because of easy degradation in the environment. on the other side, the peak at about 2.4 ppm, belonging to pyruvate, is much more intense in red garlic of nubia with respect to the other red garlic considered. in the following table (table 4), we report the molar concentration of the identified metabolites that were quantified by means of the above described procedures. in this work, we have presented our studies, by means of the powerful nmr technique known as hr - mas, on the characterization of some food products typical of the mediterranean diet. in particular, we have analyzed the metabolic profile of the pgi cherry tomato of pachino and we were able to identify few metabolites that can be considered for sample authentication. for example, in this protected foodstuff, we have found a higher concentration of gaba, sugars (fructose and glucose), glutamic compounds (glutamate and glutamine), and phenolic compounds (trigonelline, tyrosine, and tryptophan) with respect to non - pachino cherry tomatoes. furthermore, we have characterized the metabolic profile of juice from the pgi interdonato lemon of messina and compared it with that of the juice from the same hybrid cultivated in turkey. we find for both hybrids high levels of sugars (sucrose, fructose, and glucose), citric acid, vitamin c, gallic acid, and inositols (mioinositol and scyllo - inositol). the major source of fat in the mediterranean diet comes from the consumption of evoos. for this reason, we have studied the fatty composition of several sicilian cultivars (including few pdo samples) by means of two different methods both based on peaks integration. the results are consistent with those obtained by means of gas chromatography and confirm the power of nmr technique for quick quantitative chemical analysis. moreover, we were able to discriminate between cultivars grown in the same province (trapani) but coming from different nations (italy and spain), just for the different amount of oleic and unsaturated fatty acids and also for squalene content. finally, we have characterized the metabolic profile of the pat red garlic of nubia, quantifying the principal metabolites. in addition, we have compared its proton hr - mas nmr spectrum with that of another italian red garlic and have revealed that both garlics possess the same amount of carbohydrates. however, the red garlic of nubia has a bigger amount of riboflavin, pyruvate, and allylic compounds. on the other side, only the red garlic of sulmona shows a peak that could belong to diethylthiophosphate, a widely used pesticide. in conclusion, the overall results allow appreciating the enormous potential of the used technique that is able to reveal and quantify a number of metabolites (characteristic of the particular food product condition), even on few amounts of samples and without any chemical treatment. the nmr technique is a rapid (few minutes of signal acquisition), nondestructive (no need of sample treatment), and reliable methodology to be used in an official method eventually in conjunction with other traditional analytical techniques such as gc. we want to stress that the consequence of insisting on nmr spectroscopy for food products characterization leads to the reduction of chemical consumption and waste production, which is important from both the economic and environmental points of view. | nmr spectroscopy has become an experimental technique widely used in food science. the experimental procedures that allow precise and quantitative analysis on different foods are relatively simple. for a better sensitivity and resolution, nmr spectroscopy is usually applied to liquid sample by means of extraction procedures that can be addressed to the observation of particular compounds. for the study of semisolid systems such as intact tissues, high - resolution magic angle spinning (hr - mas) has received great attention within the biomedical area and beyond. metabolic profiling and metabolism changes can be investigated both in animal organs and in foods. in this work we present a proton hr - mas nmr study on the typical vegetable foods of mediterranean diet such as the protected geographical indication (pgi) cherry tomato of pachino, the pgi interdonato lemon of messina, several protected designation of origin (pdo) extra virgin olive oils from sicily, and the traditional italian food product (pat) red garlic of nubia. we were able to identify and quantify the main metabolites within the studied systems that can be used for their characterization and authentication. |
it was initially isolated from the fungus tolypocladium inflatum from a soil sample obtained by sandoz scientists at hardangervidda, norway in 1970. cyclosporine is a cyclic nonribosomal peptide of 11 amino acids and contains a single d - amino acid, which is rarely encountered in nature. the drug exhibits very poor solubility in water and is lipophilic. as a consequence, suspension and cyclosporine was originally brought to market by sandoz, now novartis, under the brand name sandimmune in 1983. it had variable bioavailability among patients with diarrhea, biliary diversion, diabetic gastroparesis, or malabsorption secondary to its high dependence on bile solubility. this was addressed by the introduction of a microemulsion formulation, neoral [3, 4 ] (novartis), in july 1995. several other similar cyclosporine formulations have subsequently been introduced in the market, and a timeline is summarized in table 1. neoral, as the branded product, has been the favored choice of physicians for many years despite facing competition from the available generics. the approval process for generics was simplified after introduction of legislation in 1984 (commonly known as the waxman - hatch amendments). the process, also known as abbreviated new drug applications (anda) has helped to increase availability of generic or ab - rated equivalents. the current fda standard of ab rating indicates that bioequivalence has been studied and demonstrated and is the standard mandated criteria for generic formulations of all prescription drugs. generics are tested against brand in healthy young volunteers by giving a single dose of the reference formulation and the generic formulation that is being tested. bioequivalence studies are performed using auc (area under the time - concentration curve) and cmax (maximum plasma concentration) to demonstrate that the generic has similar pharmacokinetics as the brand formulation. auc serves as a surrogate for the extent of absorption whereas the cmax and the time of its occurrence (tmax) together characterize the rate of absorption [57 ]. it is concluded that two pharmaceutical agents are not different from one another if the 90% confidence interval of the ratio of a log - transformed exposure measure (auc and/or cmax) falls within the range 80125%. unless otherwise indicated by a specific guidance statement, the traditional bioequivalence limit of 80 to 125 percent is the same for nonnarrow therapeutic range drugs and for narrow therapeutic range drugs. the 80125% bioequivalence acceptance range translates into a difference of 20 to + 25% in the rate and extent of absorption between two drug products for a single dose exposure. standard bioequivalence criteria do not require the generic formulation to be evaluated in target patient populations, over repeated exposure, in unhealthy people, or by administration intravenously. some physicians and patients have raised concerns over generic versions of critical drugs by claiming a difference in quality and therapeutic efficacy compared to the brand name drug [79 ]. the fda acceptance limits for generics (80125%) is too broad as there is a potential difference of as much as 45%. generic drugs are tested only in healthy volunteers and may act differently in the target disease population, resulting in uncontrolled clinical risks. every transplant physician encounters situations where a choice between brand versus generic formulation arises for financial or insurance reasons. for narrow therapeutic index drugs, this decision becomes more critical. the fda defines these products as those containing drug substances that are subject to therapeutic drug concentration or pharmacodynamic monitoring, and/or where product labeling indicates a narrow therapeutic range designation. as cyclosporine is a critical dose / narrow therapeutic index drug, a change in drug level has the potential to either cause rejection or result in renal toxicity depending on low or high levels, respectively. many pharmacies in the united states are increasingly substituting micro emulsion forms of cyclosporine, either by mandate or by choice. state regulations vary on the requirement of pharmacists to notify primary care providers of generic cyclosporine substitution, and subsequent additional therapeutic drug monitoring to verify consistency in drug exposure is frequently not undertaken. this, in turn, often leads to doubts in the physician 's as well as the patient 's mind, and ultimately leads to specifying it has been estimated that use of generics instead of brand name drugs in the year 2000 in america alone would have saved $ 8.8 billion, equivalent to 11% of total drug expenditure. another explanation for the prospering generic market is improved compliance with less expensive medications, for obvious reasons. however, in regards to cyclosporine generics, there have been concerns about the savings from their use in transplant patients. the argument given is that upfront savings are offset by the cost incurred by rejection of grafts, frequent monitoring of drug levels, or even the increased dosage requirement for the drug. the purpose of this discussion is to review evidence for and against equivalence between brand and ab - rated equivalent cyclosporine, and thereby assist the transplant community in making knowledgeable decisions. several studies have been reported from european countries claiming equivalence of generic and brand cyclosporine [13, 14 ] while one from a us center has suggested more frequent graft rejection on generic gengraf. the existing package insert for neoral includes data related to limited comparisons with generic sandimmune for transplant, rheumatoid arthritis, and psoriasis patients. in general, the mean cyclosporine area under the curve (auc) and peak blood cyclosporine concentration (cmax) were both higher for neoral, but dose - normalized trough concentrations were similar for both formulations. the main backbone of generic formulations bioequivalence has been questioned in some studies. the inability of standard testing to confirm bioequivalence in a transplant population was demonstrated in the case of the sangcya formulation of csa. sangcya had a class ii recall by fda, issued on products that have a low chance of causing major injuries or death, but where there is still the possibility of serious adverse events with irreversible consequences. it was voluntarily withdrawn from the market in 2000 after initial approval in 1998 following successful bioequivalence studies in healthy volunteers. it was found that bioavailability for sangcya was significantly different in comparison to neoral when consumed with apple juice. this finding highlighted the fact that known and unknown variables, even something as innocuous as coadministration of apple juice, may create potential confusion. in the solid organ transplant setting, additional limitations in regards to bioequivalence testing have given rise to concerns. cyclosporine absorption may differ between healthy volunteers and transplant recipients ; indeed, absorption variability in transplant recipients has been documented to be related to time after transplantation and the type of organ graft [18, 19 ]. in addition, patient characteristics such as age [20, 21 ], ethnicity [22, 23 ], or comorbid disease [24, 25 ] may affect cyclosporine absorption. isolated reports have been published that other immunosuppressive medication absorption, including sirolimus, may be affected by generic versus brand cyclosporine formulations. intestinal drug - drug interactions are a general problem in transplantation and can occur when foods, herbal drugs, and other drug formulations are taken at the same time as the immunosuppressant. this issue is obviously not unique to either brand or generic drugs. currently, there is a paucity of data relating to the efficacy and safety of transferring transplant patients from reference neoral formulations to a generic formulation or, indeed, from one generic form of cyclosporine to another. there are few studies evaluating pharmacokinetics or clinical outcomes between the cyclosporine microemulsion reference drug (neoral) and ab - rated bioequivalents (namely, gengraf). the following two studies have assessed the effect of transferring stable renal transplant patients from the neoral to a generic formulation (gengraf) of cyclosporine. roza. switched 50 stable renal allograft recipients in a multicenter, us - based study, from neoral to gengraf and then back to neoral over 35 days on a dose - for - dose basis. this trial found no significant difference in the mean pharmacokinetic measurements during the three periods of the study, and there was no need for dose adjustment in any patient. no statistically significant differences in cmax, auc0 12 hr, cmin, and t max between neoral at day 14 and gengraf at day 28 were observed. however, only mean values of pharmacokinetic parameters were provided (cmax, trough blood concentration (c0), auc, and time to maximum blood concentration (tmax)), without any individual data or ranges. a second study by carnahan and cooper involved 41 stable kidney transplant patients in an open - label clinical trial. the trial was conducted primarily to assess differences in steady - state cyclosporine concentration and serum creatinine after conversion from neoral to gengraf. secondary goals were to evaluate changes in cyclosporine dosing regimen, cyclosporine toxicity, graft rejection, hospital / emergency room admission, and changes with medications which could interact with cyclosporine. the fda acknowledges the limitations of bioequivalence studies and recognizes the need for increased assurance of the interchangeability for drug products containing narrow therapeutic index (nti) drugs. fda suggests additional testing and/or controls to ensure the quality of drug product containing immunosuppressant. concerns were raised in a study by qazi. where 6-month postrenal transplant patients with stable graft function were randomized to either remain on neoral (n = 9) or switch to gengraf (n = 73). serum creatinine and cyclosporine troughs were measured at baseline and 2 weeks after the switch. of the studied patients, 13 (18%) the mean csa trough level in all patients converted to gengraf rose from 180.5 8.4 ng / ml to 195.0 9.8 ng / ml (p < 0.05), which was statistically (but not necessarily clinically) significant. interestingly, the mean baseline csa level was 234 96 ng / ml, rose to 289 102 ng / ml after conversion, and fell back to 239 151 after decreasing the dosage. no dose changes were required among the patients who remained on neoral. in summary, the qazi study of 82 stable renal transplant recipients at least 6 months after transplant revealed that nearly 20% of patients who switched to a generic csa preparation that was considered bioequivalent required a dose adjustment to return to the preconversion cyclosporine trough level. the potential for adverse effects resulting from such conversions (especially in patients who are seen only once in several months) and when the patient or the transplant care providers are unaware of the switch is of considerable concern, but true outcome data are still not available. in 2006, hibberd. reported a study from australia comparing cysporin (generic) versus neoral kinetics. the pharmacokinetic profile of cysporin and neoral were found to be different : for cysporin the extent of absorption was lower and the rate of absorption was slower than that for neoral. the authors felt that a trial in transplant recipients (not healthy volunteers) is needed to determine the pharmacokinetics and bioequivalence of a generic immunosuppressant, particularly a critical concentration - time profile drug such as cyclosporine a. in 1999, the national kidney foundation published a white paper consensus document to suggest recommendations for the safe and effective use of generic immunosuppressants based on the expert opinion of a multi - disciplinary group of participants and their review of the literature. in summary, recommendations for improving the fda approval standards for generic immunosuppressants included the following. defining critical - dose drug characteristics and inclusion of immunosuppressive agents such as cyclosporine and tacrolimus in the list of critical - dose drugs. need for replicable pharmacokinetic studies of critical dose drugs as part of the approval process for both the innovator drugs as well as subsequent generics. bioequivalence studies in subpopulations of transplant patients (e.g., pediatric, african - american, or diabetic patients). further recommendations were made pertaining to safe and effective use of generic immunosuppressant agents, including the following. patient education and involvement in decision making before any switch from brand to generic and also from one generic to another. consistency in state regulations for pharmacist to notify the patient as well as physician prior to any substitution of an immunosuppressive medication. careful evaluation of bioequivalence data for drugs by physicians, so that appropriate prescribing decisions can be made related to generic substitution. consideration for appropriate drug monitoring techniques (including blood levels) if patients are switched from one formulation to another (e.g., brand to generic, generic to generic). patient education to identify drug formulations and to alert the physician if a drug is substituted. the american society of transplantation published a similar report in 2003 on the use of generic immunosuppressants in the transplant settings. need for consistency in use of selected immunosuppression formulation, timing of drug administration, and blood level monitoring. uniqueness among generic alternatives need for physicians and patients to be notified by pharmacists if there is any switch in dispensed brands need for patients to inform physician if any switch has taken place so that appropriate drug monitoring can be undertaken. incorporating bioequivalence studies in at - risk patient populations into generic drug approval process. most participants in the american society of transplantation forum supported de novo usage of generic cyclosporine in low - risk patients or even a switch from brand to generic as long as the patient and care providers are clearly informed about the switch, so that when indicated additional tests could be performed to ensure desired drug levels. because of insufficient bioequivalence data in the african american and pediatric population, generic substitution was not recommended in these groups. both ast and nkf recommendations for practice and policy seem rational and safe approaches while more definitive data are accumulated. the need for patient and provider education and awareness is clearly emphasized in these guidelines. in actual practice, however, changes often occur without the benefit of all relevant parties being involved in the decision making process. pharmacists, providers, and patients are intricately linked in this important area, but often overlook the importance of two - way communication and followup when formulation changes are contemplated or actually undertaken. as such, the guidelines, although not new, are still relevant for safety but have not been incorporated into routine practice. the best available evidence suggests that there are conflicting results as to whether a change from brand name product to generic equivalent will result in similar levels and outcomes. the inability to attain similar trough levels in a significant percentage of patients after a 1 : 1 switch is a concern, but this shortcoming can be easily corrected by ongoing therapeutic drug monitoring to ensure that the new steady states fall within an intended targeted range. although this may offset some cost savings in the short term, the significant risk of jeopardizing graft function could be avoided. cyclosporine is a narrow therapeutic - range drug for which small variations in exposure may have severe clinical consequences for transplant patients. in its guidance for industry on bioequivalence, the fda recommends that sponsors consider additional testing and/or controls to ensure the quality of drug products containing narrow therapeutic range drugs. the approach is designed to provide increased assurance of interchangeability for drug products containing specified narrow therapeutic range drugs. additional testing and/or controls could or should be carried out to assure the interchangeability for drug products. currently, there is a lack of comparative outcome data relating to the pharmacokinetics of the reference formulation neoral and generic formulations in transplant recipients. prospective clinical studies investigating the efficacy and safety of generic formulations in both de novo and long - term transplant patients are also awaited. for drugs such as cyclosporine, which exhibit complex absorption patterns and for which maintaining therapeutic exposure levels is critical to patient wellbeing and survival, the transplant physician must scrutinize the available pharmacokinetic and clinical data carefully before prescribing new generic formulations. until further evidence is available on the transfer of transplant patients to or between generic formulations of cyclosporine, any transfer to a different cyclosporine formulation the best available information to date, however, does not support the frequently held but unsubstantiated belief that generic preparations of immunosuppressive drugs are not as effective as brand names or that conversion from brand to generic is associated with significant danger. | the evidence for conversion from brand name to generic equivalent cyclosporine is conflicting. cyclosporine is a narrow therapeutic - range drug for which small variations in exposure may have severe clinical consequences for transplant patients. there is currently a lack of comparative outcome data relating to the pharmacokinetics of the reference formulation, neoral, and generic formulations in transplant recipients. a major common concern is the potential inability to attain similar trough levels, an issue that can be easily corrected by ongoing therapeutic drug monitoring to ensure that the new steady state falls within an intended target range. prospective clinical studies investigating the efficacy and safety of generic formulations in both de novo and long - term transplant patients are also awaited. until further evidence is available on the conversion of transplant patients to or between generic formulations of cyclosporine, any transfer to a different cyclosporine formulation should be undertaken with close supervision. the best available information to date, however, does not support the frequently held but unsubstantiated belief that generic preparations of immunosuppressive drugs are not as effective as brand names or that conversion from brand to generic is associated with significant danger. this paper attempts to initiate a discussion of these issues. |
foot ulceration occurs in approximately 1525% of people with diabetes while amputation prevalence ranges between 0.24.8%, worldwide. in addition to the morbidity, diabetic foot complications are associated with high mortality. in one study, foot ulceration was associated with a two - fold increase in mortality independent of the effect of age, diabetes type, diabetes duration, treatment and glycosylated hemoglobin. another study, in barbados, reported that five year survival after lower limb amputation was only 44% compared to compared to 82% among those without amputation. the burden of diabetic foot complications is very high in the caribbean region. in one study from barbados, hennis and colleagues documented that the incidence of lower extremity amputation among the women in barbados was second only to that of the us navajo population. previous studies in barbados had documented that on average 75% of surgical beds at the queen elizabeth hospital were occupied by patients with diabetic foot problems. in trinidad, a cross - sectional study among patients attending primary health care clinics found that 12% of persons with diabetes reported previous foot ulceration and 4% reported previous amputation. in jamaica, we have found that among patients of attending the diabetes clinic at the university hospital of the west indies (uhwi), 8.5% had an amputation and 4% had a current foot ulcer. in another study among patients with diabetes who were admitted to uhwi in 2004, the main risk factors for diabetic foot complications are peripheral neuropathy, peripheral vascular disease and foot deformity ; however improper footwear and inappropriate foot care are thought to be important contributors to diabetic foot complications. the international working group on the diabetic foot and other professional bodies have put forward recommendations for appropriate foot care and choice of footwear which may reduce the risk of foot ulceration and amputations. some features of these recommendations include daily foot examination by patients or caregivers, (including the use of mirrors to examine the sole of the feet where necessary), daily washing and careful drying of feet, use of moisturizing lotion on the feet but not between the toes, avoiding corn removal with chemical agents, wearing well - fitting shoes and avoidance of walking barefooted. patients at high risk of foot complications such as those with loss of protective sensation and those with foot deformities may require specially fitted footwear to minimise the risk ulceration. in light of these recommendations for foot care and footwear practice for persons with diabetes, we conducted this study aiming to evaluate foot care and footwear practices among patients attending uhwi diabetes clinic in order to determine the extent to which recommended practices are being followed. the study was conducted on a subset of participants in a cross - sectional survey of patients attending the uhwi diabetes clinic. the study was approved by the university hospital of the west indies, university of the west indies, faculty of medical science ethics committee. the original study was designed to estimate the prevalence of diabetic foot complications using a sex - stratified random sample of 188 patients from the clinic. a list of all 552 patients seen in the uhwi diabetes clinic in 2008 was used as the sampling frame, from which a sex - stratified random sample of 337 persons was selected, aiming to enrol 278 persons (assuming a 20% non - response rate). we successfully ascertained vital status or made contact with 253 (75%) of the potential participants of which 188 (56% of targeted sample ; 74% of contacted persons) were enrolled. participants were included in the study if they reported a history of doctor - diagnosed diabetes (subsequently confirmed by reviewing their medical records) and if they were patients of the uhwi diabetes clinic in 2008. if the invited participant did not report a history of doctor diagnosed diabetes or never attended the uhwi diabetes clinic they were excluded from the study. seventy - two of the 188 participants recruited to the study completed an additional questionnaire on foot care and footwear practices during the latter half of the study. a general questionnaire was used to obtain data on demographics, socioeconomic status, diabetes history, cigarette smoking and alcohol consumption. the footwear and foot care questionnaire collected information on foot care education, ability to care for feet and current foot care practices. the questionnaire was developed by some of the authors (tsf, mktr and srm) after reviewing a number of foot care questionnaires. some of the questions were selected from a diabetic foot care questionnaire from the american college of physicians clinical skills module on diabetic foot ulcers. table 1questionnaire items used in the study.questionnaire items for foot care and foot wear practicesfoot care education have you ever been taught how to care for your feet ? have you ever read an educational handout about foot care?ability to care for your feet can you reach and see the soles of your feet?current foot care do you or an assistant inspect your feet daily for problems ? do you always test water temperature with your hand before putting your foot in ? do you check your shoes for objects that might have fallen into them?your current footwear do you wear special shoes because you have diabetes ? do you wear any of the shoe types below at any time?[road, round toes, pointed toes, slippers (no back / heel section), open toes, athletic shoes (sneakers), leather, canvas, high heels ' plastic, working boots (for construction sites, etc.) ] socio - economic status was assessed using information on education and employment obtained by the general questionnaire. data on education level was collected in categories ranging from no schooling to tertiary education, and then was collapsed into two categories secondary or less and post - secondary for analysis. data were also collected on cigarette smoking patterns and alcohol consumption patterns and participants placed into two categories - non - smokers and past / current smokers, and never drank alcohol and past or current alcohol consumption. a capillary blood sample was collected for measurement of haemoglobin a1c (hba1c) using the nycocard reader ii (axis shield, oslo, norway). statistical analysis was performed using stata 10.1 (statacorp lp, college station, tx, usa). we obtained proportion of participants with positive responses to each of the questionnaire items and made comparison of differences in proportions for male and female participants. bivariate analyses were performed using chi square () test and fisher 's exact test where applicable. participants had a mean duration of diabetes of 17.010.2 years ; mean age at diagnosis of 40.512.4 years ; and mean hba1c of 7.71.9%. there were no significant differences in the mean values for age, diabetes duration or hba1c for the participants included in this analysis when compared with the full study sample. sixty - four percent of the men were employed compared to 36% of the women. a third of participants were current or past smokers while 32% reported current alcohol use. sixty - one percent were currently on insulin and 40% had good glycaemic control (hba1c < 7%). table 2proportion of participants with various socio - demographic characteristics for men, women and total sample. 7unemployed28.613.816.7retired / housewife / student7.150.041.7highest grade of educationsecondary or less78.665.568.1post - secondary21.434.531.9lifetime smokingnever smoked tobacco14.379.366.7past or current smoke85.720.733.3current or past alcohol usenever drank alcohol28.677.668.1current or past alcohol71.422.431.9duration of dmless than 20 years64.348.351.4twenty years or more28.627.627.8missing7.124.120.1glycemic controlgood (hba1c < 7.0%)64.334.540.3inadequate (hba1c 78.9)35.737.937.5poor (hba1c 9%)027.622.2insulin therapynever42.924.127.8current57.162.161.1past013.811.1p<0.05;p<0.01;p<0.001;p - values are from fisher 's exact test for male : female differences. fifty - three percent of participants reported ever being taught how to care for their feet with no difference between men and women. the majority of persons, who were taught foot care, were educated by doctors (29%) or nurses (25%). three participants reported being taught by a podiatrist while the others reported being taught by unspecified persons at the clinic or by a family member. only 28 women (49%) and one man reported ever reading an educational handout on diabetes foot care. the proportion of participants performing the various foot care practices is shown in table 3. almost all participants reported washing their feet daily with 90% ensuring that they dried in between toes. most participants (especially women) reported daily use of moisturizing lotion on the feet but almost 50% used lotion between the toes. most participants reported wearing shoes or slippers both indoors and outdoors, but over 40% reported walking barefooted at some time. less than 50% of participants checked the water temperature with their hands before putting their feet in, while very few (19%) reported using footwear in the shower. there were no significant associations between foot care practices and socio - demographic factors in bivariate analyses. table 3proportion of persons engaging in various foot care practices for men, women and total sample.foot care practice n=14male % n=58female% n=72total%recommended practicesdaily foot inspection by patient or an assistant71.460.362.5wash feet every day85.710097.2dry thoroughly between the toe85.791.390.2use moisturizing lotion on feet daily57.193.090.3have an assistant cut toenail or trim calluses85.770.773.6wear shoes or slippers both indoor and outdoor64.389.784.7use footwear in the shower21.419.019.4test water temperature with hand before putting feet in35.751.848.6check shoes for objects that might have fallen in them before putting shoes on78.687.986.1practices that are not recommendedput moisturizing lotion between the toes42.950.949.3walk barefooted at any time50.043.144.4use corn plaster or corn cure03.62.9p<0.05,p<0.01,p<0.001 for male : female difference;1n=71;2n=68. p<0.001 for male : female difference ; approximately 13% of participants reported wearing special shoes because of diabetes. only 10% of participants wore protective shoe inserts, while over 80% wore shoes without socks at some time. the frequency with which various types of shoes were worn is shown in figure 1. patterns were generally similar for both men and women except for more frequent use of open toe footwear for women. although much larger proportions reported wearing broad round toe shoes and leather shoes, significant proportions reported wearing pointed toe shoes, and 43% of women wore high heel shoes. there were no significant associations between footwear types and socio - demographic factors in bivariate analyses. figure 1proportion (%) of participants (men and women) who wear various types of footwear (p<0.05, p<0.01 for male : female difference). proportion (%) of participants (men and women) who wear various types of footwear (p<0.05, p<0.01 for male : female difference). fifty - three percent of participants reported ever being taught how to care for their feet with no difference between men and women. the majority of persons, who were taught foot care, were educated by doctors (29%) or nurses (25%). three participants reported being taught by a podiatrist while the others reported being taught by unspecified persons at the clinic or by a family member. only 28 women (49%) and one man reported ever reading an educational handout on diabetes foot care. the proportion of participants performing the various foot care practices is shown in table 3. almost all participants reported washing their feet daily with 90% ensuring that they dried in between toes. most participants (especially women) reported daily use of moisturizing lotion on the feet but almost 50% used lotion between the toes. most participants reported wearing shoes or slippers both indoors and outdoors, but over 40% reported walking barefooted at some time. less than 50% of participants checked the water temperature with their hands before putting their feet in, while very few (19%) reported using footwear in the shower. there were no significant associations between foot care practices and socio - demographic factors in bivariate analyses. table 3proportion of persons engaging in various foot care practices for men, women and total sample.foot care practice n=14male % n=58female% n=72total%recommended practicesdaily foot inspection by patient or an assistant71.460.362.5wash feet every day85.710097.2dry thoroughly between the toe85.791.390.2use moisturizing lotion on feet daily57.193.090.3have an assistant cut toenail or trim calluses85.770.773.6wear shoes or slippers both indoor and outdoor64.389.784.7use footwear in the shower21.419.019.4test water temperature with hand before putting feet in35.751.848.6check shoes for objects that might have fallen in them before putting shoes on78.687.986.1practices that are not recommendedput moisturizing lotion between the toes42.950.949.3walk barefooted at any time50.043.144.4use corn plaster or corn cure03.62.9p<0.05,p<0.01,p<0.001 for male : female difference;1n=71;2n=68. only 10% of participants wore protective shoe inserts, while over 80% wore shoes without socks at some time. the frequency with which various types of shoes were worn is shown in figure 1. patterns were generally similar for both men and women except for more frequent use of open toe footwear for women. although much larger proportions reported wearing broad round toe shoes and leather shoes, significant proportions reported wearing pointed toe shoes, and 43% of women wore high heel shoes. there were no significant associations between footwear types and socio - demographic factors in bivariate analyses. figure 1proportion (%) of participants (men and women) who wear various types of footwear (p<0.05, p<0.01 for male : female difference). proportion (%) of participants (men and women) who wear various types of footwear (p<0.05, p<0.01 for male : female difference). in this study we found that although approximately 60% of patients at the uhwi diabetes clinic engaged in some recommended foot care practices, fairly high proportions reported foot care or footwear choices that should be avoided ; in particular, walking barefooted, wearing shoes without socks and wearing pointed (narrow) toe shoes. in addition, almost a half of the study participants reported that they had never been taught about foot care. two studies, one from trinidad and the other from barbados have reported some data. in trinidad 49% of patients attending primary care diabetes clinics reported walking barefooted inside the house and 23% walked barefooted outside the house. in barbados, 39% of cases of persons who had amputations reported walking barefooted in the garden compared to 18% of controls who did not have amputations. in that study, walking barefooted was associated with an almost two - fold increase in the odds of amputation. the barbadian study also showed that while the majority of participants wore broad leather shoes, sneakers, rubber sandals and fashion shoes were also frequently worn. in both studies the questions were asked in a different manner from the current study, hence the data are not directly comparable. however, both studies highlight that foot care and footwear practices were sub - optimal and the barbados study made evident the effect of improper foot care and footwear practices in increasing amputation risk. overall these studies highlight the need for greater emphasis on foot care education to improve knowledge and practice among patients with diabetes, as the problems highlighted in this study are not limited to the caribbean. this is supported by the finding of similar gaps in knowledge and practice in studies from disparate countries, including the usa, nigeria and india. for example, in one study from india 44.7% of patients reported receiving no previous foot care education and 45% walked barefooted indoors, while in a study from nigeria only 40.9% of patients practiced daily foot inspection and 38% usually walked barefooted. another study among veterans in the united states found that a majority of participants felt that they did not know enough about foot self - care, with only 32% reporting that they looked at the bottom of the feet and 33% checking shoes for objects, while 41% of patients reported walking barefoot indoors. it is therefore apparent that patients with diabetes, particularly those without overt complications, receive little information about foot complications and how these might be avoided. we therefore support recommendations that all patients with diabetes should be offered foot care education aimed at improving foot care related knowledge and practice and thus reducing the risk for foot complications. the findings of this study are limited by the small sample size and the fact that it was conducted in a specialist diabetes clinic and as such may not be broadly generalizable. the findings are however consistent with other studies as cited above and therefore suggest that the situation may be similar among persons with diabetes in jamaica and the caribbean. the small sample size also limited our ability to demonstrate any associations between foot care practices or footwear and socio - demographic factors. despite these limitations, these data highlight the need for further studies with larger, representative, samples to better understand the problem at a national level. we also believe that publication of these findings will serve as a catalyst for further studies in the subject area, where clinicians and researchers can evaluate the extent to which appropriate practices are being followed in their setting. additionally prospective studies that evaluate the impact of foot care and footwear practices on outcomes such as foot ulcers and amputations would further help to determine the potential for interventions to improve practice and reduce complications. overall, the study highlights the need for greater emphasis on foot care education for patients with diabetes in jamaica and the need to identify barriers to foot care practice, both as it relates to the physician and to the patient. this would then lead to studies evaluating the efficacy of various intervention strategies in order to provide evidence - based guidelines for practice. | this study aimed to estimate the proportion of patients at the university hospital of the west indies (uhwi) diabetes clinic who engage in recommended foot care and footwear practices. seventy - two participants from the uhwi diabetes clinic completed an interviewer - administered questionnaire on foot care practices and types of footwear worn. participants were a subset of a sex - stratified random sample of clinic attendees and were interviewed in 2010. data analysis included frequency estimates of the various foot care practices and types of footwear worn. participants had a mean age of 57.014.3 years and mean duration of diabetes of 17.010.3 years. fifty - three percent of participants reported being taught how to care for their feet, while daily foot inspection was performed by approximately 60% of participants. most participants (90%) reported daily use of moisturizing lotion on the feet but almost 50% used lotion between the toes. approximately 85% of participants reported wearing shoes or slippers both indoors and outdoors but over 40% reported walking barefoot at some time. thirteen percent wore special shoes for diabetes while over 80% wore shoes without socks at some time. although much larger proportions reported wearing broad round toe shoes (82%) or leather shoes (64%), fairly high proportions reported wearing pointed toe shoes (39%), and 43% of women wore high heel shoes. in conclusion, approximately 60% of patients at the uhwi diabetic clinic engage in daily foot inspection and other recommended practices, but fairly high proportions reported foot care or footwear choices that should be avoided. |
autoimmune diseases (aids) are chronic disorders originated by the loss of immunological tolerance to self - antigens. this heterogeneous group of conditions present common genetic risk factors and share several pathophysiological mechanisms leading to overlapping clinical manifestations targeting specific organs or multiple organ systems. there is evidence that they share similar immunogenetic mechanisms, even though they exhibit varying epidemiological features and clinical manifestations [2, 3 ]. underlying these diverse clinical phenotypes is a deregulated immune system with an enriched ability to respond against self - tissues. the fact that aids share several clinical signs and symptoms (i.e., subphenotypes) and also share physiopathological mechanisms and genetic factors has been called autoimmune tautology and indicates that they may have a common origin. the immune system is in charge of the defence against external pathogens. for this purpose, t and b lymphocytes are responsible for the immune response through regulated cell - cell interactions and secretion of cytokines, chemokines, and other inflammatory mediators. this defence against external pathogens must occur without causing unnecessary harm to self. to achieve this delicate balance, the majority of self - reactive t and b lymphocytes are destroyed in the thymus and bone marrow through negative selection. nevertheless, this process is far from perfect, and self - reactive lymphocytes escape into the periphery. consequently, peripheral tolerance mechanisms are necessary to keep these self - reactive cells in check. activated self - reactive t and b cells promote autoimmunity when the effector and regulatory balance of the immune response is disturbed. major histocompatibility complex (mhc) molecules are widely distributed surface membrane glycoproteins that present antigenic peptides to t cell receptors (tcrs). developing thymocytes encounter a highly heterogeneous repertoire of self (endogenous) peptide - mhc (pmhc) complexes on thymic epithelial cells, the main thymus antigen presenting cells. the affinity / avidity with which these thymocyte tcrs bind self pmhc determines if it is destined to perish or if it will survive. in this way, a repertoire of peripheral t cells that is essentially self - tolerant several hypotheses have been put forward to explain how mhc polymorphisms influence autoimmunity risk or protection. they must do so, somehow, by shaping the central or peripheral t cell repertoires toward autoimmune resistance or proclivity. a protective mhc profile could achieve this by the selection of a t cell repertoire with diminished pathogenicity. on the other hand, protective mhc molecules may keep autoimmunity in check by favouring the negative selection of particular self - reactive t cells [1214 ]. the functional basis of the association between specific hla alleles and development of aids can be classically explained by two possible etiopathogenic models. the molecular mimicry hypothesis proposes that certain hla alleles are more efficient in presenting pathogen epitopes that share structural features with self - peptides to mature t cells. once the response to the pathogen is initiated the self - antigen is also recognized and disease ensues. central selection failure proposes that certain hla alleles are less efficient at presenting self - peptides to developing t cells in the thymus, so negative selection fails. a different hypothesis proposes that different alleles can modulate the immunologic profile of an individual, through antigen - independent mechanisms, resulting in either promoting a higher autoimmune predisposition or, in opposition, a more efficient immune regulation. given the consistent association of hla - drb1 alleles with different autoimmune diseases (table 1), we explored the idea that the same hla - drb1 alleles could be influencing several different autoimmune diseases. to this end this study includes four autoimmune systemic diseases, namely, systemic lupus erythematosus (sle), rheumatoid arthritis (ra), psoriasis or psoriatic arthritis (ps + psa), and systemic sclerosis (ssc). patients with multiple sclerosis (ms) and myasthenia gravis (mg) were also included. a total of 1228 patients with aids, 213 patients with sle and 153 patients with ra diagnosed according to the american college of rheumatology (acr) criteria, 166 patients with ps + psa, 67 with ssc, 536 with definitive diagnosis of ms according to the revised mcdonald criteria, and 93 with mg, were recruited from the neurology and medicine outpatient clinic of centro hospitalar do porto - hospital de santo antnio (chp - hsa). the hla - drb1 frequencies of patients were compared with the ones of a control group consisting of 282 unrelated individuals without disease and from the same geographic origin (north of portugal). genomic dna was obtained from proteinase - k treated peripheral blood leukocytes by using a salting - out procedure. drb1 locus (i.e., 2-digit hla nomenclature) was performed using polymerase chain reaction and sequence - specific primers (pcr - ssp), based on methods previously described. in order to produce pcr - ssp reactions able to detect and discriminate each of the known hla - drb1 genes, primers were designed using sequence alignments comprising all hla - drb1 variants and were validated by the twelfth international histocompatibility workshop. pcr products were visualized under ultraviolet light after running in a 1.5% agarose gel containing ethidium bromide. to identify the hla - drb1 genes contributing to the six different aids, we applied stepwise logistic regression on an allelic level, using forward selection which involves starting with no variables in the model, testing the addition of each variable using a chosen model comparison criterion, adding the variable (if any) that improves the model the most, and repeating this process until none improves the model. it should be noted that odds ratios (ors) obtained in a multivariable logistic regression analysis are adjusted for all the other genes included in the model and therefore differ from those obtained when a given gene is compared with all other genes. a total of 1228 cases and 282 controls were analysed and different types of association between alleles and aids were found (table 2). these included three risk alleles for two or more aids, two protective alleles for two or more aids, and three risk alleles for a particular aid. hla - drb113 was a protective allele for four aids : sle (18.3% versus 29.8%, p = 0.016, or = 0.58, and 95% ci = 0.370.90), ps + psa (19.3% versus 29.8%, p = 0.050, or = 0.621, and 95% ci = 0.391.00), ra (16.3% versus 29.8%, p = 0.044, or = 0.58, and 95% ci = 0.340.98), and ssc (11.9% versus 29.8%, p = 0.035, or = 0.42, and 95% ci = 0.190.94). hla - drb103 was found to be a risk factor for sle (34.3% versus 15.6%, p = 4.2 10, or = 2.49, and 95% ci = 1.613.86), ms (22.9% versus 15.6%, p = 0.003, or = 1.81, and 95% ci = 1.232.67), and mg (35.5% versus 15.6%, p = 6.1 10, or = 2.98, and 95% ci = 1.755.07). there were two risk alleles associated with two aids : hla - drb108 was positively associated with ms (12.1% versus 8.5%, p = 0.033, or = 1.73, and 95% ci = 1.052.87) and ssc (22.4% versus 8.5%, p = 0.004, or = 3.01, and 95% ci = 1.436.31) and hla - drb101 was found to be a risk factor for ra (32.7% versus 23.4%, p = 0.017, or = 1.79, and 95% ci = 1.112.88) and ssc (41.8% versus 23.4%, p = 0.006, or = 2.28, and 95% ci = 1.274.09). hla - drb109 was negatively associated with sle (1.0% versus 5.0%, p = 0.027, or = 0.18, and 95% ci = 0.040.83), ms (1.0% versus 5.0%, p = 0.004, or = 0.22, and 95% ci = 0.080.63), and ra (0.0% versus 1.0%, p = 0.003, or = 0.95, and 95% ci = 0.930.98). three risk disease - specific alleles were found : hla - drb104 for ra (47.7% versus 24.5%, p = 6 10, or = 2.81, and 95% ci = 1.794.39), hla - drb107 for ps + psa (39.8% versus 25.5%, p = 0.006, or = 1.79, and 95% ci = 1.182.72), and hla - drb115 for ms (32.7% versus 19.9%, p = 2 10, or = 2.17, and 95% ci = 1.533.10). considering aids as a group, hla - drb103 frequency was significantly higher (23.9% versus 15.6%, p = 0.022, or = 1.51, and 95% ci = 1.02.15) compared with controls ; conversely hla - drb113 (20.0% versus 29.8%, p = 0.004, or = 0.58, and 95% ci = 0.430.79) and hla - drb109 (1.4% versus 5.0%, p = 1 10, or = 0.23, and 95% ci = 0.110.49) frequencies were significantly lower. through a systematic review of published works, cruz - tapias and collaborators, in 2012, identified some common hla class ii alleles that contribute to susceptibility to aids in latin americans. the present study is, to date and to the best of our knowledge, the only one that addresses the hypothesis that a hla - drb1 allele could influence different autoimmune diseases, using a new cohort, encompassing six different autoimmune diseases. in this study we confirmed positive and negative associations in ms [24, 25 ], sle [1618 ], ps + psa [19, 20 ], ra, ssc [22, 23 ], and mg, previously reported in our or other populations. when aids studied were considered as a group, hla - drb103 allele was significantly overrepresented, as already described. it has been shown that this allele has low affinity for clip (class ii - associated invariant chain peptide) and may not require hla - dm to ensure peptide presentation, preventing efficient peptide selection and allowing the binding of low stability peptides. concerning the observed negative association with hla - drb109, we think that this is likely a spurious association, as this is a rare allele and the frequency found in controls is, for some reason, double the one reported for the portuguese population. our observations suggest that the presence of hla - drb113 allele may confer protection for aids. hla - drb113 is a high frequency allele in the general population both in portugal and worldwide. our results confirm that the lower frequency of hla - drb113 in every individual aids group is not secondary to the deviations granted by the concurrent positive associations. when the data obtained from previous studies are taken into consideration, the hla - drb113 allele seems to be a universal protective allele for ra. it was reported as protective against ra in asian [32, 33 ], turkish, and several european populations [3537 ]. recently this allele was also described to be protective in sle in the japanese population and for anca - associated vasculitis in the dutch population. specific amino acid patterns at the peptide binding cleft are involved in disease susceptibility, such as the well - known shared epitope first described in the ra susceptibility alleles hla - drb101 and hla - drb104 [37, 39 ]. similar to the shared epitope classification of susceptibility alleles, protective hla - drb1 alleles have been categorized according to several models. one of the most accepted classifications proposes that protection against ra is conferred by the deraa sequence at positions 7074 of the hla - drb1 allele. other models suggest that protection is conferred by an aspartic acid at position 70 (d70 allele) or an isoleucine at position 67 (i67 allele) of the hla - drb1 molecule. because it was unclear which hla - drb1 alleles were protective a meta - analysis was performed involving four european populations with > 2,700 patients and > 3,000 control subjects. the objective was to investigate exhaustively which hla - drb1 alleles were associated with protection against ra. interestingly, this study showed that the protective effect attributed to deraa and d70 was no longer present after the exclusion of hla - drb113. the authors concluded that this evidence indicates that hla - drb113 rather than deraa, d70, or i67 is associated with protection. in a recent study van heemst and collaborators identify citrullinated vinculin, present in the joints of acpa ra patients, as an autoantigen targeted by acpa and cd4 t cells. these t cells recognize an epitope with the core sequence deraa, which is also found in many microbes and in protective hla - drb113 molecules, presented by predisposing hla - dq molecules. intriguingly, deraa - directed t cells were not detected in hla - drb113 donors, indicating that the deraa epitope from hla - drb113 could mediate thymic tolerance in these donors and explain the protective effects associated with hla - drb113. they suggest that subjects born with hla - drb113 will present the hla - drb113-derived deraa - peptide in the thymus, leading to tolerization of the deraa - reactive t cell response. the negative association we describe here supports the idea that the hla - drb113 allele, possibly by its specific structural features, may as well confer resistance to several other aids. the protective effect of hla - drb113 could be explained by a more proficient antigen presentation by these molecules [43, 44 ], favouring an efficient thymic selection. as a result, negative selection and development of dr - driven autoreactive regulatory t cells a different model would relate hla molecules with the presence of specific endophenotypes indirectly associated with autoimmunity. other studies of our group suggest that the hla genotype may primarily influence the general activation state of cd4 t cells. the protective effect of hla - drb113 curiously, several reports have suggested an association of hla - drb113 and/or hla - dqb106 with slow disease progression in human immunodeficiency virus (hiv) infected individuals, meaning that among hiv controllers there is an association between the presence of certain class ii hla alleles and strong cd4 t cell responses [46, 47 ]. although different alleles are associated with particular aids, the same allele, hla - drb113, was underrepresented in all six diseases. this observation suggests that this allele confers protection to aids in general and particularly to rheumatic diseases. | autoimmune diseases (aids) are characterized by a multifactorial aetiology and a complex genetic background, with the mhc region playing a major role. we genotyped for hla - drb1 locus 1228 patients with aids-213 with systemic lupus erythematosus (sle), 166 with psoriasis or psoriatic arthritis (ps + psa), 153 with rheumatoid arthritis (ra), 67 with systemic sclerosis (ssc), 536 with multiple sclerosis (ms), and 93 with myasthenia gravis (mg) and 282 unrelated controls. we confirmed previously established associations of hla - drb115 (or = 2.17) and hla - drb103 (or = 1.81) alleles with ms, hla - drb103 with sle (or = 2.49), hla - drb101 (or = 1.79) and hla - drb104 (or = 2.81) with ra, hla - drb107 with ps + psa (or = 1.79), hla - drb101 (or = 2.28) and hla - drb108 (or = 3.01) with ssc, and hla - drb103 with mg (or = 2.98). we further observed a consistent negative association of hla - drb113 allele with sle, ps + psa, ra, and ssc (18.3%, 19.3%, 16.3%, and 11.9%, resp., versus 29.8% in controls). hla - drb113 frequency in the aids group was 20.0% (or = 0.58). although different alleles were associated with particular aids, the same allele, hla - drb113, was underrepresented in all of the six diseases analysed. this observation suggests that this allele may confer protection for aids, particularly for systemic and rheumatic disease. the protective effect of hla - drb113 could be explained by a more proficient antigen presentation by these molecules, favouring efficient clonal deletion during thymic selection. |
acute inflammation produces a condition known as hyperalgesia, which is characterized by enhanced pain sensation and reduced pain threshold. this abnormal sensory state is brought about, at least in part, by sensitization of peripheral nociceptors. accumulating evidence indicates that inflammatory mediators including prostaglandin e2 (pge2) and pgi2 are responsible for the sensitization of nociceptors [1, 2 ]. recent studies further clarified the molecular mechanism of sensitization : pge2 and pgi2 enhance or sensitize the activity of the capsaicin receptor transient potential receptor v1 (trpv1) through the activation of pge2 ep1 and pgi2 receptors, respectively. protein kinase c and protein kinase a are involved in the activation of trpv1 by these prostaglandins. in contrast, cyclopentenone prostaglandins, including 15-d - pgj2, pga2, and pga1, which are metabolites of pgd2, pge2, and pge1, respectively, directly activate the irritant receptor trpa1. for pharmacological control of inflammatory hyperalgesia, it is important to understand how these prostaglandins are produced in inflamed tissues. prostaglandins are generated through the arachidonic acid cascade, which involves three enzymatic steps : first phospholipase a2 (pla2) cleaves membrane phospholipids and releases arachidonic acid ; second, cyclooxygenase (cox) converts arachidonic acid into pgh2 ; and, third, various types of prostaglandin isomerases convert pgh2 to bioactive prostaglandins, including pge2 and pgi2. recent studies have shown that cox-2, an inducible isozyme of cox, and microsomal prostaglandin synthase-1 (mpges-1), an inducible isozyme of pge synthase, are responsible for the generation of pge2 in inflamed tissues [6, 7 ]. in these studies, administration of a cox-2 inhibitor to rats or disruption of the mpges-1 gene in mice lowered pge2 content in inflamed tissues and eased pain - related behaviors. thus, cox-2 and mpges-1 could be pharmacological targets for the treatment of inflammatory pain. however, it is not clear which isozyme of pla2 is responsible for the production of prostaglandins and development of hyperalgesia in inflamed tissues. pla2 has over ten isozymes which are classified into three categories based on their structural and functional similarities [8, 9 ]. first, cytosolic pla2s (cpla2 or group iv pla2) are located in the cytoplasm and are activated by low calcium ion concentrations (m levels). second, secretory pla2s (spla2 or group ib, ii, v, or x pla2) are released into the extracellular space where they are activated by high concentrations (mm levels) of calcium ions. third, calcium - independent pla2s (ipla2 or group vi pla2) are present in the cytoplasm and do not require calcium ions for their enzymatic activity, although the precise mechanisms for activation are unclear. among these pla2s, a significant role for ipla2 in the production of prostaglandins in carrageenan - induced pleuritis in rats was demonstrated. it is also reported that, in the spinal cord, cpla2 seems to be involved in inflammation - induced hyperalgesia. in the present study, we examined if ipla2 and/or cpla2 are responsible for the production of prostaglandins and development of hyperalgesia in carrageenan - induced inflammation in the rat hind paw. male sprague - dawley rats (nine weeks old, 300320 g) were purchased from charles river laboratories (yokohama, japan). all chemicals were obtained from commercial suppliers : bromoenol lactone (bel ; ipla2 inhibitor), arachidonyl trifluoromethyl ketone (aacocf3 ; cpla2 inhibitor), pge2 enzyme immunoassay (eia) kit, and 6-keto - pgf1 eia kit from cayman chemical (ann arbor, mi) ; isogen (rna extraction solution) from nippon gene (tokyo, japan) ; reverse transcription kit from invitrogen (carlsbad, ca) ; pcr sybr green master mix, lightcycler taqman master, and taqman probes from roche diagnostics (indianapolis, in) ; and rnalater (rna stabilization solution) from ambion (austin, tx). all experiments were carried out according to protocols approved by the institutional animal care committee of kyoto prefectural university of medicine. rats were housed four per cage and maintained on a 12 h light / dark cycle (light on 8:0020:00) with controlled temperature (25 3c) and humidity (55 15%). the plantar surface of the left paw received a subcutaneous injection of either 3 mg type carrageenan (sigma - aldrich, st. just after carrageenan injection, one of the two pla2 inhibitors (30 nmol) or vehicle (100 l of 0.1% dimethyl sulfoxide (dmso) in saline) was injected into the same site. for biochemical analyses, each rat was anesthetized with pentobarbital (60 mg / kg, intraperitoneal injection) 3 h after subcutaneous injection, and the left hind leg was cut at the knee, quickly frozen in dry ice powder, and kept at 80c until further processing. this time point was determined based on the following nociceptive behavior study, in which a pla2 inhibitor was effective between 1 h and 3 h after injection. for the assessment of nociceptive behavior response, rats were injected with the carrageenan / saline and pla2 inhibitors / vehicle in the manner described above. thermally evoked paw - withdrawal response was assessed using a device developed in yaksh 's lab. the paw - withdrawal latency was measured for the left hind paw before and every 60 minutes after the subcutaneous injection for a total of 360 minutes. for each time point, the latencies were measured 3 times in each rat and averaged. paw - withdrawal latencies were expressed as ratios to the baseline value of each rat. for analyses of pge2 and 6-keto - pgf1 (a metabolite of pgi2), the hind paws were coronally cut into 50 m thick frozen sections in a cryostat at 20c. twenty of these sections were collected in a plastic tube containing precooled ethanol (1 ml) and indomethacin (10 g) which prevents the synthesis of prostaglandins during tissue processing. after measuring wet tissue weight, the sections were homogenized with polytron for 30 s followed by sonication for 20 s. the homogenates were centrifuged (15000 rpm for 20 min at 4c) and the supernatant was collected. pge2 and 6-keto - pgf1 were measured using eia kits according to the manufacturer 's instructions. tissue pellet remaining in the plastic tube was heated in a heat block to completely evaporate the ethanol. the weight of dried pellet was considered to be the dry tissue weight of the paw from which the prostaglandins were extracted. twenty of these sections were placed into a vial containing rna later (1 ml) and stored at 30c until further processing. for rna extraction, the samples were homogenized in 1 ml phenol - based rna extraction solution (isogen) with polytron for 30 s followed by sonication for 20 s. total rna was isolated according to the manufacturer 's instructions. cdna was prepared from total rna using m - mlv reverse transcriptase and random hexamer as the primer. the reverse - transcribed cdna was amplified using a light cycler (roche diagnostics). mrnas of cox-2, mpges-1, ipla2, ipla2, and gapdh were quantified with the sybr green protocol. for the quantification of mrnas of prostaglandin i synthase (pgis), interleukin-1 (il1), and interleukin-6 (il6), the taqman probe protocol was used. primer pairs used for the pcr reaction were as follows : cox-2 : 5-ctcactttgagtcattc-3, 5-gattagtactgtagggttaatg-3, mpges-1 : 5-aatgaacccacgcattcgct-3, 5-cagccttcatggctccgtct-3, ipla2 : 5-caaggaactgggcaagatgg-3, 5-agagggcgttgaccagcact-3, ipla2 : 5-gaataccacaacatacacga-3, 5-acctaaaatacgtgtcagca-3, gapdh : 5-tgaacgggaagctcactgg-3, 5-tccaccaccctgttgctgta-3, pgis : 5-atgccatcaacagcatcaaa-3, 5-gctccaggtcgaaatgagtc-3, taqman probe (upl # 18), il1 : 5-tgtgatgaaagacggcacac-3, 5-cttcttctttgggtattgtttgg-3, taqman probe (upl # 78), il6 : 5-cccttcaggaacagctatgaa-3, 5-acaacatcagtcccaagaagg-3, taqman probe (upl # 20), gapdh : 5-agctggtcatcaatgggaaa-3, 5-atttgatgttagcgggatcg-3, taqman probe (upl # 9). cox-2 : 5-ctcactttgagtcattc-3, 5-gattagtactgtagggttaatg-3, mpges-1 : 5-aatgaacccacgcattcgct-3, 5-cagccttcatggctccgtct-3, ipla2 : 5-caaggaactgggcaagatgg-3, 5-agagggcgttgaccagcact-3, ipla2 : 5-gaataccacaacatacacga-3, 5-acctaaaatacgtgtcagca-3, gapdh : 5-tgaacgggaagctcactgg-3, 5-tccaccaccctgttgctgta-3, pgis : 5-atgccatcaacagcatcaaa-3, 5-gctccaggtcgaaatgagtc-3, taqman probe (upl # 18), il1 : 5-tgtgatgaaagacggcacac-3, 5-cttcttctttgggtattgtttgg-3, taqman probe (upl # 78), il6 : 5-cccttcaggaacagctatgaa-3, 5-acaacatcagtcccaagaagg-3, taqman probe (upl # 20), gapdh : 5-agctggtcatcaatgggaaa-3, 5-atttgatgttagcgggatcg-3, taqman probe (upl # 9). relative expression levels of each gene were calculated with the following formula : (1)relative expression of mrna=2cycle number of gapdh2cycle number of gene of interest. specificity of the pcr products was checked by the melting curve and by agarose gel electrophoresis in the sybr green protocol. the significance of differences among multiple groups was determined by multiple t - tests with bonferroni correction. we examined the effects of pla2 inhibitors on pge2 and 6-keto - pgf1 (a metabolite of pgi2) levels in inflamed foot pad. carrageenan and pla2 inhibitors / vehicle were injected into the right foot pad at the same time. three hours after the injection, carrageenan significantly elevated pge2 and 6-keto - pgf1 levels compared to injection of saline alone in vehicle-, bel- and aacocf3-coinjected groups (n = 4 in each group, p = 0.00020.014) (figure 1). bel, an ipla2 inhibitor, significantly suppressed carrageenan - induced increases in pge2 by 57% (p = 0.009) and 6-keto - pgf1 by 49% (p = 0.017) compared to vehicle. on the other hand, aacocf3, a cpla2 inhibitor and less potent ipla2 inhibitor, did not suppress the prostaglandin levels compared to the vehicle - treated rats. the two inhibitors did not exert significant effects on the prostaglandin levels in the saline - injected foot pad. real - time rt - pcr studies revealed that mrnas of both ipla2 isozymes, that is, ipla2 and ipla2, were present in the noninflamed foot pad, and their levels did not change following carrageenan injection (figure 2). we then asked if bel influences the induction of cox-2, mpges-1, and pgis, which are possibly involved in carrageenan - induced prostaglandin synthesis. cox-2 mrna, mpges-1 mrna, and pgis mrna were increased by carrageenan injection though the increases did not reach the statistically significant level except for cox-2 mrna and pgis mrna in carrageenan + bel group (figure 3). there was no significant effect of bel on the cox-2 mrna, mpges-1 mrna, or pgis mrna levels (figure 3). we also examined the effects of bel on carrageenan - induced proinflammatory cytokine mrnas (figure 4). bel slightly but significantly elevated carrageenan - induced il1 mrna and did not change carrageenan - induced il6 mrna. therefore, bel seemed to act solely on the enzyme activity of ipla2s resulting in suppression of prostaglandin levels in inflamed tissue. lastly, we studied the effects of pla2 inhibitors on carrageenan - induced thermal hyperalgesia. when carrageenan was injected with vehicle, foot withdrawal latency to radiant heat was reduced by approximately 70% compared to the preinjection level (at time point 0), indicating the induction of thermal hyperalgesia (figure 5(a)). the thermal hyperalgesia was statistically significant throughout the observation period of 6 h (n = 5, p < 0.005). carrageenan - induced thermal hyperalgesia was ameliorated by bel by 44% at 1 h, 61% at 2 h, and 46% at 3 h after injection compared to vehicle - treated group. at these 3 time points, the difference between carrageenan + bel group (n = 6) and carrageenan + vehicle group (n = 5) was statistically significant (p = 0.026, 0.014, and 0.015, resp.). on the other hand, aacocf3 did not influence the thermal hyperalgesia (figure 5(a)). both bel and aacocf3 did not show significant effect in saline - injected foot pad. pge2 and pgi2 are known to sensitize nociceptors and develop hyperalgesia during inflammation [1, 2 ]. however, which isoform of pla2 is involved in the synthesis of these prostaglandins in inflammatory tissues was unclear. in the present study, we showed that bel, a potent inhibitor of ipla2, lowered these prostaglandins by approximately 50% in rat foot pads that were inflamed with carrageenan. the inhibitor also halved carrageenan - induced thermal hyperalgesia during the first 3 h after injection. we confirmed that mrnas of ipla2 and ipla2 were constitutively expressed in the rat foot pad. this result is in line with the study by gilroy., who demonstrated that bel reduced pge2 content in the cell - free inflammatory exudates of the pleural cavity 3 h after carrageenan injection. it should be noted that bel is also a potent inhibitor of phosphatidic acid phosphohydrolase. reported that bel suppressed cox-2 expression in lps - activated macrophages through the inhibition of phosphatidic acid phosphohydrolase-1.. however, in the present study, carrageenan - induced cox-2 mrna was not affected by bel. this was also the case for mrnas of mpges-1 and pgis, the final enzymes in pge2 and pgi2 synthesis, respectively. in line with these results, bel did not reduce carrageenan - induced proinflammatory cytokines, which are strong inducers of cox-2 and other enzymes in the arachidonic acid cascade. these results strongly suggest that bel did not significantly suppress inflammation but inhibited ipla2 enzyme activity to reduce carrageenan - induced pge2 and pgi2 synthesis and, consequently, thermal hyperalgesia. therefore, we conclude that ipla2 is responsible for the synthesis of prostaglandins in carrageenan - induced tissue inflammation. nonsteroidal anti - inflammatory drugs (nsaids) are widely used for the treatment of inflammatory pain. nsaids potently inhibit cox but do not suppress the supply of its substrate, that is, arachidonic acid. thus, free arachidonic acid may enter lipoxygenase or epoxygenase pathways to be various potent eicosanoids such as leukotrienes. since inhibitors of pla2, such as bel, reduce the supply of free arachidonic acid, pla2 inhibitors may be more ideal in the treatment of symptoms induced by eicosanoids than nsaids. this fact suggests that other pla2 isoforms, that is, cpla2 or spla2s, also participated in carrageenan - induced prostaglandin synthesis. traditionally, cpla2 has been considered the major pla2 that cleaves arachidonic acid from the sn-2 position of membrane phospholipids during inflammation. in the present study injection of aacocf3, a cpla2 inhibitor, into the foot pad neither reduced pge2 and pgi2 nor suppressed thermal hyperalgesia, suggesting that cpla2 does not play a significant role in these responses this result is consistent with those of two previous studies, the first of which by gilroy. showed no effect of aacocf3 on the pge2 content of cell - free inflammatory exudates of the pleural cavity 3 h after carrageenan injection. the second study by lucas. reported that intraplantar injection of aacocf3 did not suppress carrageenan - induced thermal hyperalgesia even with a dose approximately 20 times higher than that used in the present study. on the other hand, it is reported that a new type of cpla2 inhibitor reduced carrageenan - induced pge2 production in the rat air pouch. the reason for the discrepancy is unclear at present, and we can not completely exclude the possibility that cpla2 is partly involved in pge2 and pgi2 production in inflammatory tissues. pge2 and possibly pgi2 augment nociceptive responses by acting in multiple sites along the pain processing neural pathway. in each site, distinct isoforms of pla2 seem to be involved in prostaglandin synthesis. in the peripheral inflammatory site, these prostaglandins are produced by the action of ipla2 and sensitize nociceptors, as shown in this study. in the spinal cord, pge2 is produced in response to nociceptive neural inputs from the primary sensory neurons and possibly enhances the pain signal transmission. in this site, cpla2 and spla2 but not ipla2 are reported to play major roles in pge2 synthesis and hyperalgesic response [11, 16 ]. furthermore, in the spinal cord and brain, peripheral inflammation induces prostaglandin synthesis through the action of circulating inflammatory cytokines such as interleukin-6. this is evidenced by an elevation of pge2 in the cerebrospinal fluid and induction of cox-2 in endothelial cells throughout the brain and spinal cord after intraplantar injection of carrageenan.. however, little is known about which isoform of pla2 is activated upstream of cox-2. further studies are necessary to thoroughly determine which pla2 isoforms are involved in prostaglandin synthesis in the central nervous system under inflammatory conditions. the present study has indicated the importance of ipla2 in prostaglandin synthesis at the inflammatory site and suggested that ipla2 is a possible target for the treatment of inflammatory hyperalgesia. | prostaglandin (pg) e2 and pgi2 are essential to hyperalgesia in inflammatory tissues. these prostaglandins are produced from arachidonic acid, which is cleaved from membrane phospholipids by the action of phospholipase a2 (pla2). which isozyme of pla2 is responsible for the cleavage of arachidonic acid and the production of prostaglandins essential to inflammation - induced hyperalgesia is not clear. in this study, we examined the effects of two pla2 isozyme - specific inhibitors on carrageenan - induced production of pge2 and pgi2 in rat hind paw and behavioral nociceptive response to radiant heat. local administration of bromoenol lactone (bel), an inhibitor of calcium - independent pla2 (ipla2), significantly reduced carrageenan - induced elevation of prostaglandins in the inflamed foot pad 3 h after injection. it also ameliorated the hyperalgesic response between 1 h and 3 h after carrageenan injection. on the other hand, aacocf3, an inhibitor of cytosolic pla2, suppressed neither prostaglandin production nor the hyperalgesic response. bel did not suppress the mrna levels of ipla2, ipla2, cyclooxygenase-2, microsomal prostaglandin e synthase, prostaglandin i synthase, or proinflammatory cytokines in the inflamed foot pad, indicating that bel did not suppress inflammation itself. these results suggest that ipla2 is involved in the production of prostaglandins and hyperalgesia at the inflammatory loci. |
the ethics committee of gois federal university approved this study under protocol # 020/2008. recombinant mycobacterium smegmatis production : m. smegmatis mc 155 (kindly provided by dr. luciana leite of the butantan institute, brazil) was grown in middlebrook 7h9 broth (himedia, mumbai, india) supplemented with 0.05% of tween 80 at 37c for three days. cells were washed in 10% glycerol, and electrocompetent cells were aliquoted in 100 l volumes in cryotubes at 80c until use. the recombinant plasmid containing the gene of the fusion protein cmx constructed in our lab (pla71/ cmx) and the empty plasmid (pla71, kindly provided by dr. brigitte gicquel, from pasteur institute, france) were introduced into m. smegmatis mc 155 (mc - cmx and mc - pla71, respectively) by electroporation. construction of the recombinant m. smegmatis - cmx (mc - cmx) was described by junqueira - kipnis.. briefly, the dna sequence containing the immunodominant epitopes from ag85c and mpt51 and the entire hspx gene, described previously by de sousa., was used as template for pcr amplification using a set of primers to create flanking restriction enzyme sites and facilitate posterior cloning. the product of this amplification was cloned into pgem - t easy vector (promega, madison, wis, u.s.a.). the recombinant pgem - t easy vector containing the fused gene (cmx) was digested with kpni and noti for elution of the cmx gene. the eluted cmx / kpni / noti gene was ligated to the mycobacteria / e. coli shuttle vector pla71 digested with the same enzymes, the size of which was 12,248 pb, and as a selective marker, pla71 contains a kanamycin resistance gene. the cmx gene was inserted downstream of the -lactamase gene (blaf) promoter. this promoter codes the 32 amino acids of the mature -lactamase signal sequence and the first 5 amino acids of the mature protein for expression and export of cmx protein. pla71/cmx and pla71 vectors were transformed into mc and screened on media with kanamycin, to produce the recombinant vaccine mc - cmx and mc - pla71 control vaccines, respectively. animals and experimental design : thirty nelore calves that ranged in age from 10 to 12 months and were negative for the tuberculin skin test (from the goiano federal institute uruta campus) were separated into three groups randomly. group 1 received 1.0 ml (1 10 cfu / ml) of the recombinant vaccine (m. smegmatis mc - cmx) ; group 2 received 1.0 ml (1 10 cfu / ml) of the empty vector vaccine (m. smegmatis mc - pla71) ; and group 3 received 1.0 ml of pbs. prior to each immunization, blood collection was performed such that there were blood samples corresponding to the basal level, the levels at 21 days after the first and second immunizations and the level at 30 days after the last immunization. in order to test the maintenance of memory immune responses, the animals were revaccinated a fourth time at 200 days after the third vaccination. blood was collected from the tail artery / vein without an anti - coagulant. sera were obtained after centrifugation at 2,500 g for 10 min at 4c and stored at 20c until use. blood was also collected with heparin as an anti - coagulant for cytometric analysis. peripheral blood mononuclear cell (pbmc) was obtained after centrifugation at 2,500 g for 15 min at 4c, treatment with erythrocytes lysis solution and resuspension in rpmi 1640 media (gibco, carlsbad, ca, u.s.a.) supplemented with 24 mm of sodium bicarbonate, 10% heat - inactivated fetal calf serum, 2 mm of l - glutamine (sigma - aldrich, st louis, mo, u.s.a.), 1% of penicillin - streptomycin (sigma - aldrich), 1 mm sodium pyruvate (sigma - aldrich) and nonessential amino acids (sigma - aldrich). immunoenzymatic assay (elisa) : ninety - six - well polystyrene plates (santa cruz biotechnology, dallas, tx, u.s.a.) were coated with the lysates of mc - cmx vaccine or purified recombinant protein (rcmx, rag85c, rmpt51 or rhspx, 10 g / ml) diluted in 15 mm carbonate / bicarbonate buffer (ph 9.8). after 18 hr of incubation at 4c, the plates were blocked with carbonate / bicarbonate buffer containing 1% gelatin for 2 hr at 37c. serum, diluted (1:320) in pbs containing 0.1% gelatin, was added and incubated for 2 hr at 37c. extensive washing with pbs tween (0.05%) was performed followed by addition of an anti - bovine igg conjugate (jackson immunoresearch laboratories, inc., london, u.k.) diluted in pbs 0.1% gelatin (1:10,000) and incubation for 1 hr at 37c. after additional washing with pbs tween (0.05%), citrate buffer (ph 5.0) containing o - phenylenediamine (opd) and hydrogen peroxide was added. samples were analyzed in a multiskan plus (thermo scientific, walthan, ma, u.s.a.) elisa reader at 492 nm. flow cytometry : flow cytometry analysis was performed after stimulation of 10 pbmcs with rcmx (10 g / ml) or phytohemagglutinin (pha 1 g / ml) for 6 hr at 37c in 5% co2. cells were further incubated with monensin (ebioscience,, san diego, ca, u.s.a.) for 4 hr at 37c in 5% co2. cell staining was performed with antibodies conjugated with surface membrane or intracellular markers, pe - cd8, percp - ifn- and apc - cd4, for 30 min, washed with pbs containing 0.1% sodium azide, fixed and permeabilized with perm fix / perm wash (bd pharmingen, sanjose, ca, u.s.a.). cell selection after flow cytometry acquisition of 50,000 events (bd biosciences, san diego, ca, u.s.a. facscanto ii) was based on size (fsc) and granularity (ssc) to gate lymphocytes using bd facsdiva software. histopathological analysis : ninety days after the booster immunization, superficial cervical lymph node biopsies were performed at the site of vaccine injections. biopsy samples were conditioned in 10% buffered formalin, posteriorly submitted to routine processing and stained with hematoxylin eosin (he) and fite s acid fast staining. single cervical tuberculin skin test : all animals were submitted to tuberculin skin testing prior to beginning the experiments and after the end of all immunizations. the test was performed by intradermally injecting 0.1 ml of m. bovis standard ppd (0.1 mg of m. bovis strain an5) in the mid - cervical area. the animals were examined for a hypersensitivity response 72 hr post injection. statistical analysis : the results were analyzed using the microsoft excel (version 14.3.4, 2011) and graphpad (prism 4.0) software. differences between groups were analyzed using anova test. individual groups were compared by the nonparametric kruskal - wallis test followed by dunn s test. results with a p - value of less than 0.05% were considered statistically significant. in order to verify if mc - cmx was able to induce an immune humoral response in bovine, an elisa was developed to detect the presence of vaccine - specific antibodies (fig. (a) serum levels of anti - mc - cmx antibodies among bovine immunized with mc - cmx, mc and pbs. (b) serum levels of anti - rcmx antibodies among bovine immunized with mccmx, mc and pbs. animals were immunized with 1 10 cfu / ml of each vaccine subcutaneously. sera were obtained at different time points following vaccination and analyzed by elisa. each time point represents the mean and standard error of the mean of the optical density from 10 animals per group. statistically significant difference between the mc - cmx and pbs groups (p<0.05). statistically significant difference between the mc and pbs groups (p<0.05).). a progressive increase in igg antibody levels was observed as immunizations were performed, and there was a significant difference in mc - cmx - specific antibody levels at the latter time points of immunizations compared with at the initial time points, as well as compared with the control groups immunized with mc or pbs. an elisa was also performed to detect the presence of rcmx - specific antibodies (fig. 1b), and the results were similar to those of the first test. a progressive increase in igg antibodiy levels occurred, with higher titers being observed at latter time points of immunization. however, no response was observed to the individual recombinant antigens (rag85c, rmpt51 or rhspx, data not shown). (a) serum levels of anti - mc - cmx antibodies among bovine immunized with mc - cmx, mc and pbs. (b) serum levels of anti - rcmx antibodies among bovine immunized with mccmx, mc and pbs. animals were immunized with 1 10 cfu / ml of each vaccine subcutaneously. sera were obtained at different time points following vaccination and analyzed by elisa. each time point represents the mean and standard error of the mean of the optical density from 10 animals per group. statistically significant difference between the mc - cmx and pbs groups (p<0.05). statistically significant difference between the mc and pbs groups (p<0.05). to evaluate if vaccination altered the number of cd4 t cells in the peripheral blood, pbmcs were analyzed, and the total number of cd4 t cells from each animal was obtained. cattle were revaccinated 200 days after the third immunization, and pbmcs were obtained. (a) total cd4 t cells from the vaccinated group were quantified by flow cytometry. the dots represent the number of cd4 t cell / ml from each animal. (b) pbmcs from mc - cmx - vaccinated animals were stimulated in vitro with medium, pha or cmx, and the percentage of cd4ifn--positive t cells was analyzed. no significant statistical difference was observed.). since it is well known that the cellular immune response to btb is crucial for protection, after the booster vaccination to induce reactivation of memory cells, ifn--producing cd4 t lymphocyte populations were quantified. stimulation of pbmcs from mc - cmx - vaccinated bovine with recombinant rcmx did not reveal an increase in the amount of ifn--producing cd4 t lymphocytes, regardless of the animal immunization status (fig. cattle were revaccinated 200 days after the third immunization, and pbmcs were obtained. (a) total cd4 t cells from the vaccinated group were quantified by flow cytometry. the dots represent the number of cd4 t cell / ml from each animal. (b) pbmcs from mc - cmx - vaccinated animals were stimulated in vitro with medium, pha or cmx, and the percentage of cd4ifn--positive t cells was analyzed. no significant statistical difference was observed. after the second immunization, animals that received the mc - cmx vaccine presented accentuated lymphadenomegaly in superficial cervical lymph nodes. neither necrosis nor inflammatory infiltrate or granuloma formation was found within the analyzed biopsy samples (fig. 3fig. 3.histopathological analysis of a superficial cervical lymph node from the mc - cmx vaccinated group. the lymph node from a representative animal was sectioned and stained with hematoxylin eosin (h&e) (a, c and d) and fite s acid fast stain (b) 90 days after the last immunization of the group. note the absence of inflammatory infiltrate or granuloma formation or necrosis (a). fite s acid fast staining revealed no presence of acid - fast bacilli (b). the structure of a normal lymph node with a germinal center, medular region and blood vessels can be seen (c and d).). histopathological analysis of a superficial cervical lymph node from the mc - cmx vaccinated group. the lymph node from a representative animal was sectioned and stained with hematoxylin eosin (h&e) (a, c and d) and fite s acid fast stain (b) 90 days after the last immunization of the group. note the absence of inflammatory infiltrate or granuloma formation or necrosis (a). fite s acid fast staining revealed no presence of acid - fast bacilli (b). the structure of a normal lymph node with a germinal center, medular region and blood vessels can be seen (c and d). all animals remained negative for the tuberculin skin test until the end of the experimental period. in the present study, we investigated the capacity of a recombinant vaccine, mc - cmx, to induce specific igg antibodies in bovine. the cervical lymph nodes of mc - cmx - vaccinated animals presented follicular hyperplasia characteristic of cellular proliferation. the animals remained negative in the tuberculin skin test throughout the duration of the study. in recent years, several approaches have been made to increase the protective efficacy of bcg, in addition to the search of new immunogenic antigens from mtb as well as from other mycobacteria, in order to develop improved vaccines. an effective vaccine against tb or btb must be capable of preventing infection and establishing a long - lasting protection. consequently, it is essential that a robust induction of effector and memory cells take place and that a significant level of antibodies is raised following vaccination. it is known that antibodies play an important role in mycobacterial infections, as they can induce microbicidal activities of macrophages and attract monocytes, neutrophils and nk cells to the site of infection, in addition to activating dendritic cells and macrophages, enabling efficient antigen processing and presentation by these cells [19, 25, 39, 41 ]. in this study, a significant increase in specific igg levels against mc - cmx and rcmx was observed in mc - cmx - vaccinated animals (fig. 1). despite the observation of an initial response among animals vaccinated with m. smegmatis (mc) alone, there was a significant increase in the antibody level in animals receiving mc - cmx after the second time point of vaccination (p<0.05). this cross - reaction observed among the control groups could be explained by the presence of ag85c and mpt51 analog proteins in m. smegmatis and by the constant exposure of animals to environmental mycobacteria as well. bovines vaccinated with mc - cmx could not produce igg antibodies against individual ag85c, mpt51 or hspx recombinant antigens when tested in an indirect elisa (data not shown), reflecting the immunogenicity of the rcmx protein in the bovine model. similar results were observed when mice were immunized with mc - cmx or rcmx [15, 24 ]. a possible explanation for these observations could involve the tertiary structure of the secreted fusion protein produced by the recombinant m. smegmatis vaccine hiding or exposing unique epitopes not present in the single antigens. to circumvent the absence of response observed to the individual wild - type antigens, further engineering of the recombinant fusion protein is needed, such as replacing the hinge regions with bigger and/or different amino acid sequences to allow independence of each epitope exposition and folding. similarly, we have shown that rcmx, a recombinant fusion protein, induces production of igg1 and igg2a in mice when used as a subunit vaccine in addition to the ability of this recombinant protein to detect high levels of specific igg and igm in the sera of active tb individuals. these results corroborate with immunogenic potential of the recombinant protein in the context of the vaccine. a recent report showed the ability of a recombinant m. smegmatis vaccine expressing a fusion protein comprised of the antigens esat- 6 and cfp-10 from mtb to induce a high titer of antibodies in the mouse. thus, the fusion protein cmx can be considered a candidate to be used in the production of recombinant or subunit vaccines, as they presented antigenicity and immunogenicity in different study host models. further development of the fusion protein should be performed before this vaccine is considered a candidate to protect btb. the essential role of cell - mediated immune response in the protection against tb has been well characterized [13, 30, 34 ]., as they produce ifn- and tnf-, which are key cytokines in the immune response against mycobacterial infections. in addition, cd8 t lymphocytes also contribute to a long - lasting protection against tb [28, 31, 33 ], as they can use a diverse repertoire of mechanisms against infected cells, especially phagocytes, the main target of mycobacteria, and non - phagocytic cells that can also be infected by this pathogen. here, we show that there was no significant increase in specific cd4 ifn--positive t cells (fig. this phenomena could be explained by a) the majority of the effector/ memory t cell being localized in the draining lymph nodes instead of in peripheral blood [26, 35 ], b) 30 days after the booster possibly being too long for the persistence of this population of cells in the periphery [26, 39 ] and c) cmx not having been presented by bovine antigen - presenting cells. it is well known that mycobacteria can be drained into the lymph node by infected resident macrophages. consequently, we sought to understand if the observed lymphadenomegaly in superficial cervical lymph nodes was an inflammatory process caused by m. smegmatis proliferation. the histopathological analysis revealed neither necrosis nor inflammatory infiltrate or granuloma formation within the analyzed biopsy samples. additionally, no acid - fast bacilli were detected within the analyzed tissues (fig. the observed lymphadenomegaly was probably due to cellular proliferation as a result of a lymphocytic activation. it is possible that the observed hyperplasia was strictly correlated with higher levels of antibodies induced against cmx, although activation of cd4 or cd8 t cells was not observed. previous studies stated that bovine have a dichotomous response to mycobacteria, that is, alternating high levels of antibody production with cellular immune response [35, 38, 50 ]. in line with this, we can infer that a humoral immune response was elicited in response to the fusion protein cmx and that a cellular response was directed only toward m. smegmatis, and thus, it was not observed in our assays. an inflammatory response or the presence of bacteria was not observed in the lymphoid tissue (fig. furthermore, the maintenance of a negative tst status among animals indicates that the vaccine did not elicit a cross - reaction with the test. explanations for pitfalls in human and animal tb vaccines are encountered throughout the literature. some studies affirm that previous contact with environmental mycobacteria can reduce the immune response induced by tb vaccines. different animal model studies have already proven the interference of sensitization to other mycobacteria in the immune response against tuberculosis [7, 14, 30 ]. a study with cattle demonstrated that, prior to bcg immunization, bovines exhibited high levels of ifn- in response to ppd from mycobacterium avium, and these animals showed an ifn- response to ppd from m. bovis for only one week after vaccination with bcg ; on the other hand, groups that received a newly derived attenuated m. bovis strain maintained this response for two weeks. failure in bcg vaccination among animal models is consistent with studies in humans, where it has been shown that previous contact with other mycobacteria could block multiplication of the vaccine and induction of an immune response. regarding humans, a study with populations from malawi and the united kingdom showed that people from malawi had lower levels of ifn- one year after bcg vaccination, which was attributed to a preexposure to environmental mycobacteria. the development of new therapies against infectious diseases that affect human populations is a constant challenge for the scientific community. in regard to tb, a zoonosis causing a significant death toll in humans and important economic losses worldwide, these challenges are even greater. an important limitation of this study was the necessity to validate the real potential of mc - cmx as a vaccine in a protective study in bovines challenged with m. bovis. this pitfall does not diminish the importance of our findings, as we showed for the first time the bovine immune response to a recombinant fusion protein used as a btb vaccine. in conclusion, this work shows that the mc - cmx vaccine was able to induce a humoral immune response and did not induce cross - reactivity in the tst, indicating that it can potentially be further tested as a vaccine for bovine tuberculosis. | abstractthis study aimed to evaluate the immunogenicity of a recombinant mycobacterium smegmatis vaccine expressing the cmx fusion protein composed of immunodominant epitopes ag85c, mpt51 and hspx of mycobacterium tuberculosis, which are important mycobacteria virulence factors. a group of nelore heifers that were 10 to 12 months of age and negative for the tuberculin skin test (tst) were immunized with four doses of the recombinant vaccine mc2-cmx (m. smegmatis - ag85c - mpt51-hspx) during a period of one year. before each immunization, blood was collected to obtain sera for antibody analysis. serological analysis demonstrated that mc2-cmx was able to induce a humoral response with increased levels of specific igg antibodies against cmx, despite minimum antibody levels being detected for individual ag85c, mpt51 or hspx recombinant antigens. however, there was no significant increase in specific cd4 + ifn--positive t cells. lymphadenomegaly was observed in superficial cervical lymph nodes adjacent to the site of vaccination among mc2-cmx - vaccinated bovines, and the histopathological analysis demonstrated follicular hyperplasia without inflammatory infiltrate or granuloma formation. animals remained negative for the tst until the end of the experiments, showing no cross - reactivity with the recombinant vaccine and tuberculin proteins. we discuss the potential of mc2-cmx to induce an immune response in cattle. |
cerebral palsy (cp) is a severe childhood disability, characterized by a nonprogressive motor disorder of posture and movement due to a lesion in the developing brain. the prevalence of this clinical condition among children aged 310 years has been reported to be 2.4 in every 1000 live - borns, being significantly higher in males and black people. the most common disorders associated with cp are mental retardation, sensory limitations, epilepsy, speech disorders and hearing loss. the more common oral conditions in individuals with cp include higher mean decayed, missing and filled surfaces index, higher plaque index, tendency for delayed eruption of permanent molars, malocclusion, as well as high rates of bruxism. this rhythmic grinding can cause masseter hypertrophy, headaches, temporomandibular joint destruction, and tooth wear. the incidence of bruxism in the general population has been reported to be as high as 21%, but its incidence in pc is still unknown. many factors may be involved in the etiology of this parafunctional activity such as spasticity ; unbalanced oral myofunctional disturbance[3, 5 ], backbone dysfunction with the head projected forward, which changes the contact between the teeth and predisposes hyperactivity of the main masticatory muscles (temporal and masseteric), lack of control of the mandibular posture which can worsen in periods of emotional stress ; sleep disorders ; use of neuroleptics and malocclusion. the mastigatory musculature spasticity of cp can interfere with daily activities such as tooth brushing, cleaning of the oral cavity and eating. the treatment for this parafunctional activity includes restorative treatment, occlusal adjustment, the use of oral splints, pharmacological treatments and dental extraction. the most severe cases require a multidisciplinary input, including pediatricians, psychiatrists, paediatric dentists, and/or oral surgeons. the following paper reports a severe case of bruxism in a child with cerebral palsy and discusses the treatment given. a seven - year - old boy with spastic cerebral palsy (type quadriplegia, the severest cp) was brought to the pediatric dentistry department of the federal university of rio de janeiro, brazil because his parents complained of the repeated grinding which was damaging his teeth and gums. the parents reported during anamnesis that the pregnancy and parturition were normal and he was born in 40th week of gestation. when he was 5 days old he spent 11 days in the neonatal intensive care unit because of icterus with moderate bilirubin levels and he needed a blood transfusion. the child was under medical treatment during his first year of life because of convulsive crises. the child has been followed up by a multidisciplinary team including pediatrician, physiotherapist, psychologist and neurologist. the enamel had been worn away on large areas of anterior cusps and mastigatory primary molar surfaces. because of the poor participation of the patient, the size of movements could not be measured. he had no salivary drooling and had an adequate swallow in spite of evidence of facial and hypoglossal nerve dysfunction. his parents reported he used to eat ordinary food, but in the last six months they had noticed he appeared in pain while eating so he was only able to eat pured food. as the molars had extensive teeth surface losses, stainless steel crowns were the proposed treatment for the primary molars under local anesthesia and nitrous oxide - induced sedation (n2o). at the first appointment, the treatment of the right molars was carried out and four stainless steel crowns were cemented on the primary molars. a week later a protective oral appliance was designed for covering all the maxillary molars, in order to reduce the bruxism and prevent injuries to the soft tissues. impressions were made with a silicone material to obtain a working model (figure 1) and two acrylic resin appliances were made (figures 2 and 3) under n2o sedation. at the following appointment, the protective appliance was fixed on both sides of the mandibular teeth by means of ionomer cement. the parents were educated on the proper oral hygiene measures to be adopted and the need for regular dental visits in the future. after a period of two weeks, grinding behavior had decreased significantly and no further damage to the dentition were seen. his parents reported that the child had received the appliance well and he no longer ground his teeth (the bruxism had been controlled with concomitant alleviation of symptoms) ; feeding had improved, because he was able to consume solid foods instead of the semisolid diet that he had before treatment. the patient was periodically followed up in order to remove and clean the appliances, apply topical fluoride and instruct the parents on oral hygiene. six months later, the eruption of permanent central incisors of both jaws could be noted and at the one year (figure 4) review the crowns of these teeth were more exposed. individuals with cp tend to develop accentuated involuntary muscle tonus in orofacial muscles and other muscles and often show various types of stereotypy, especially when they lack any other occupation, as in this case, where the child occupied himself by grinding his teeth for a large part of the day. lindqvist and heijbel observed that abnormal dental wear is closely related to a low level of mental development and severe dental wear indicates that cp children have more pronounced bruxism than normal children [8, 9 ]. in this case, clinical signs as lip biting or small ulcers were not found, even though they have been reported as clinical signs of bruxism [6, 18].on the other hand, the patient presented limited mouth opening or trismus, an inherent characteristic of bruxism. there is contradictory information in the literature regarding the incidence of oral diseases in patients with cp. according to brown and schodel, these controversies are due to the failure of the criteria used to choose the population to be studied as well as the absence of control groups. however, no carious lesions were diagnosed in the patient described in the present case. this may be explained because the parents had received oral hygiene and diet instructions in a pediatric clinic when the child was very young and they have always been very careful with the child 's oral hygiene. they are often treated under general anesthetic or deep sedation, which has many disadvantages.yoshida. presented a study pointing out the beneficial properties of nitrous oxide-(n2o-) induced sedation performed during dental treatment on cp patients. the authors decided to carry out the dental procedures on the patient using n2o sedation considering the procedure was of short duration, the difficulties involved in local hospital admission and the safety of the technique. in studies of bruxism in mentally retarded children in the literature, the term abnormal abrasion has varying implications ; so treatment for the oral injuries caused by this parafunctional activity may include restorative techniques, dental extractions, and the use of oral protective appliances. the first choice was to restore the primary molars with stainless steel crowns because of their high degree of dental wear. this procedure was not sufficiently effective, since the patient continued to grind his teeth with great force. although the appliance was cemented, the parents were instructed to verify the stability of the appliance in order to avoid the risk of aspiration. although the appliance was cemented, before choosing this treatment option, the authors discussed the risk of aspiration with the child 's parents. they were instructed to verify the stability of the appliance and to keep it cleaned. although there are few studies concerning the use of oral devices directly with children, an oral appliance applied to prevent the self - mutilation of the lower lip in a mentally handicapped patient with moebius syndrome, has been described, with positive results [15, 23 ]. the bruxism was reduced and the parents related that activities like eating and sucking became easier for the patient. it probably occurred because the resin appliance increased vertical dimension and the occlusal parameters had been established, so the patient was able to have a better functional occlusal activity. in a later followup, the eruption of permanent first molars and incisors was observed, indicating the success of the treatment. as the child gets older, the appliance can be extended to other teeth if necessary, as his future neurological condition can not be predicted. early diagnosis and interventions of parafunctional habits in individuals with cp is important to reduce invasive treatment and worse complications for the patient. this paper also highlights that oral protective appliances can be clinically viable and effective for the prevention of bruxism. furthermore, this case illustrates the importance of the treatment by a dental team in patients with cerebral palsy. | cerebral palsy (cp) is one of the most severe childhood disabilities due to a lesion in the developing brain. oral conditions often observed in this pathogenic are a tendency for the delayed eruption of permanent molars, higher percentages of malocclusion and parafunctional habits, including bruxism. the significance of oral conditions observed in cp patients demonstrates the need for intensive home and professional care for these individuals. this paper presents a 7-year - old boy, with cerebral palsy, severe mental retardation, who had high abrasion wear of the primary teeth related to bruxism. dental care was carried out under oxide - induced sedation, and management of the bruxism was achieved after the use of a resin acrylic protective appliance fixed on both sides of the mandibula. the treatment performed offered efficiency advantages, was clinically viable, and should be a valuable option to practitioners considering appliance therapy to control parafunctional behavior. |
lactic acidosis (la) is a high anion gap metabolic acidosis with serum lactate levels > 5 mm / l and acidemia (ph < 7.35) caused by over - production or underutilization of lactic acid from the body. thiamine deficiency is one of the causes and cases of refractory la treated successfully with thiamine were reported in the literature. though chronic kidney disease (ckd) and hemodialysis are proven risk factors for occult thiamine deficiency, we are not aware of any case report describing reversal of acute refractory la with intravenous (iv) thiamine in this patient population. a 48-year - old female with end - stage renal disease, hypertensive since past 7 years was electively posted for robotic assisted laparoscopic renal transplantation from a living related donor. she was on maintenance hemodialysis twice a week since 6 months, and her hypertension was well controlled with one antihypertensive agent. her fasting, postprandial blood sugar and glycated hemoglobin levels were within normal limits in more than three examinations preoperatively. preoperative hemodialysis was done within 24 h of the scheduled surgery. on the day of surgery her baseline vitals were heart rate (hr) 94/min, mean arterial pressure (map) 103 mm of hg, spo2 100%, central venous pressure (cvp) 12 cm of h2o, and core temperature 98.6f. arterial blood gas (abg) analysis after induction of anesthesia (10 am) was unremarkable with a lactate of 1.9 mm / l [table 1 ]. volume controlled ventilation was adjusted to maintain etco2 between 35 mm and 40 mm of hg. three hours after induction of anesthesia hemodynamic parameters deteriorated with hr of 122/min, map of 72 mm of hg and cvp of 16 cm of h2o. abg analysis showed metabolic acidosis with elevated lactate levels, hyperglycemia, elevated anion gap and fall in hemoglobin [table 1 ]. blood sample for ketone bodies tested negative and it was repeated every fourth hourly to rule out diabetic ketoacidosis as a cause of the acidosis. considering occult blood loss, iv fluids and two units of packed red blood cells were transfused. as vascular anastomosis of the graft kidney was being carried out, noradrenaline infusion [figure 1 ] was started to maintain a map of 90 mm of hg. abg analysis of the patient since the initiation of surgery till the next 22 h trend of lactate / blood glucose levels / mean arterial pressure from the initiation of surgery till the next 22 h depicting various interventions the patient was shifted to intensive care unit (icu) 7 h after induction of anesthesia with noradrenaline infusion (0.12 g / kg / min) for elective ventilation and possible need for continuous renal replacement therapy to correct acidosis. abg analysis in the icu revealed progressively increasing la with hyperglycemia refractory to bicarbonate and insulin infusion [figure 1 ]. considering the possibility of sepsis, procalcitonin levels were obtained (14.6 ng / ml). suspecting thiamine deficiency, empirical thiamine 300 mg was administered intravenously 3 h after surgery. abg analysis 2 h after thiamine administration showed rapidly decreasing lactate and sugar levels with improvement in map. abg analysis, 8 h after the first dose of thiamine injection was within normal limits [table 1 ]. lactic acidosis is classified into two types : type a is caused by tissue hypoperfusion, hypoxia, etc., whereas type b is caused by hereditary metabolic diseases, drugs, systemic disorders (thiamine deficiency, liver / kidney failure, malignancy etc.,) and release of endogenous catecholamines from stress response. among all the above - mentioned causes, numerous case reports of unexplained la in patients who were on parenteral nutrition without thiamine supplementation have been published in the past. ckd, uremia and chronic hemodialysis have variable effects over thiamine activity and metabolism in the body : diminished intestinal and mitochondrial thiamine transporters in spite of normal serum thiamine levels, diminished transketolase (one of the enzymes requiring thiamine as a co - factor) activity, chronic hemodialysis itself can lead to accelerated removal of thiamine from the body. thiamine requirement increases during stressful periods such as critical illness and major surgery etc., which can precipitate symptoms of thiamine deficiency in such individuals. thiamine pyrophosphate is the cofactor for three important enzymes including pyruvate dehydrogenase (pdh). pdh catalyzes the oxidative decarboxylation of pyruvate to acetyl coenzyme a (coa) which enters the kreb 's cycle. in thiamine deficiency, when pyruvate can not undergo this conversion, it is then converted to lactate by the action of lactate dehydrogenase [figure 2 ]. this conversion also results in the release of protons in equal number to the molecules of lactate produced leading to acidemia. hyperglycemia can be an associated sign as excess lactate is metabolized by the liver into glucose through gluconeogenesis and thiamine deficiency per se can cause defective insulin secretion. lactate production and metabolism our patient was suffering from ckd and was on maintenance hemodialysis for 6 months, history and physical examination did not reveal the poor nutritional status or obvious signs and symptoms of thiamine deficiency. septicemia was ruled out as a probable cause as the patient was afebrile, normal total leukocyte counts and she did not have significant hypotension when the la was discovered. although her postoperative pro - calcitonin level was high, it is not diagnostic as she received t - cell antibody anti - thymocyte globulin as induction therapy intraoperatively. hence, acute thiamine deficiency was suspected, which was supported by dramatic improvement in her clinical and biochemical parameters after iv thiamine supplementation. probably, surgical stress stimulated accelerated glycolysis and enhanced pyruvate production, which increased her thiamine requirement unmasking occult thiamine deficiency and/or defective thiamine activity leading to severe la with hyperglycemia. intramuscular / iv thiamine 100 mg once a day is the recommended dose in suspected thiamine deficiency. we administered 300 mg of iv thiamine as the excess dose has no proven adverse effects in humans. if thiamine deficiency is suspected, erythrocyte transketolase activity and thiamine pyrophosphate effect can be measured for an objective documentation of the deficiency. we were not able to perform these biochemical tests as they were not available in local laboratories. however, the definitive test for vitamin b1 deficiency is an improvement of symptoms with thiamine administration. thiamine deficiency should be considered in the differential diagnosis of unexplained severe la with hyperglycemia in patients with ckd. physicians caring for these patients should maintain a low threshold for infusing a therapeutic dose of thiamine as treatment is safe, inexpensive, readily available and potentially life - saving. | a 48-year - old female patient with end - stage renal failure developed unexplained severe lactic acidosis (la) associated with hyperglycemia during robotic - assisted laparoscopic renal transplantation. initial treatment with sodium bicarbonate and insulin infusion were ineffective in treating acidemia. postoperatively, intravenous administration of thiamine resulted in rapid improvement of la and blood sugar levels. uremia and chronic hemodialysis might be the causes behind the quantitative / qualitative deficiency of thiamine unmasked during the surgical stress. though a rare entity, acute thiamine deficiency should be considered in the differential diagnosis of unexplained severe la in patients with chronic kidney disease and hemodialysis who undergo major surgery or admitted to critical illness care units. |
large airway obstruction may be due to intraluminal disease, stenosis, tracheobronchial collapse and extinsic compression. surgical resection and anastomosis is the accepted approach to short focal airway disease. in many cases this may not be feasible because of the site and extent of the stenosis, the cause of the underlying disease and the general state of the patient. nonsurgical palliative methods have been developed and include laser photocoagulation, balloon dilatation, cryotherapy, alcohol injection therapy and prosthesis. so, another palliative treatment method, self - expandable metallic stent, was used in the narrowed airway where surgical resection is inadvisable. this procedure had been also performed successfully in tuberculous bronchial stenosis that does not respond to medical therapy. after insertion of stents in the large airway, previous reported short - term complications are granuloma formation, stent migration, localized inflammation, fatal massive hemoptysis, wall perforation and respiratory distress. to our best knowlege, there was no report about long - term complication after stent insertion in tuberculous bronchial stenosis. a 29-year - old woman had had left pleuritic chest pain and dyspnea for 6 months. three years earlier, bronchoscopic examination revealed focal narrowing of the left main bronchus with severe fibrotic changes suggestive of sequelae of endobronchial tuberculosis. but her symptoms increased over the next 2 months, and it was believed that further treatment was needed for her stenosis. after the procedure, her clinical symptoms were improved. however, 30 months after stent placement, dyspnea on exertion developed and we noted obstruction at the site of the metallic stent by bronchoscopic examination. on physical examination, bronchoscopic examination revealed total obstruction of left main bronchus with granuloma - appeared polypoid mass and proximal portion of migrated metallic stents (fig. pulmonary function tests showed the forced expiratory volume in 1s (fev1) of 0.74l(26% of predicted), fev1/fvc 50%, the forced vital capacity (fvc) for 1.48l (41% of predicted). resection of the left lung, inclunding the stenotic segments of the left bronchus, was performed. after operation, her pulmonary function tests shows fev1, of 1.32l (48% of predicted), fev1/fvc 68%, fvc for 1.80l (50% of predicted). in resected specimen, biopsy finding of left main bronchus shows granulomatous inflammation by foreign body reaction which is composed of epithelioid cells and lymphocyte. at 6 months after the operation, techuiques used to treat bronchial stenosis include surgical resection, cryotherapy, laser photo - resection and balloon dilatation. balloon dilatation to treat tuberculous bronchial stenosis was first reported in english literature by nakamura. balloon dilatation is easier to perform, less invasive and less costly than surgery, but complications such as restenosis, bleeding, airway rupture and acquired bronchomalacia frequently occur. another palliative method is insertion of rigid silicone t tube (montgomery) but this type of prosthesis is poorly tolerated historically. the other palliative method, self - expandible prosthesis, nas introduced in the narrowed airway where surgical resection is inadvisable. after the first use of gianturco self - expanding metallic stents in the vascular and biliary systems, an expandable stainless steel stent was formulated for use in bronchogenic tumor, postoperative stenosis, tracheomalacia and airway collapse following tracheal reconstruction, relapsing polychondritis and stenosis in secondary lung transplantation. the stent can restore the patency of the airway promptly and supports the airway against increased thoracic pressure during expiration, in one physiolgic study, fvc increased fron 64% predicted to 73% predicted, fev1 from 49% predicted to 72% predicted, and the ratio of the fev1/fvc from 59% predicted to 78% predicted after tracheobronchial stents insertion. the advantage of the self - expandig metallic stent is that it can be used with the patient under local anesthesia, it is easy to perform and does not occlude tributaries of the airway, even if the stents override the opening of the branches of the airways. the complications are granuloma formation, stent migration, localized inflammation, dysphagia, suction catheter entrapment, fatal massive hemoptysis, rupture of the metallic mesh, obstruction, wall perforation and respiratory distress. the most frequently recovered isolates in granulation tissue were streptococci viridans, pseudomonas aeruginosa, nonhemolytic streptococcus, staphylococcus aureus. in tuberculous bronchial stenosis, expandable metallic stent is used if restenosis occurs after several attempts with balloon dilatation. the first case of successful tueatment with gianturco self - expanding metallic stents in tuberculous bronchial stenosis we had successfully treated one patient with tuberculous bronchial stenosis with a gianturco - type stent. the dyspnea on exertion was developed and we notified the obstruction at the site of the metallic stent by bronchoscopic examination. the self - expanding metallic stent was introduced as a good method in tuberculous bronchial stenosis. however, expandible metallic stent does not seem to be safe in the treatment of endobronchial tuberculosis in the long - term effect. | expandable metallic stents seemed to be a good method in tuberculous bronchial stenosis that does not respond to medical therapy. but there was no long - term follow - up study after stents insertion in tuberculous bronchial stenosis. we report a case of obstruction after successful gianturco metallic stents insertion due to tuberculous bronchial stenosis. |
hypomurocin (hm) peptides are the members of peptaibol family [13 ], which were originally isolated from the fungus hypocrea muroiana (synonymous to trichoderma atroviride), and they showed antibiotic and hemolytic activities. in a recent study, the hm molecules were also detected in other hypocrea spp., and based on the results, it was suggested that the production of peptaibols by trichoderma / hypocrea species may contribute to the colonization and defense of ecological niches for these fungi. based on their sequence length, the hypomurocins can be divided into two distinct groups, namely, the short - sequence hm a peptides composed of 11 amino acids and the long - sequence hm b peptides consisting of 18 amino acid residues. the former group of hm molecules covers six different peptides (see table 1), which contain a nonproteinogenic amino acid (i.e., -aminoisobutyric acid, aib), and additionally, one of them (i.e., hm a-2 molecule) contains a further nonproteinogenic residue (i.e., d - isovaline, d - iva). as it can be seen, the n - terminal amino acid of hm a peptides is acetylated, and an amino alcohol (i.e., leucinol, leuol) is linked by an amide bond at their c - terminal end. among the hm a molecules, for the hm a-1 peptide, the nmr measurement performed in dimethyl sulfoxide (dmso) revealed that this molecule adopted a mixed helical conformation, which contained - and 310-helices, as well as type i -turn structure. nevertheless, this nmr measurement led to the observation that the hm a-1 peptide could be characterized predominantly by 310-helical structure in the presence of micelles. in a subsequent nmr study, the structural features of hm a-3 and hm a-5 molecules were investigated in dmso solution, and based on the results, it was suggested that these peptides possessed also a mixed helical conformation containing not only - and 310-helical parts, but also type i -turn structure. in our theoretical study, a detailed conformational analysis was performed on all six hm a peptides, in order to identify their characteristic structural properties, focusing on the backbone and side - chain conformations, as well as on the evolving intramolecular h - bonds. the hm a molecules contain nonproteinogenic amino acids, such as aib and d - iva residues, and furthermore, they contain an amino alcohol, that is, leuol ; thus these nonstandard residues were parameterized by means of quantum chemical calculations. for the parameterization, the second - order mller - plesset perturbation method and the 6 - 311 g basis set were applied. in order to derive the partial atomic charges for the above - mentioned nonstandard residues, the restrained electrostatic potential (resp) method was used. to perform the conformational analysis of hm a peptides, the simulated annealing (sa) method was applied, as it was previously used for the short - sequence trichobrachin molecules. the sa calculations were carried out with the amber 9 program, in the course of which the amber 99sb force field and the generalized born implicit solvent model [1113 ] were applied, and no - cutoff was used for the nonbonding interactions. in the case of each hm a molecule, first of all an initial energy - minimization was performed, and using this geometrically optimized structure, the sa procedure composed of three consecutive stages was applied, as follows : (1) heating to 1000 k for 1000 fs, (2) equilibration at 1000 k during 4000 fs, and (3) cooling from 1000 k to 50 k for 10000 fs. for the cooling stage, a near - exponential protocol was used, which consisted of the following linear phases, successively : (1) from 1000 k to 500 k during 1000 fs, (2) from 500 k to 200 k for 2000 fs, (3) from 200 k to 50 k during 7000 fs. the sa procedure, composed of heating, equilibration, and cooling stages, was carried out 1000 times ; thus 1000 conformers were obtained in the case of each peptide, for which a final energy - minimization was performed. this geometry optimization was carried out by the steepest descent method applied for the first 100 steps, which was followed by the conjugated gradient method, using the gradient convergence criterion of 0.001 kcal mol and the maximum number of iterations of 10000. in order to identify the characteristic structural features of hm a peptides, the backbone and side - chain conformations were studied, and the evolving intramolecular h - bonds were investigated, based on the conformers derived from the sa calculations. to characterize the backbone conformations of hm a molecules, the presence of types i and iii -turns was examined along the entire sequence of peptides. for the determination of -turn structures, the typical ranges of and torsion angles with regard to the i + 1th and i + 2th residues of a tetrapeptide unit were used [14, 15 ]. they were as follows : (1) in the case of type i -turn : i+1 = 60 30 and i+1 = 30 30, i+2 = 90 30 and i+2 = 0 30 ; (2) in the case of type iii -turn : i+1 = 60 30 and i+1 = 30 30, i+2 = 60 30 and i+2 = 30 30. the populations of types i and iii -turns identified in certain tetrapeptide units of hm a molecules are represented in table 2 for the conformers obtained by the sa simulations. based on these results, it could be concluded that types i and iii -turns could be detected for almost all tetrapeptide units of each molecule. in the case of five hm a peptides (i.e., hm a-1, hm a-3, hm a-4, hm a-5, and hm a-5a), a similar conformational pattern could be observed with regard to the populations of -turn structures. accordingly, for the gln xaa xaa aib and pro leu xaa aib tetrapeptide segments, larger amount of types i and iii -turns could be found, in comparison with those detected in the case of other four tetrapeptide units (i.e., xaa xaa, xaa aib pro leu, aib pro leu xaa, and xaa aib pro leuol). on the basis of data shown in table 2, it could be seen that, for the hm a-2 molecule, the relationship between the populations of -turn structures was the opposite, considering the above - mentioned two groups of tetrapeptide segments. consequently, types i and iii -turns appeared with lower frequency in the case of former two tetrapeptide units than for the latter four tetrapeptide segments. nevertheless, taking into account the different tetrapeptide units, it could be seen that, in the case of hm a-2 molecule, the amount of -turn structures found in the gln xaa xaa aib and pro leu xaa aib segments were lower, while the populations of types i and iii -turns observed in the xaa leu, aib pro leu xaa and xaa aib pro leuol units were larger, as compared to those detected in the corresponding tetrapeptide segments of other five hm a molecules. on the whole, these results indicated that the hm a peptides could be characterized by types i and iii -turn structures. in order to further describe the backbone conformations of hm a molecules, the occurrence of 310- and -helical conformations was also investigated. to identify these helical structures, the following characteristic ranges of and torsion angles were applied : (1) for the 310-helix, = 60 30 and = 30 30 ; (2) for the -helix, = 60 30 and = 50 30. in the case of two types of the helical conformations mentioned above, the typical ranges of and torsion angles overlap with each other ; thus the cumulative helical content was also calculated, using the combined ranges of and torsion angles characteristic to the 310- and -helices. in order to characterize the evolving helical structures, the percentages of helical contents (i.e., helicities) were calculated for each conformer derived from the sa calculations, applying the following formula [17, 18 ] : (1)f = nhn100, where f is the helicity and nh is the number of amino acids satisfying the torsion angle criteria for the 310- and -helical conformations and the cumulative helical content, respectively. for simplicity, in this study, n is not equal to the number of all amino acids (i.e., 11 residues) found in the sequences of hm a peptides, as it was previously used [17, 18 ]. in the present study, the n = 10 was applied, and the reasons for this were as follows : (1) the short sequence length of hm a molecules ; (2) the torsion angle could not be defined in the case of leuol residue. based on the conformers obtained by the sa simulations, the distributions of helicity values were calculated, and the populations of conformers characterized by certain helical contents were determined. as a representative sample, the distributions of helicity values are illustrated in figure 1 for the hm a-1 peptide, and these distributions were found to be similar in the case of other five molecules. nevertheless, the populations of conformers possessing different helical contents are shown in table 3 for each hm a peptide. since the values regarding the cumulative helical content proved to be equal to the values concerning the 310-helical content, the distribution of these helicity values was not demonstrated in figure 1, and these helicity values were not included in table 3. as the distribution plot and the values with regard to the 310- and -helical contents indicated, differences could be observed between these two types of helicities. accordingly, larger populations of conformers characterized by at least 30% helicity could be detected for the 310-helical content, as compared to those found in the case of -helical content. moreover, on the basis of data presented in table 3, it could be concluded that a similar distribution of helicity values could be observed for all the hm a molecules, taking into account the 310- and -helical contents, respectively. on the basis of these results, it could be suggested that the hm a peptides showed a preference for the 310-helical structure over the -helical conformation. for the side - chains of amino acids, except for the aib and pro residues, the proportions of three rotamer states (i.e., g(+), g(), and trans) were determined (see table 4), and the preferred rotamers were identified, on the basis of conformers derived from the sa calculations. the side - chain of the d - iva amino acid of hm a-2 peptide showed a preference for the g(+) rotamer over the other two rotamer states ; nevertheless, the ratios of g() and trans rotamers proved to be similar. in contrast, for the side - chains of gln, val, and val residues, mainly the g() rotamer state was favored, and similar proportions of g(+) and trans rotamers could be observed in the majority of cases. for the side - chains of ile, ile, and ile amino acids, a decreasing tendency could be detected with respect to the ratios of three rotamer states, as follows : g(+) > trans > g(). in the case of the side - chains of leu, leu, and leu residues, similar proportions of g() and trans rotamers could be observed in the majority of cases, and both of them were found to be preferred over the g(+) rotamer state. for the side - chains of leuol amino acids, large, moderate, and small ratios were identified concerning the g(), trans, and g(+) rotamers, respectively. since various intramolecular h - bonds could contribute to the stability of the conformational states of peptides, the appearance of h - bonds evolved between the backbone nh donor and co acceptor groups was investigated. among them, the i i + 3 h - bonds formed between a nh group of i + 3th and a co group of ith amino acids play an important role in the structural stabilization of -turns, as well as of 310-helical conformation. the other type of intramolecular h - bonds, such as the i i + 4 h - bonds formed between a nh group of i + 4th and a co group of ith residues, contributes to the structural stability of -helical conformation. the occurrence of i i + 3 and i i + 4 h - bonds was examined along the entire sequence of hm a peptides. an intramolecular h - bond was assumed to exist if the n o distance between the n atom of nh donor group and the o atom of co acceptor group was within 3.5, and if the n h o angle subtended at the h atom by the bond to the n atom and the line joining the h and o atoms was larger than 120. the populations of the different types of i i + 3 and i i + 4 h - bonds observed for the conformers of each hm a molecule are represented in table 5. these results indicated that the i i + 3 h - bonds appeared in all the tetrapeptide units, for which types i and iii -turns were identified. the i i + 4 h - bonds were also detected along the entire sequence of molecules ; however, their populations proved to be much smaller, as compared to those in the case of i i + 3 h - bonds. based on these results, it could be also suggested that the hm a peptides could be characterized by -turn or 310-helical structure rather than by -helical conformation. in the present study, a conformational analysis was performed for all the hm a peptides, and the structural properties of these short - sequence peptaibols were characterized comprehensively. taking into account the backbone conformations, types i and iii -turns were identified in certain tetrapeptide units of each hm a molecule, and additionally, the 310- and -helical contents were determined. these results indicated that the hm a peptides could be characterized by -turn structures or 310-helical conformation rather than by -helical structure. nevertheless, the side - chain conformations of amino acids were investigated, and the ratios of three rotamer states, as well as the preferred rotamers, were identified. for the hm a peptides, the appearance of i i + 3 and i i + 4 h - bonds was examined, and the results led to the conclusion that the i i + 3 h - bonds could contribute to the stability of -turn and 310-helical structures. as it was mentioned previously, based on the data derived from the nmr measurements, it was suggested that the hm a-1, hm a-3, and hm a-5 peptides adopted a mixed helical conformation, for which - and 310-helices, as well as type i -turn structure, appeared [6, 7 ]. the results obtained by our structural investigation indicated that the hm a molecules could be characterized by types i and iii -turns or 310-helical conformation, and these peptides showed a preference for these two secondary structural elements over the -helical structure. in summary, our theoretical study provided a detailed description of the three - dimensional structure of short - sequence hm a peptides. | in this theoretical study, a conformational analysis was performed on short - sequence hypomurocin a peptides, in order to identify their characteristic structural properties. for each hypomurocin a molecule, not only the backbone conformations, but also the side - chain conformations were examined. the results indicated that certain tetrapeptide units could be characterized by types i and iii -turn structures, and considering the helical conformations, it could be concluded that the hypomurocin a peptides showed a preference for the 310-helical structure over the -helical structure. beside the backbone conformations, the side - chain conformations were investigated, and the preferred rotamer states of the side - chains of amino acids were determined. furthermore, the occurrence of i i + 3 and i i + 4 intramolecular h - bonds was studied, which could play a role in the structural stabilization of -turns and helical conformations. on the whole, our theoretical study supplied a comprehensive characterization of the three - dimensional structure of short - sequence hypomurocin a peptides. |
phyllodes tumor of the breast accounts for 0.3%1% of all primary breast tumors and for 2.5% of all fibroepithelial tumors of the breast. they are circumscribed biphasic fibroepithelial tumors, with classic histology showing hypercellular mesenchymal stroma and double - layered epithelium in clefts. based on stromal cellularity, atypia, mitosis and nature of tumor margins, phyllodes tumor is classified into benign, borderline, and malignant. coexistence of invasive carcinoma in the proximity of malignant phyllodes tumor as synchronous collision tumors and skin ulceration by malignant phyllodes tumor is extremely rare. we report one such case in a 51-year - old female presenting as a fungating mass. a 51-year - old female presented with a history of breast lump for past 6 months. there was a history of rapid increase in size with pain and ulceration for 1 month. there was no history of breast malignancy in the family. on examination, left breast showed a huge foul smelling fungating mass with absent nipple and areola, occupying almost entire left breast. positron emission tomography scan revealed large hypermetabolic mass in the left breast and few hypermetabolic lymph nodes in the left axilla. there was no evidence of metastasis elsewhere in the body. with the diagnosis of phyllodes tumor probably malignant with skin ulceration, mastectomy with axillary dissection an another firm tumor measuring 2.5 cm with spiculated margin was seen at the periphery of large tumor, abutting it [figure 1 ]. histology of the larger tumor showed stromal predominance occupying a low power field [figure 2 ], increased cellularity, spindle cells with moderate atypia, and increased mitosis [figure 3 ] of 13/10 high power field (hpf)., an epithelial tumor is seen without transition zone in between [figure 4 ], consists of cells in tubules and trabeculae with stromal desmoplasiar [figure 5 ]. a diagnosis of malignant phyllodes tumor with ulceration with coexisting invasive carcinoma of no special type was rendered. gross specimen showing large lobulated phyllodes tumor (gray arrow) and a spiculated tumor at its proximity (black arrow) low - power view of phyllodes tumor showing stromal predominance and epithelial cleft (h and e, 10) phyllodes tumor showing hypercellular stroma and increased mitosis (arrows) (h and e, 40) phyllodes tumor merging with invasive carcinoma of no special type (h and e, 10) invasive carcinoma showing tumor cells in nests and tubules (h and e, 40) phyllodes tumor is a biphasic fibroepithelial tumor of the breast, accounting for 0.3%1% of all primary breast tumors and for 2.5% of all fibroepithelial tumors of the breast. clinically, patients present with unilateral firm breast mass, not attached to skin. large tumors might stretch the skin, but ulceration is very rare (seen in our case). phyllodes tumor is classified based on stromal cellularity, stromal atypia, stromal overgrowth, mitosis and tumor margins into benign, borderline, and malignant. any phyllodes tumor can harbor ductal carcinoma in situ or invasive carcinoma, although it is an uncommon finding. the epithelial tumor can be seen either with or outside the phyllodes tumor, synchronously, or metachronously, in the same or distinct breast. very rare cases of malignant phyllodes tumor with coexisting invasive carcinoma outside the phyllodes tumor in the same breast were described. our patient had invasive carcinoma in the proximity of phyllodes tumor without a transition zone synchronously in the same breast as a collision tumor. genetic changes with intratumoral heterogeneity have been reported, which might lead to the transformation of epithelial and mesenchymal elements, resulting in the development of phyllodes tumor and the association of breast carcinoma inside the phyllodes tumor. the coexistence of both tumors can be made out by meticulous examination of the specimen and extensive sampling. phyllodes tumor have homogenous surface with lobulated margins, while breast carcinoma usually has firm consistency with spiculated margins. histologically, malignant phyllodes tumor reveal stromal overgrowth occupying more than a low - power field, hypercellularity, stromal atypia, mitosis of more than 10/10 hpf, and invasive margins local recurrence is usual and can metastasize to lungs, liver, and bone by hematogenous spread. it is graded based on the amount of tubule formation, nuclear atypia, and mitosis per 10 hpf. it usually metastasize to axillary lymph nodes, and to other organs such as lungs and bone. the behavior of the tumor and treatment depends on the higher grade of either of the tumors, mitotic activity, resected margin status, skin involvement, and metastasis. malignant phyllodes tumor with coexisting invasive breast carcinoma in the same breast outside the phyllodes tumor is a rare occurrence. there were two case reports of malignant phyllodes tumor ulcerating the skin with coexisting invasive breast carcinoma as collision tumor and presenting as a large fungating mass in the english literature. early diagnosis and complete surgical removal prevent the recurrence of such tumors and improve patient outcome. treatment of these rare cases has to be standardized with a multimodal approach, including complete surgical removal with axillary clearance, and adjuvant treatment with chemotherapy and radiotherapy as well. | phyllodes tumor is a rare fibroepithelial biphasic tumor of the breast composed of hypercellular mesenchymal stroma and double - layered epithelial component, arranged in clefts with leaf - like projections. phyllodes tumor with coincidental presence of invasive carcinoma or in situ ductal carcinoma in the same or distinct breast is a rare occurrence, documented by some reports. invasive carcinoma can be seen within or outside the phyllodes tumor. skin ulceration by malignant phyllodes tumor with coexisting invasive carcinoma as collision tumor is extremely rare. here, we report an extremely rare presentation of malignant phyllodes tumor as a giant fungating mass with distinct invasive carcinoma in the same breast in a 51-year - old female. |
dermatophytes, yeasts, and moulds are all potential causes on onychomycosis worldwide. often, non - dermatophytes are considered contaminants or secondary pathogens in onychomycosis, invading the already damaged by trauma or disease. onychomycosis is a frequent clinical entity encountered in dermatological practice worldwide. accounting for about 50% of nail infections, its incidence is rising owing to a number of factors including an ageing population and expanding number of immunocompromised patients. the clinical picture is characterized by alterations in nail architecture like changes in color, thickness, onycholysis, and onychodystrophy. the main types are distal and lateral subungual onychomycosis, superficial onychomycosis, proximal subungual onychomycosis, endonyx onychomycosis, and total dystrophic onychomycosis. in addition although not life - threatening, patients with onychomycosis suffer from serious physical, psychosocial, and occupational effects. most commonly, onychomycosis is caused by various species of filamentous fungi like the dermatophytes or yeasts of the genus candida. however, non - dermatophytic molds with known habitats mostly in soil and decaying plant debris are now being increasingly recognized as pathogens in fungal nail infection. prevalence rates of onychomycoses caused by non - dermatophyte molds range between 1.45% and 17.60%. the most common non - dermatophytes molds causing nail disease are scopulariopsis, scytalidium, fusarium, aspergillus, and onychocola canadensis. onychomycosis due to dermatophytes and non - dermatophytes is clinically indistinguishable, hence underlining the need of relevant laboratory investigations before starting the treatment. a 35-year - old man, laborer by occupation, presented with discolored and thickened fingernails of both the hands. he gave a history of loss of weight for the past 1 year and development of multiple crusted to scaly, erythematous papules and plaques all over the trunk, both arms, and lower limbs. about 2 - 3 months after the appearance of skin lesions, he noticed discoloration and brittleness of the nail of little finger of the left hand. gradually, the rest of the finger nails of both the hands got involved. cutaneous examination revealed hyperpigmented macules, scaly erythematous papules, and few lichenoid plaques with adherent scales diffusely present over the trunk and extremities. the fingernails showed yellowish discoloration, subungual hyperkeratosis, and pitting shown in clinical figures 1 and 2. with this clinical history and physical examination, possibility of underlying immunosuppression the patient was advised for hiv testing, which revealed that he was hiv - positive. he was started on anti - retroviral therapy (art) at the art clinic and referred back to the dermatology department for management of skin lesions. further, he was sent for histopathological investigations, which demonstrated psoriasiform hyperplasia of the epidermis, focally diminished granular layer, suprapapillary thinning and congested vessels in the dermis. koh examination of the scrapings revealed wide, ribbon - like hyphae typical for zygomycetae. slide culture revealed non - septate broad hyphae with internodal rhizoids, irregularly branched sporangiophores with sporangia at their apices. after the first diagnosis, a second sample was taken, the laboratory procedures were repeated, and the diagnosis was confirmed. clinician dermatologists were informed about the diagnosis, and the patient was started on fluconazole. it is a saprophytic fungi, found in soil and decaying vegetation and isolated from environmental sources worldwide. limited available in vitro susceptibility testing demonstrates r. pusillus susceptibility to amphotericin b but resistance to flucytosine, fluconazole, miconazole, and ketoconazole. only 6 of the 19 patients with rhizomucor infection described in the literature have responded to treatment. however, there has been no known case report on onychomycosis caused by rhizomucor species. in our patient, ribbon - like hyphae in direct koh mount and microscopic morphology of the cultured fungus consistent with rhizomucor was found. same findings obtained on mycological investigations conducted on two different occasions in the absence of dermatophyte growth suggest that rhizomucor is the causative agent of nail disease in this patient. dystrophic psoriatic nails are easier for the fungi to penetrate as the nail plate is already compromised, hence there is a higher prevalence of onychomycosis in psoriatics compared with non - psoriatics. both these factors could have been involved in the occurrence of fungal nail infection by rhizomucor, a non - dermatophytic mold. to the best of our knowledge, there has been no data on onychomycosis due to rhizomucor species, although it has been reported to cause systemic infection in immunocompromised individuals. this unique case report underlines the fact that fungal species appearing as environmental contaminants in the nail samples should be further investigated with suspicion in view of appropriate clinical history and examination. non - dermatophytic molds must be kept as one of the differential diagnosis while investigating and treating a case of onychomycosis and the common practice of discarding them, considering them to be contaminants should be avoided. regarding laboratory diagnosis, it is extremely important to confirm if the fungus is real etiological agent by repetition of the tests on a new collected sample. | onychomycosis is frequently seen in dermatological clinical practice worldwide. the causative agents are usually two pathogenic groups of fungi namely, dermatophytes and yeasts of the genus candida. in some cases, non - dermatophytic molds belonging to different genera and species may be the etiological agents. we report an unusual case of onychomycosis in an hiv - positive psoriatic patient caused by rhizomucor pusillus, which has not been mentioned in the literature before. our finding underline the fact that fungal species appearing as contaminants should be evaluated by proper clinical - mycological correlation to ensure an accurate diagnosis. |
altman and das (1965) proposed in 1965 the revolutionary idea that new neurons are born in the adult brain. for decades, the concept has been ignored until it was rediscovered in the early 1990s. it is now well accepted that new neurons are born throughout life, even in humans (eriksson., 1998 ; jin., 2004 ; d'alessio., 2010) and in aged animals (kuhn., 1996 ; kempermann., adult neurogenesis has been clearly demonstrated and confirmed in two brain regions : the subventricular zone (svz) of the lateral ventricles and the subgranular zone (sgz) of the dentate gyrus (dg) of the hippocampal formation (kempermann and gage, 1999). cells born in the adult svz migrate through the rostral migratory stream and become granule neurons and periglomerular neurons in the olfactory bulb. cells born in the adult sgz migrate into the granule cell layer of the dg and become dentate granule cells (dgc). it is only in the last decade that hippocampal adult neurogenesis has become a hot topic. many studies have been designed to characterize these newly generated neuronal cells (kempermann., 2004 ; zhao., 2006), to unravel the regulation of the different maturation stages (zhao., 2008) and to unveil the potential meaning of this phenomenon (zhao., 2008 ; the newly generated cells undergo different maturation stages to become functional mature neurons. as a consequence of neurons being born in a continuous manner, the dg is composed of a heterogeneous population of dividing and non - dividing, immature and mature, neuronal and non - neuronal cells. (2004) have developed a model of six developmental milestones of hippocampal adult neurogenesis based on their basic morphology and the combinatorial expression of six fairly stage - specific markers. they are characterized by their radial process extending to the inner molecular layer where they ramify, and by the expression of both nestin and glial fibrillary acidic protein (gfap). nestin - positive cells that do not express gfap nor doublecortin are classified as type-2a putative progenitor cells. doublecortin is indeed expressed in type-2b, type-3 cells, both putative progenitor cells, and by immature granule cells. postmitotic immature neurons are further characterized by the emergence of dendrites and the coexpression of calretinin or neun. finally, mature dgc have developed a dendritic arborization and are calbindin- and neun - positive. recent studies have suggested that the electrophysiological and morphological properties of the newborn cells gradually emerge during neuronal maturation. these properties are needed by the young neurons for successful integration into the existing synaptic circuits (zhao., 2006, 2008 ; the idea that new neurons are needed to build new memories has been quick to emerge. the concept implies hippocampal adult neurogenesis as being a new form of structural plasticity correlated to the processes of synaptic learning and memory formation. should this be the case, animals with impaired neurogenesis should, compared to wildtype animals, show alterations in acquisition, retention, recall or extinction of some kinds of information like spatial or contextual information. 2010) have used the synras transgenic mouse model in order to better understand the role of adult neurogenesis in learning and memory. the mouse has a synapsin1 promoter - driven overexpression of the constitutively activated g - protein p21ras in neurons. intriguingly, this mouse has significantly depressed rates of hippocampal adult neurogenesis, and this is associated with impaired performance in an object recognition task (lafenetre., 2010). both, impaired neurogenesis and impaired object recognition could however be rescued by exposing synras mice to free access to a running wheel (lafenetre., 2010 ; proliferation, assessed by the number of bromodeoxyuridine (brdu)-labeled cells, brain - derived neurotrophic factor levels, dendritic arborization of doublecortin - labeled cells and object recognition performance have been studied in wildtype (wt) and transgenic (synras) mice under basal conditions and with free access to a running wheel. (2010), starting by presenting the regulation of adult neurogenesis by physical activity as an epigenetic factor and the possible implication of neurotrophins as molecular mediators. adult neurogenesis has been shown to be a dynamic process that can be regulated both positively and negatively by many factors, including epigenetic factors. indeed, in the last decades, five of them have clearly been identified and studied. whereas stress and aging downregulate adult neurogenesis, physical activity, environmental enrichment, and learning and memory have beneficial effects (figure 1). regulation of adult neurogenesis can modulate learning and memory. whereas aging and stress downregulate adult neurogenesis (pink arrows), experience, enriched environment and physical activity (blue arrows) stimulate the generation of new neurons. (2010) to rescue the reduced adult neurogenesis of the synras mice due to reduced proliferation. voluntary physical activity is indeed the most potent enhancer of adult proliferation (van praag., 1999a ; olson., 2006 ; fabel and kempermann, 2008). the increase in neurogenesis is however region - specific and occurs only in the hippocampus and does not stimulate the olfactory bulb neurogenesis (brown., however, the modalities could vary from one experiment to another and could induce differential effects. first, the access to the wheel could be free or restricted to some hours per day or night (holmes., 2004). second, running is limited to physical activity when rodents are housed individually but could also be considered as a social activity when the animals are housed per group. indeed, when rodents are isolated, the effects may be delayed or prevented (stranahan., 2006 ; leasure and decker, 2009) but this is still under debate (kannangara., 2009, 2010). thirdly, grouped animals could use the running wheel according to two modes : some are very active and seem to become addicted to running whereas other individuals are passive users. in the study by lafenetre. (2010), grouped mice had free access to the running wheel : this paradigm gave the greatest opportunity to enhance adult neurogenesis. 2010) have thus confirmed the positive regulation of adult neurogenesis by physical activity that had been described earlier in wild type rodents (van praag. this paradigm is also efficient in stimulating the neurogenesis of synras mice characterized by a reduced basal adult neurogenesis in the hippocampus (figure 2 ; lafenetre., 2010 ; manns., the ways by which physical activity promotes proliferation must thus differ from the regulation of proliferation in basal conditions (lafenetre., 2010). it has earlier been shown that neurogenesis could be partially rescued in aged animals by voluntary physical activity. this also suggests the regulatory mechanisms could be modified during aging but that the cells could still respond to external stimuli (van praag. doublecortin - labeled cells in the dentate gyrus of wildtype controls (upper left), wildtype runners (upper right), synras controls (bottom left), and synras runners (bottom right). wheel running has also been shown to improve hippocampal - dependent spatial learning in rodents in the morris water maze (fordyce and farrar, 1991 ; van praag., 1999a ; vaynman., 2004) and in the radial maze (anderson., 2000). other tasks like contextual fear conditioning have also been used to show the running - induced improvement in cognitive performance (baruch., the beneficial effect of physical activity could be mediated by increased synaptic plasticity (van praag., 1999b ; farmer., 2004), neurotransmission and growth factor expression (cotman and berchtold, 2002) that are observed both in running mice and rats. in their study, lafenetre., (2010) have focused on the involvement of brain - derived neurotrophic factor (bdnf) as one of the potential mediators of these effects. brain - derived neurotrophic factor is a neurotrophin that is highly expressed in the hippocampus and has been implicated in synaptic plasticity and neuronal development (binder and scharfman, 2004). bdnf is known for its survival - promoting effects on new neuroblasts through the trkb receptor (bath., 2008). environmental factors like environmental enrichment and physical activity induce an increase in bdnf expression level even after a short exposure to a running wheel (cotman and berchtold, 2002). moreover, running wheel activity increases the levels of the phosphorylated form of the bdnf receptor trkb (vaynman., 2003). the functional role of bdnf in the regulation of the hippocampal adult neurogenesis is quite controversial. 2010) showed a running - induced increase in bdnf protein level that was associated with increased level of bromodeoxyuridine (brdu)-labeled cells and doublecortin - labeled cells in both wildtype and synras mice. bdnf acts through the trkb receptor which is present on doublecortin - expressing cells (donovan., 2008 ; lafenetre., 2010 the authors thus suggested that bdnf could not only promote the proliferation of doublecortin - expressing neuronal precursor cells but also stimulate the dendritic arborization of the immature neurons and facilitate their survival (figure 3). free and social access to the running wheel would induce an increase in bdnf release that could act on (1) the proliferation of doublecortin - immunoreactive precursor cells, (2) on the dendritic arborization of doublecortin - immunoreactive immature neurons, stimulating their maturation and their differentiation, (3) on the selective recruitment of immature cells. the infusion of bdnf for 2 weeks directly into the hippocampus increases neurogenesis of granule cells (scharfman., 2005). however, classical genetic studies manipulating directly the expression of bdnf do not lead to such clear results. indeed, enhanced and reduced cell proliferation in heterogeneous bdnf knockout (bdnf+/) mice have been reported (lee., 2002 ; sairanen., the survival rate of the neurons must however be dependent on bdnf expression as impaired levels of cell survival were observed in both studies (lee., 2002 ; sairanen., 2005). moreover, enriched environment does not enhance the survival of newborn cells in bdnf+/ heterogenous knockout mice (rossi., 2006). in accordance with the role of bdnf in promoting the dendritic differentiation of mature neurons (mcallister., 1996 ; wirth., 2003), the dendritic arborization of the dgc is more developed in bdnf - overexpressing transgenic mice (tolwani., 2002). in a recent study, the role of bdnf in different stages of adult neurogenesis has been assessed in conditional knockout mice that lack the expression of bdnf in mature neurons of the adult hippocampus, resulting in 50% of the bdnf levels. bdnf has been suggested to play a critical role in the regulation of the survival and of the dendritic development of neuronal precursor cells but seems less important for exercise - induced proliferation of the cells (choi., 2009). the conditional deletion of the trkb receptor in progenitor cells leads to impaired basic organization of synaptic connections and compromises the survival and the integration of the newborn neurons (bergami., thus, whereas the role of bdnf in the proliferation of neuronal progenitor cells is still unclear, the survival and the integration of these newborn neurons rely on the good functioning of bdnf / trkb signaling. due to the cellular heterogeneity of the dg, the understanding of the role of bdnf and other growth and neurotrophic factors, like vegf, igf1, erythropoietin, remains difficult. presumably, they have to be tightly regulated and can act differentially on different developmental stages of the various cellular subsets. the functionality of the newly generated neurons has remained incompletely understood. with regard to the well - characterized role of the hippocampus in learning and memory, hippocampal adult neurogenesis has been proposed to be a new form of plasticity that underlies these processes. three technical approaches have been used to study the involvement of the newly generated neurons in learning and memory processes : the neural progenitor cells are ablated by pharmacological treatment, by irradiation or by genetic tools.. injections of the dna methylating agents, methylazoxymethanol (mam), or more recently of temozolomide (tmz), significantly reduce the rate of adult neurogenesis. x - ray irradiation has also been a very powerful, even stronger, tool. however, these techniques may induce side - effects that could cause other detrimental effects on brain physiology and function (bruel - jungerman., 2007). with the generation of new genetic tools, it has been possible to specifically target the neural progenitor cells in the hippocampus and better assess the role of the new neurons in learning and memory processes. besides, the use of other transgenic mice has helped in the better characterization of some molecules that could be involved in the regulation of adult neurogenesis. as in lafenetre., (2010), many studies have used environmental stimuli that could affect both, the animals behavior and adult neurogenesis (figure 3). (2010) have recently reviewed the different behavioral tasks that have been performed with animals subjected to an ablation of progenitor cells. for instance, the involvement of adult neurogenesis in the acquisition of the morris water maze test, the most commonly used paradigm to test hippocampal functions, has been reported by dupret. by contrast, other studies have reported that adult neurogenesis is not required for the acquisition of the morris water maze test (shors., 2002 ; madsen. similarly, is adult neurogenesis necessary for long - term retention of the location of the hidden platform ? when progenitor cells are ablated by irradiation (rat ; snyder., 2005), viral disruption of the wnt signaling (rat ; jessberger., 2009) and genetic disruption (mouse ; dupret., 2008 ; however, the retention is only affected 1 week after the viral injection in the genetic model of deng., (2009) and only the retention of the reversal is affected by tmz treatment (garthe., 2009). other studies have not found any positive correlation between reduced adult neurogenesis and impaired cognitive performance in the morris water maze (shors., 2002 ; meshi., 2006 ; saxe., 2006) and it has even been revealed that inhibiting adult neurogenesis could lead to better learning and memory performances (kerr., 2010). similar contradictory results have been obtained in the contextual fear conditioning paradigm which is also a key test to assess the role of the hippocampus. some studies have found deficits in freezing behavior (saxe., 2006 ; winocur., 2008 ; hernandez - rabaza., 2009 ; snyder., 2009) but no effect on freezing were reporting by others (dupret., 2008 ; zhang., 2008 ; snyder., while specific effects on the formation but not the extinction of contextual fear memory have been found in strongly irradiated mice, mice that have received mam injections (ko., 2009) or mice that have been irradiated to a lesser extent (kitamura., 2009 ; ko., 2009) are not impaired. by contrast, in nestin - tk mice with ablation of actively dividing progenitor cells by ganciclovir, impairment in freezing or fear conditioning is not observed, yet they are not able to extinguish their fear response to the context as efficiently as the wildtype mice (deng., 2009). the synras mouse has a reduced adult neurogenesis and is impaired in exploring and discriminating a novel object compared to a familiar object. however, stimulating adult neurogenesis by physical activity rescues these deficits (table 1). similar deficits have been observed in rats that have been injected with a lentiviral virus targeting the wnt signaling (jessberger., 2009). several other studies employing hippocampus - dependent or independent tasks have also led to inconclusive results (see deng., 2010 for review). recent studies have shown how dramatic hippocampal activity varies between standard laboratory mouse lines including those used for genetic engineering. electrical activity of neuronal networks is a driving force, e.g., for the production, release and action of bdnf, the structural differentiation of neurons and the ability to form meaningful circuits. in particular, mouse lines differ in the patterns of spontaneous and evoked gamma oscillations, which are considered to be central to cognitive performance (jansen., 2009). strains also differ in the rates of proliferation and/or survival of newly formed cells (kempermann and gage, 2002) and this correlates with deficits in learning tasks (kempermann., 1998b). even substrains of the frequently used c57 strain differ dramatically in many behavioral aspects (siegmund., 2005 ; matsuo., the different ablation protocols may also affect different populations of cells that would be more vulnerable according to their maturation stage (deng., 2010). equally important are the collateral side - effects, the parameters used to assess the different phenotypes, the cognitive demand of the test, age, sex, and presumably the life history of the individual animals. it is thus difficult to integrate the results of different studies with so many variables. it would thus be needed to standardize protocols to target stage - specific cells in specific conditions. an often overlooked aspect is that most experimental paradigms are primarily designed to assess the role of the hippocampus with all its input, intrinsic, and output networks. are the tests adequate for understanding the role of the newly generated granule cells in the dg ? of course, the dg is considered as the information gateway to the hippocampus and has been specifically shown to be involved in pattern separation (gilbert., 2001 ; computational models based on this function of the dg have been proposed for explaining either the addition of the new neurons in the network or the replacement of old dgc by these new neurons (aimone., 2009, 2010 ; new born cells would thus help avoiding interferences between new and already established memories and promoting the individuals behavioral adaptation. studies directly assessing this role are thus needed to better understand the real involvement of adult neurogenesis in learning and memory processes. according to a recent study, the newly generated cells in the dg must have a functional implication in pattern separation only when stimuli are presented with little separation in space, but not when they are widely separated (clelland., 2009). similarly, voluntary running improves the performance of the subtle discrimination of the location of two adjacent identical stimuli, which is tightly correlated to adult neurogenesis (creer. adult new neurons may thus be important for pattern separation and for encoding fine spatial distinctions (clelland. lafenetre., (2010) have been able to show that voluntary physical activity could rescue two main phenotypes, the reduced adult neurogenesis and the impaired performance in an object recognition test, of a genetically modified mouse via an increase of bdnf. these results thus support the idea that adult neurogenesis is a dynamic process that is under the influence of environmental changes and that this form of plasticity is regulated by neurotrophic factors like bdnf. epigenetic factors, physical activity, enriched environment, aging, stress, and learning induce changes in both, the rate of adult neurogenesis and the behavior of the individual. however, the causal relationships are still not clearly determined and the role of adult neurogenesis in learning and memory processes remain obscure due to the many discrepant results. more studies are needed to better determine the effects of the various extrinsic factors on the progenitors and the subsequent stages of differentiation of the newly generated neurons, and their ability to integrate into the adult dg. furthermore, the functional involvement of the newly generated granule cells must be further studied in the restricted context of the direct role of the dg. computational models will thus help in elaborating theories that could be tested with animal models. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. | neurogenesis occurs in two neurogenic zones in the adult brain : new neurons are born at the subventricular zone of the lateral ventricles and then migrate to the olfactory bulb, and at the subgranular zone to integrate the granular cell layer of the dentate gyrus. the hippocampus is involved in learning and memory and the generation of new hippocampal neurons has been suggested to be a new form of plasticity implicated in these processes. in the last decades, diverse intrinsic and epigenetic factors have been identified to influence adult neurogenesis but the underlying mechanisms remain unclear. in a recent study, lafenetre. (2010) showed the beneficial influence of physical voluntary activity on adult neurogenesis and cognitive performance in a transgenic mouse, the synras mouse via brain - derived neurotrophic factor. here we review how hippocampal neurogenesis can be regulated by environmental factors and the possible role of the newly generated cells in learning and memory. |
man 's yesterday may ne'er be like his morrow ; nought may endure but mutabililty. percy bysshe shelley (1816) mutability the first hint that the cytosine - guanine (cpg) dinucleotide might constitute a hotspot for pathological mutations in the human genome came nearly 25 years ago with the finding that two different cga tga (arg term) nonsense mutations had recurred quite independently at different locations in the factor viii (f8) gene causing haemophilia a. the potential generality of this phenomenon was supported by the finding that 12 of the 34 (35 per cent) single base - pair (bp) substitutions then known to cause human inherited disease were c t and g a transitions within cpg dinucleotides. further studies soon confirmed that the cpg dinucleotide was a mutation hotspot in a variety of different human disease genes, including pah, f9, ldlr, rb1, hprt1 and dmd. as mutation data accumulated, cga tga transitions were encountered particularly frequently as a cause of human genetic disease ; such nonsense mutations are inherently more likely to come to clinical attention than missense mutations. from the outset, it was realised that the hyper - mutability of the cpg dinucleotide was related to its role as the major site of cytosine methylation in the human genome. the reason traditionally put forward to explain this association has been that, while cytosine spontaneously deaminates to uracil (which is efficiently recognised as a non - dna base and removed by uracil - dna glycosylase), the spontaneous deamination of 5-methylcytosine (5mc) yields thymine, thereby creating gt mismatches whose removal by methyl - cpg binding domain protein 4 (mbd4) and/or thymine dna glycosylase followed by base excision repair is error prone [12 - 16 ]. it remains possible, however, that mcpg transitions are not exclusively caused by the spontaneous deamination of 5-methylcytosine and may also arise through the action of other mechanisms and processes [17 - 19 ]. (1998) estimated that the rate of cg tg (and cg ca on the other strand) transitions was five times the base mutation rate. subsequent estimates of 5mc hypermutability -- derived from various studies of polymorphism, disease mutations or evolutionary divergence -- have ranged between four - fold and 15-fold [20 - 26 ]. it has been known for some time that cytosine methylation also occurs in the context of cpnpg sites in mammalian genomes, where n represents any nucleotide. since the intrinsic symmetry of the cpnpg trinucleotide would support a semi - conservative model of replication of the methylation pattern (as with the cpg dinucleotide), it comes as no surprise that both maintenance and de novo methylation occurs at cpnpg sites in mammalian cells. in their recent paper on the human methylome, lister. reported abundant dna methylation in cphpg trinucleotides (where h = a, c or t). specifically, some 17.3 per cent of 5mc in embryonic stem cells was found to occur within cpapg, cpcpg and cptpg, with a further 7.2 per cent of 5mc occurring in cphph. although lister. suggested that non - cpg methylation is almost entirely lost upon differentiation (a conclusion based solely upon the analysis of foetal lung fibroblasts), others have noted cpnpg methylation within human genes in a variety of different somatic tissues. although the extent of non - cpg methylation in the germ - line remains unclear, if we were to assume not only that cphpg methylation occurs in the germline, but also that 5mc deamination can occur within a cphpg context, then it is very likely that methylated cphpg sites would constitute mutation hot - spots. indirect evidence that this might indeed be the case has come from a disproportionately high number of c t and g a transitions at cpnpg sites in studies of the human nf1 and brca1 genes. in the light of the above, we have revisited the question of cpg dinucleotide hyper - mutability and explored the potential contribution that cphpg transitions might make to human inherited disease. according to the april 2010 release of the human gene mutation database (hgmd ; http://www.hgmd.org), 56,457 pathological mutations have been reported in a total of 2,242 human genes. a subset of 54,625 pathological missense and nonsense mutations in 2,113 genes, with 2 bp genomic dna sequence flanking the site of mutation, was retrieved from the hgmd. the numbers of c t and g a mutations in this mutation dataset that were located within either cpg dinucleotides or cphpg trinucleotides were counted and termed ' mutations in di / trinucleotide ' (table 1). only these c t and g a transitions, found in the context of a cpg dinucleotide or cphpg trinucleotide, would be compatible with a model of methylation - mediated deamination of 5mc. the remaining mutations in this hgmd dataset that were located in non - cpg or non - cphpg di / trinucleotides within the genes in question were also counted and termed ' mutations not in di / trinucleotide ' (table 1). thus, 18.2 per cent of the studied missense / nonsense mutations causing human inherited disease are located in the cpg dinucleotide, while the corresponding proportion for the cphpg trinucleotide is 9.9 per cent. to assess the significance of these figures, the number of all possible c t and g a mutations within either cpg dinucleotides or cphpg trinucleotides within the coding regions of the mutated genes, termed ' possible mutations in di / trinucleotides ', all possible single bp substitutions that occurred in a non - cpg dinucleotide or non - cphpg trinucleotide context (as well as mutations other than c t and g a in cpg and cphpg) within the coding regions of the mutated genes were counted as ' possible mutations not in di / trinucleotide ' (table 1). a weak positive correlation was noted between the number of cpg mutations in the 2,113 genes analysed and the number of possible cpg mutations in these genes (pearson 's correlation 0.129, p = 2.45 10), implying that the cpg mutation frequency is influenced to some extent by the frequency of occurrence of the underlying cpg dinucleotide. unsurprisingly, a significantly greater proportion (approximately ten - fold) of observed pathological missense / nonsense mutations within these genes were c t and g a transitions within cpg dinucleotides than would have been expected (by chance alone) for all possible mutations (table 1 ; p < 10). a weak positive correlation (pearson 's correlation 0.251, p = 1.01 10) was also noted between the number of cphpg - located mutations and the number of cphpg trinucleotides in these genes, implying that the cphpg mutation frequency is influenced to some extent by the frequency of occurrence of the underlying cphpg trinucleotide. once again, a greater proportion (approximately two - fold) of observed pathological missense / nonsense mutations within these genes were c t and g a transitions within cphpg trinucleotides than would have been expected by chance alone for all possible mutations (table 1 ; p < 10). numbers of c t and g a mutations found in cpg dinucleotides and cphpg trinucleotides in a dataset of 54,625 missense and nonsense mutations in 2,113 different human genes (hgmd) and the numbers of possible c t and g a mutations in cpg dinucleotides and cphpg trinucleotides within the coding regions of the mutated genes. from the data presented in table 1, we estimate that ~11.8 per cent of the 9,947 cpg mutations (ie 1,176) occurred within this dinucleotide by chance alone and hence would not necessarily have originated via the methylation - mediated deamination of 5mc. in a similar vein, we estimate that ~46 per cent (2,460) of the cphpg mutations (5,402) occurred within these trinucleotides by chance alone and hence may not have originated via methylation - mediated deamination of 5mc. the other side of this particular coin, however, is that the remaining 54 per cent of the 5,402 observed cphpg mutations in hgmd (ie the excess 2,842 over expectation, or ~5 per cent of all the missense / nonsense mutations analysed) may well be attributable to methylation - mediated deamination of 5mc within a cphpg context. as far as we are aware, this is the first (albeit crude) estimate of the potential impact of cphpg mutations on human inherited disease. a similar analysis was performed for 1,766 regulatory mutations (identified in the promoters of 191 human genes) retrieved from the hgmd. the numbers of actual and possible cpg and cphpg mutations were counted as before, using the promoter sequences of each gene. in order to determine the total numbers of possible cpg / cphpg and non - cpg / cphpg mutations, the wild - type promoter sequences for each gene (total length, 22,051 bp) were used (table 2). as with the missense / nonsense mutations, an approximately twofold higher proportion of observed pathological regulatory mutations within these genes were c t and g a transitions within cpg dinucleotides than would have been expected by chance alone for all possible mutations (table 2 ; p = 6.03 10). we estimate that ~55 per cent of the 94 cpg mutations (ie ~52) probably occurred within these dinucleotides by chance alone rather than via methylation - mediated deamination of 5mc. by contrast, a lower than expected proportion of c t and g a regulatory mutations located in cphpg trinucleotides was observed (p = 0.011). the absence of any excess of c t and g a mutations located in cphpg trinucleotides indicates that most, if not all, the promoter cphpg mutations probably occurred by chance alone, making it unnecessary to invoke methylation - mediated deamination of 5mc to account for them. since neither cpg nor cphpg were found to be under - represented in the examined promoter regions as compared to the coding regions, we surmise that the reduced (cpg) or absent (cphpg) preponderance of c t and g a promoter mutations in the methylatable di / trinu - cleotides may be due to the relative paucity of cytosine methylation within the promoter regions that would render unmethylated cpg and cphpg di / trinucleotides no more mutable than any other di / trinucleotide. numbers of c t and g a mutations found in cpg dinucleotides and cphpg trinucleotides in a dataset of 1,766 regulatory mutations of 191 gene promoters (hgmd) and the numbers of possible c t and g a mutations in cpg dinucleotides and cphpg trinucleotides. although two specific examples of non - cpg methylation altering the binding of transcription factors to promoter elements within human genes have so far been reported, the functional role of most non - cpg methylation in the human genome is still unclear. irrespective of the functionality or otherwise of this specific type of post - synthetic dna modification in the human genome, it would appear that methylation of the cphpg trinucleotide may leave a significant imprint on the spectrum of missense / nonsense mutations causing human genetic disease. | the cytosine - guanine (cpg) dinucleotide has long been known to be a hotspot for pathological mutation in the human genome. this hypermutability is related to its role as the major site of cytosine methylation with the attendant risk of spontaneous deamination of 5-methylcytosine (5mc) to yield thymine. cytosine methylation, however, also occurs in the context of cpnpg sites in the human genome, an unsurprising finding since the intrinsic symmetry of cpnpg renders it capable of supporting a semi - conservative model of replication of the methylation pattern. recently, it has become clear that significant dna methylation occurs in a cphpg context (where h = a, c or t) in a variety of human somatic tissues. if we assume that cphpg methylation also occurs in the germline, and that 5mc deamination can occur within a cphpg context, then we might surmise that methylated cphpg sites could also constitute mutation hotspots causing human genetic disease. to test this postulate, 54,625 missense and nonsense mutations from 2,113 genes causing inherited disease were retrieved from the human gene mutation database http://www.hgmd.org. some 18.2 per cent of these pathological lesions were found to be c t and g a transitions located in cpg dinucleotides (compatible with a model of methylation - mediated deamination of 5mc), an approximately ten - fold higher proportion than would have been expected by chance alone. the corresponding proportion for the cphpg trinucleotide was 9.9 per cent, an approximately two - fold higher proportion than would have been expected by chance. we therefore estimate that ~5 per cent of missense / nonsense mutations causing human inherited disease may be attributable to methylation - mediated deamination of 5mc within a cphpg context. |
the present staging system for melanoma, using breslow thickness, ulceration, mitotic rate, and the presence of regional and distant metastases, stratifies patients into heterogenous groups, with wide variability in outcome or response to therapy. this results in applying more aggressive surgical and adjuvant therapies to large populations, diluting the impact of therapy while exposing more patients to toxicity. better biomarkers in melanoma are needed to target both surgical and adjuvant therapies, but to date have been elusive. for many solid tumors, the large - scale analysis of gene expression at the rna level can provide patterns of gene expression that may stratify patients better than tnm staging and help guide therapy. however, this approach requires fresh tissue from a large number of primary tumors, a unique challenge in melanoma where the primary is often only a few millimeters in size, with no residual tissue after the diagnosis has been made. for this reason, we chose to examine serum protein markers, hypothesizing that antibody discovery was ideal for the patient with malignant melanoma, as primary tumor tissue is not required and the presence of an immune response to melanoma - associated antigens has been well documented [14 ]. the investigation of humoral response provides new perspective to focus on melanoma - associated antibodies, which are more sensitive and stable to become diagnostic biomarkers for early - stage melanoma. we focused on glycoproteins, as most of the tumor - associated antigens are cell surface proteins or released to the extracellular matrix, where glycosylation is the major type of posttranslational modifications [5, 6 ]. moreover, glycoproteins are considered to be the linkage between t cells and antigen - presenting cells to help the orientation of binding, and play important roles in the generation and loading of antigenic peptides into mhc class i and mhc class ii [57 ]. using this approach we sought biomarkers that correlated with the presence of regional metastases among melanoma patients. using dual - lectin affinity chromatography and a natural protein microarray - based analysis to select a subproteome of target glycoproteins which were then used as baits to profile the antibodies against melanoma - associated antigens. this significantly improved technology using lectin affinity chromatography allows us to concentrate low abundant glycoproteins which are typically undetectable in whole cell lysate. this approach led us to the discovery of antibodies to 5 interesting melanoma - associated antigens (75 kd glucose - regulated protein (grp75), 94 kd glucose - regulated protein (grp94), acid ceramidase (asah1), cathepsin d (ctsd), and lactate dehydrogenase b (ldhb)) that correlated with the presence of melanoma within the regional lymph nodes. a member of the heat shock protein-70 family, it also inactivates the tumor suppressor p53. grp94, also known as heat shock protein-90, is a chaperone protein that is involved in the function and stability of many cell - signaling molecules. asah1 is a catabolic lysosomal enzyme that deacylates ceramide, which when phosphorylated forms the poten mitogen s1p. ctsd is a lysosomal acid proteinase which is involved in regulation of programmed cell death. lactate dehydrogenase (ldh) is an enzyme that catalyzes the conversion of lactate to pyruvate, and serum levels are associated with outcome in stage iv melanoma. we proposed that these autoantibodies may form the basis of a serum test that could select patients for sentinel lymph node biopsy. however, many prognostic factors show limited utility when used clinically in the context of known prognostic factors. we therefore sought to examine the potential clinical utility of these novel serum markers for predicting regional involvement among patients with melanoma in our previous work, we identified serum autoantibodies that recognized glycoproteins from a melanoma cell line and distinguished between 27 node - negative patients and 16 node - positive patients. in that work, we subsequently validated these results using recombinant proteins among a larger sample set of 79 patients. for this university of michigan institutional review board approved project, we used this latter sample set to examine the clinical utility of these serum autoantibodies as a predictor of regional node involvement. serum samples were obtained from patients being evaluated at our melanoma multidisciplinary clinic, a few weeks after the diagnostic biopsy, but 2 to 3 weeks prior to undergoing wide local excision and lymph node surgery (sln biopsy for clinically node - negative patients (n = 71) or lymph node dissection for clinically node - positive patients (n = 8)). blood was allowed to clot at room temperature, after which the tubes were centrifugated at 2500 g for 10 minutes. our initial discovery (using extracted glycoproteins) and validation (using recombinant proteins) of these serum autoantibodies have been previously described. for this study we used the results obtained with the recombinant proteins. 75 kd glucose - regulated protein (grp75), 94 kd glucose - regulated protein (grp94), cathepsin d (ctsd), and lactate dehydrogenase b (ldhb) were purchased from abcam (cambridge, ma, usa). these 5 recombinant proteins were chosen because the amino acid sequences described in the manufacturers ' instructions are perfectly matched with the sequences acquired from swiss - prot database. the sequences and the purity of purchased recombinant proteins were reconfirmed by maldi - qit (shimadzu, ca, usa). to summarize our previous work, the recombinant proteins were dissolved in the printing buffer (62.5 mm tris - hcl (ph 6.8), 1% w / v sodium dodecyl sulfate (sds), 5% w / v dithiothreitol (dtt), and 1% glycerol in 1x pbs) to reach a final concentration of 100 g / ml, respectively. each protein solution was then transferred to a well in a 200 l 96-well clear printing plate (bio - rad). the recombinant proteins from the printing plate were spotted onto nitrocellulose (whatman, usa) slides using a noncontact piezoelectric printer (nanoplotter 2 gesim). each spot contains five spotting events of 500 pl each so that the total volume of each protein solution was 2.5 nl. each spot was found to be ~450 m in diameter, with the distance between spots maintained at 600 m. printed slides were dried on the printer deck overnight and stored in a refrigerator desiccated at 4c if the slides were not used immediately. each recombinant protein was printed in triplicate, and 14 identical blocks were printed on each slide. the slides were washed three times with 0.1% tween-20 in pbs buffer (pbst) and then blocked with 1% bovine serum albumin (roche) in pbst for 1 hr. the blocked slides were dried by centrifugation and inserted into a simplex (gentel bioscience) multiarray device which divides each slide by 16 wells. each serum sample was diluted 1 : 200 in probe buffer which consisted of 1% bsa, 0.05% triton x-100, 0.1% brij-30 (sigma - aldrich, usa) in 1x pbs. the sample hybridization was totally randomized on each slide in no specific order to prevent bias. each block was hybridized in 100 microliter of diluted serum for 2 hrs at 4c. then goat - anti - human igg (h+l) conjugated with alexa fluor 647 (1 g / ml, invitrogen, carlsbad, ca) was applied to each block to bind with the antibodies attached on the protein array. anti - human igg was printed on the array as positive control and printing buffer served as the negative control. all processed slides were immediately scanned using an axon 4000b microarray scanner (axon instruments, foster city, usa). genepix pro 6.0 was used to extract the numerical data from each spot on the slides. the background subtracted median intensity of each spot was taken as a single data point. then the mean intensity of each protein from the triplicate was used for the further analysis. for this study, patient and tumor characteristics were collected for our sample of 79 melanoma cases, it included patient 's age at surgery, gender, tumor thickness (breslow), mitotic rate, presence of ulceration, and nodal status. for use as a potential clinical test, the sample distribution of each auto - antibody classified as over- or underexpressed, defined as a serum antibody level one standard deviation increment above the sample mean value. association between the levels of the autoantibodies is summarized by the spearman rank correlation coefficient with p values testing for significant correlations reported. the associations between patient, tumor, and antibody covariates with nodal disease was compared using the two - sample t - test for continuous covariates and the chi - square or fisher 's exact test for categorical covariates. the magnitude of the association between each serum antibody level and nodal disease is reported categorically as the odds ratio and 95% confidence interval for cases with overexpression versus cases without. odds ratios and confidence intervals were reported separately for the univariate associations and after adjustment for the patient and tumor characteristics using standard logistic regression. all statistical analyses were conducted using sas version 9.2 (sas institute, inc., cary, nc, usa) with p values less than 5% considered statistically significant. in the previous work, we used the native proteins extracted by a dual - lectin column from the melanoma cell line as bait to detect the presence of autoantibodies in the sera of melanoma patients, identifying 5 antigens including 75 kd glucose - regulated protein (grp75), 94 kd glucose - regulated protein (grp94), acid ceramidase (asah1), cathepsin d (ctsd), and lactate dehydrogenase b (ldhb), and we investigated the humoral response against the recombinant proteins using a larger sample set of 79 melanoma patients (figure 1). the clinical characteristics of the patient population are shown in table 1 for the total population and stratified by nodal disease status. of note, one patient with a negative sln subsequently recurred in a regional basin, changing the population from 48 node - negative and 31 node - positive to 47 node - negative and 32 node - positive patients. among the 32 node - positive patients, 8 were clinically node - positive, 23 were sln positive, and 1 represented a regional recurrence after false - negative sln. each glycoprotein was summarized by over- (> 1sd), standard (1sd), and underexpression (1 sd above the mean) of anti - asah1, anti - ctsd, or anti - ldhb was highly significantly associated with being sln negative, with an odds ratio of 0.05 (0.010.31, p = 0.002) after adjusting for age, gender, ulceration, mitotic rate, and breslow thickness. elevation of one of these three antibodies was not uncommon ; among the 70 clinically node - negative patients in this study, 22 patients (33%) had elevation of one of these antibodies. if it were validated that patients with elevation of one of these antibodies had a very low risk of sln metastases, then potentially these patients could be treated by wide excision alone, reducing the cost and morbidity of melanoma treatment. in contrast, elevation of anti - grp94 was associated with an increased risk of regional metastases, and was elevated in both patients with clinically evident disease (5 of 8 patients) and microscopic disease (4 of 23 patients). while detection of serum anti - grp94 levels would be less useful clinically, it could potentially identify some patients with thin melanoma for whom sln might otherwise be omitted. a bigger question is the role these proteins may play in the development and progrsion of melanoma. beyond the development of serum - based diagnostic tests, proteomics may identify targets for therapeutic intervention. indeed, 3 of the 4 proteins have strong associations with melanoma progression and prognosis. lactate dehydrogenase (ldh) is an enzyme that catalyzes the conversion of lactate to pyruvate, and serum levels of ldh are strongly associated with melanoma prognosis. serum ldh levels strongly correlate with outcome among stage iv patients and serum ldh measurements are part of the american joint cancer commission (ajcc) staging system for melanoma [913 ]. however, serum ldh levels are rarely elevated and of no clinical utility in nonmetastatic melanoma [1416 ]. cathepsin d (ctsd) is a lysosomal acid proteinase which degrades proteins, peptides, and peptide precursors. in addition, it appears to be involved in other biological processes including regulation of programmed cell death, tissue remodeling and renewal, activation of proteolytic enzymes, and fibrinolysis. many tumors have altered processing, secretion, and activity levels of ctsd, and they are often associated with aggressive behavior, stimulating tumor cell proliferation, invasion, and metastases [17, 18 ]. immunohistochemical studies have shown that ctsd is markedly expressed in melanoma cell lines and tissue biopsies from primary and metastatic melanoma, and these correlate with poor outcome [1924 ]. as with ldh, measuring plasma levels of ctsd was not of clinical value for identifying patients at risk of recurrence [17, 25 ]. as we can detect very low levels of antibodies in the serum, measuring antibody levels may be more sensitive than measuring protein levels, allowing transition of known serum markers from utility in stage iv disease only to the setting of early - stage disease. prior to this publication, the third protein, acid ceramidase (asah1), had not been strongly associated with melanoma progression, but has been associated with cancers of the breast, prostate, and thyroid [2629 ]. asah1 is a catabolic lysosomal enzyme that deacylates ceramide and yields sphingosine, which when phosphorylated, forms the potent mitogen s1p. the cellular levels of ceramide, sphingosine, and s1p are integral in determining cell survival and growth [3032 ]. targeting this pathway holds promise for anticancer therapies [3234 ]. in the case of these three proteins, expression of the proteins is associated with advanced stage, but the presence of antibodies to these proteins is associated with lower stage. the presence of the antibody may be due to increased exposure of the proteins (overexpression), immune recognition of protein alteration, or the antibodies may be functionally blocking critical pathways. in the case of these three proteins / antibodies, it remains unclear whether this represents specific functional inhibition by the antibodies, or increased progression in the face of decreased immune recognition of overexpressed proteins (disease advancement in the face of decreased immune surveillance). as these proteins are identified, further analysis of their role in melanoma progression, and their posttranslational structure is necessary. in contrast to these three proteins, for which the detection of autoantibodies was a favorable prognostic sign, the presence of autoantibodies to grp94, or heat shock protein-90 (hsp90), was associated with an increased risk of regional metastases. although this protein is highly conserved (and should not trigger a significant immune response), our primary data and validation studies using the recombinant proteins demonstrate the presence of anti - grp94 antibodies in the serum of nearly between 1/4 and 1/3 of node - positive patients. hsp90 is a chaperone protein that is crucially involved in the function and stability of many oncogene products and cell - signaling molecules, including craf, erb - b2, bcr - abl, cdk4, cdk6, akt, mutated p53, mek, vegfr, and importantly to melanoma mutated (but not wildtype) braf. hsp90 protects these proteins from deterioration caused by environmental stress, which includes cancer therapy. expression of hsp90 is elevated in melanoma, correlates with increasing breslow thickness, and is associated with advanced disease. because hsp90 chaperones so many proteins implicated in carcinogenesis, inhibiting hsp90 could inhibit several pathways at once, hsp90 inhibitors are presently in clinical trial in metastatic melanoma. while our data suggest that autoantibodies do little to inhibit hsp90 functionally, their presence as a response to overexpression is clearly related to melanoma progression. in conclusion, the creation of a glycoprotein microarray to screen melanoma patient serum samples for autoantibodies yielded four autoantibodies that show promise in predicting regional metastases, and could potentially form the basis of a blood test to select clinically node - negative patients for sln biopsy. if validated, this test could greatly minimize the cost and morbidity associated with the surgical treatment of melanoma, as well as identify patients with thin melanomas who should undergo the procedure. in addition, glycoproteins recognized by these antibodies may have important roles in the development and progression of melanoma and may serve as targets for intervention. on a broader scale, this approach could be used to identify additional serum autoantibodies that can identify patients at high risk of distant metastases and those unlikely to respond to treatment, allowing a more tailored use of adjuvant therapies. | better prognostic and predictive markers in melanoma are needed to select patients for therapy. we utilized a dual - lectin affinity chromatography and a natural protein microarray - based analysis to select a subproteome of target glycoproteins to profile serum antibodies against melanoma associated antigens that may predict nodal positivity. we identified 5 melanoma - associated antigens using this microarray coupled to mass spectrometry ; grp75, grp94, asah1, ctsd and ldhb. we evaluated their predictive value for nodal status adjusting for age, gender, breslow thickness, mitotic rate and ulceration using standard logistic regression. after adjustment, asah1, ctsd and ldhb were significantly negatively associated with nodal status (p = 0.0008) and grp94 was significantly positively associated (p = 0.014). our best multivariate model for nodal positivity included breslow thickness, presence of serum anti - asah1, anti - ldhb or anti - ctsd, and presence of serum anti - grp94, with an area under the roc curve of 0.869. if validated, these results show promise for selecting clinically node negative patients for sln biopsy. in addition, there is strong potential for glycoprotein microarray to screen serum autoantibodies that may identify patients at high risk of distant metastases or those likely or unlikely to respond to treatment, and these proteins may serve as targets for intervention. |
cardiovascular diseases and related disorders are a major cause of mortality both in men and women all over the world. they are commonly characterized by high levels of total cholesterol, triglyceride, and low density lipoprotein (ldl) cholesterol in the blood. large amount of triglyceride and total cholesterol, more importantly ldl cholesterol in the blood, is often associated with the etiology of cardiovascular diseases and is seen as primary risk factors. high level of lipids in the blood has been associated with hypertension, stroke, and lipid peroxidation. epidemiological studies support the view that consuming diets rich in soluble fibers (fruits, grains, nuts, and vegetables) reduce the incidence of chronic diseases such as cardiovascular disorders, obesity, and diabetes. a significant correlation between consumption of fibers and serum concentration of lipids has been noted. meals rich in fiber have been associated with the propensity to reduce the risk of developing cardiovascular diseases and related disorders. apples, oats, and wheat bran are food products with high fiber content and are likely to reduce the total cholesterol, tg, and ldl cholesterol as well as possibly increase hdl cholesterol in the blood, a condition that lowers the risk of cardiovascular diseases and attendant mortality [5, 7 ]. there is an inverse correlation between hdl cholesterol and cardiovascular disorders, the higher the hdl cholesterol in the blood the more these lipids are in the blood, the more prone an individual is to cardiovascular diseases, atherosclerosis, and hypertension. the present study therefore sought to compare the antilipidemic and anticholesteremic effects of oat, apple, and wheat bran in physiologically normal wistar rats. twenty (20) wistar rats of opposite sex were obtained from a local breeder in ibadan, southwest of nigeria. egg yolk was obtained by separating the yolk from the albumin and dried in a hot air oven at 80c for 3 hours to constant moisture content. healthy apples, canned oats, and wheat bran were purchased from appropriate commercial centers in ibadan, southwest of nigeria. twenty (20) male and female wistar rats weighing between 150 and 160 g were randomly assigned to four groups (a, b, c, and d), n = 5. they were housed in individual cage and fed with grower 's mash for two (2) weeks after which the rats in different groups were fed as follows for another two weeks. rats in groups a, b, c, and d were fed with apple, oats, wheat bran, and egg yolk, respectively. the daily amounts of food intake by the rats in all the groups were determined and their body weights were measured on weekly basis. after the last food treatment, the rats were fasted for 12 h ; blood samples were collected from the retro orbital sinus of the eye by ocular puncture into nonheparinised tubes and allowed to clot at room temperature for 30 min. the blood samples were then centrifuged at 3,000 rpm for 15 min and the serum obtained in each case was used for lipid profile analysis. the concentrations of high density lipoprotein cholesterol, low density lipoprotein cholesterol, trilglyceride, and total cholesterol were determined using commercial kits from randox laboratories, united kingdom. the principle underlining each assay the indicator quinoneimine is formed from hydrogen peroxide and 4-aminoantipyrine in the presence of phenol and peroxidase. ldl cholesterol and vldl cholesterol are precipitated from serum by the action of a polysaccharide in the presence of divalent cations, after which the hdl cholesterol present in the supernatant is determined. ldl cholesterol is determined as the difference between total cholesterol and cholesterol content of the supernatant after precipitation of the ldl cholesterol fraction by polyvinyl sulphate (pvs) in the presence of polyethylene glycol monomethyl ether. the indicator is a quinone imine formed from hydrogen peroxide, 4-aminophenazone and chlorophenol under catalytic influence of peroxidase. the mean gains in body weight and food intake of the experimental rats at the end of four weeks are shown in table 1. rats fed with egg yolk, oats, and wheat bran increased in body weight by 32.6 28.6 and 25.7 g, respectively, while those fed with apple reduced in body weight by 24.1 g. the relative higher gain in body weight of rats fed with egg yolk is probably due to the high fat content of egg yolk. this tends to explain the loss in body weight observed in rats fed with apple in this study. animals fed with oats consumed the highest amount of food, and those placed on with wheat bran consumed the lowest amount of food. similar quantities of food were consumed by animals in the control group fed with egg yolk and those fed with apple (table 1). the palatability of the diets obviously affected the rate of consumption of each food and partly accounts for the trend in the final body weights of the animals. as anticipated, the group of rats fed with egg yolk expressed the highest levels of ldl cholesterol (96.4 mg / dl), total cholesterol (117.4 mg / dl), and triglycerides (109.8 mg / dl) as well as the lowest level of hdl cholesterol (18.5 mg / dl). egg yolk is rich in cholesterol (117.1 mg / dl) and its involvement in the incidence of cardiovascular diseases and atherosclerosis is popular. on the contrary, rats fed with oats recorded the lowest level of total cholesterol (82.9 1.8 mg), low density lipoprotein (ldl), cholesterol (49.3 1.4 mg), and triglycerides (tg) (75.1 1.7 mg), as well as the highest level of hdl cholesterol (33.9 0.9 mg). there was no significant difference (p < 0.05) between oats and apple in their effects on blood lipid profile of wistar rats, with the chief constituent of these fibers being pectin and responsible for the hypocholesterolemic effect [10, 11 ]. rats fed with apple exhibited increased level of triglycerides (96.9 mg / dl) but exhibited lower levels of ldl cholesterol (57.9 mg / dl) and total cholesterol (88.5 mg / dl) when compared with rats fed with oats egg yolk. lipogenesis may have accounted for the high triglycerides level observed in rats fed with apple when compared with those fed with oats and wheat bran. the results observed with oat and apple in this study corroborate previous reports [6, 7 ]. wheat bran, though has the highest amount of crude fiber (9.9%) (table 2), had the least effect in improving the lipid profile of rats when compared to oats and apple. the reason for this is obvious in that wheat is chiefly composed of cellulose and lignin which are insoluble dietary fibers. this observation agrees with the report of jenkins and his colleagues, who reported in one of their studies that wheat bran did not have any significant effect on the serum lipids of individuals fed with it. soluble dietary fibers have been demonstrated to be beneficial in the management or treatment of diabetes and cardiovascular disorders. possibly, foods rich in soluble dietary fiber either reduced the quantity or facilitate the elimination of other foods which may be risk factors for these diseases. moreover, diets that are high in fiber tend to be low in energy and these diets can be useful in the control of body weight [12, 13 ], a critical factor in individual 's susceptibility to hyperlipidamia related disorders. overall, feeding of oats and apple to rats significantly improves the serum lipid profile in this study. consumption of foods rich in soluble dietary fibers such as oats and apple is highly encouraged. dietary fiber is found only in plant foods such as fruits, vegetables, nuts, and grains. milk, meat, and egg do not contain dietary fibers ; hence their intake should be minimized especially among adults. the removal of seed coat, peel, or hull reduces the fiber content of foods. for instance whole tomatoes have more fiber than peeled tomatoes ; likewise, whole wheat bread contains more fiber than white bread. | excess consumption of egg especially its yolk has been implicated in hyperlipidaemia (high level of cholesterol and triglyceride in the blood). conversely, soluble dietary fibers, probably due to their ability to bind free lipid molecules, appear to play an important role in protecting against hyperlipidaemia. this study sought to evaluate the comparative effects of selected sources of fibers : apple, oats, and wheat bran, on serum lipid profile in physiologically normal wistar rats. twenty rats were used for the study and were randomized into four groups, with each containing five animals (n = 5). a group which serves as control was fed with egg yolk while the other three groups were fed with apple, oats, and wheat bran, respectively. after two weeks of feeding, the animals were fasted overnight and blood samples from the retro - orbital sinus of the eye were collected for analyses of lipid profile. the results obtained showed that the group fed with oats had the lowest level of total cholesterol (82.9 1.8 mg), low density lipoprotein (ldl) cholesterol (49.3 1.4 mg), and triglycerides (tg) (75.1 1.7 mg), as well as the highest level of hdl cholesterol (33.9 0.9 mg). on the contrary, the group fed with egg yolk showed the highest level of total cholesterol (117.1 4.4 mg), ldl cholesterol (96.4 1.5 mg), and triacylglyceride (109 2.6 mg), as well as the lowest level of hdl cholesterol (18.5 0.9 mg). there was no significant difference (p < 0.05) between oats and apple in their effects on blood lipid profile of wistar rats. wheat bran, being an insoluble dietary fibre, had less significant (p < 0.05) effect on the blood lipid profile when compared to oats and apple. findings from this study may assist physicians and dieticians in recommending appropriate diet for individuals desiring to normalize their blood lipids levels. |
the incidence of vascular complications of injecting illicit drugs is growing due to the new kinds of drugs and diverse way of consumption. these facts have contributed to the increasing risk of venous thromboembolism.[24 ] moreover, patients with hiv / aids have more frequent episodes of deep venous thrombosis (dvt) than the general population. preventive measures against venous thrombosis must involve a higher awareness about hiv / aids and the risks associated with intravenous drug use (ivdu). we report a patient with ivdu and hiv / aids who developed recurrent venous thromboembolism associated with injections in vessels of lower limbs and groin regions. a 44-year - old female was admitted because of an insidious edema in the right lower limb, associated with moderate local pain, with 2 days of evolution. she denied any antecedent of trauma and related that three similar episodes had occurred during the last 18 months. her diagnosis of hiv-1 infection and aids was established in 2000, and she was in regular use of efavirenz (600 mg / day) plus lamivudine (150 mg / day) and zidovudine (600 mg / day). she was cigarette smoker (pack - year : 15) and has been in use of injecting cocaine for 12 years, in addition to smoke crack, marijuana, and paste of cocaine for about 6 years. admission examination showed body mass index (bmi) : 19.4 kg / m, edema in the right leg, moderate pain on palpation of the right thigh and right calf, and an abscess in the dorsum of the right foot. doppler ultrasound scan (uss) showed thrombosis with signs of recanalization in right popliteal vein [figure 1a ] and calcified thrombi in the right common femoral vein and in the superficial femoral vein [figure 1b ]. blood determinations of a 44-year - old woman hiv positive and injecting drug user presenting recurrent venous thrombosis (a) thrombus in the right popliteal vein with signs of recanalization ; (b) calcifi ed thrombus in the right superfi cial femoral vein although this middle - aged and ivdu woman had been under antiretroviral treatment and presented three events of dvt (2008, 2009, and 2010), thromboprophylaxis was not done. her diagnosis of recurrent dvt was characterized by clinical data and comparative imaging studies by uss, and a full anticoagulation schedule was utilized with success. complications related with ivdu may affect veins, arteries, and lymphatic vessels, including ischemia and pseudoaneurism due to intraarterial injection, vasculitis, artery dissection, arterial - venous fistulae, compartment syndrome, dvt, thromboflebitis, soft - tissue infections, bacteraemia, and sepsis.[246 ] recently, low serum levels of the c and s proteins and high levels of homocysteine were reported by basavanagowdappa. in a 27-year - old indian male with hiv infection and normal cd4. although the involved mechanisms are not entirely clear, hiv is an independent risk factor for venous thrombosis. the prothrombotic state observed in hiv - infected patients has been associated with antiphospholipid antibodies, aspartyl protease, endothelial and platelet activation, low levels of c and s proteins, and lupus anticoagulant.[57 ] in spite of controversies, the highly active antiretroviral therapy and protease inhibitors may cause thromboembolic events. the risk of thrombosis can increase in patients with aids due to systemic inflammatory response and comorbidities such as immobility, infections, and malignancies.[47 ] cooke and fletcher reviewed the data about dvt among 109 patients in england ; 33 of the ivdu group and 76 of the non - ivdu group (median ages were 29 and 51 years, respectively). although the exact rate of recurrences could not be monitored, the recurrent dvts were more frequent in patients from the ivdu group. one possible explanation was the shorter duration of anticoagulation to avoid hemorrhagic events in this group of patients. irish. reviewed data from the united kingdom department of health about skin, soft tissue, and vascular complications among drug users and found a conspicuous increase in hospitalization. they emphasized the role of injections in femoral vessels (groin injecting), which may cause superficial phlebitis, dvt, and arterial pseudoaneurisms in lower limbs. saber. reviewed data of 45 patients in new york with hiv / aids and dvts in the lower limbs and found recurrent episodes of thrombosis in 26.7% of the cases. the patient 's mean age was 43 years and 55.5% of the dvts had caused femoral or iliofemoral obstructions. the authors concluded that hiv / aids constitutes a main risk factor for dvts in lower limbs, and this condition occurs near 10 times more often than in general population. preventive measures against dvt should include higher awareness about the prothrombotic risks of hiv / aids and ivdu ; subcutaneous, intramuscular, and groin injecting should be avoided by drug users ; and accurate examination of the lower limbs must be routinely performed in all patients with antecedent of ivdu in primary care setting. with limitations inherent to a single case study, this report may contribute to increase the awareness about the higher tendency to venous thromboses among drug users and hiv - infected individuals. | we report a case of recurrent deep venous thrombosis in a 44-year - old woman, intravenous drug user and hiv - infected, who injected cocaine in the groins and veins of the dorsum of the feet. she suffered several episodes of deep venous thrombosis and soft - tissue infections in the lower limbs. images of doppler ultrasound scan revealed thrombosis in the right popliteal vein with partial recanalization and calcified thrombi in the territory of the right femoral vein. after use of heparin and oral anticoagulation, her clinical evolution was uneventful, and she was asymptomatic at the occasion of the hospital discharge. this report calls for better awareness about injections in the groins and superficial femoral veins, which are part of the deep venous system. thrombosis related to hiv infection is highlighted. |
prevention of blood loss is a major concern during liver resections as it is the major determinant of operative outcome. bleeding along with bile leak and hepatic failure is one of the major postoperative complications following liver resection [13 ]. lin. introduced the finger fracture technique which involves crushing of liver parenchyma by surgeon 's finger under inflow occlusion so as to isolate vessels and bile ducts for ligation. this technique was subsequently improved through the use of small kelly clamp for blunt dissection which gives better control, namely, clamp crushing or kellyclasia [57 ]. people have also used finer versions of clamps similar to kelly like pean, halstead, heiss, or bengolea clamps [6, 8 ]. these include ultrasonic dissector, harmonic scalpel, ligasure, dissecting sealer using radiofrequency, and staplers [911 ]. however, the clamp crushing technique is the most widely used method [3, 9, 1214 ] and has multiple advantages over other more advanced methods including safety, speed, and cost - effectiveness. thumb forceps including debakey forceps has significant advantages in terms of its design and ergonomics over kelly clamp which were reported previously when compared for their usage for diathermy. but so far its use in liver parenchymal transection has not been tried or reported in literature. we have been using debakey forceps for liver transections in all our liver resections for the past 8 years. the purpose of this study is to present our experience of 100 consecutive elective liver resections with debakey forceps crushing technique. we also highlight its ergonomic advantages over clamp crushing method. to the best of our knowledge this is the first such study reporting the usage of debakey forceps for hepatic parenchymal transection. this is a retrospective study of prospectively collected data of consecutive liver resections. during the period of january 2006 to october 2013 we performed a total of 146 liver resections in three hospitals under supervision of the main author (sundeep jain). of these 46 were performed in emergency setting (trauma n = 44 ; liver necrosis types of liver resection, according to brisbane terminology, in these 100 patients are presented in table 2. these patients were classified in 4 groups according to the type of underlying liver parenchyma into group a normal liver ; group b these four groups were compared in terms of age, gender, comorbid conditions, transection time, total operative time, postoperative length of hospital stay, blood transfusion rates, morbidity, and mortality to evaluate the effect of type of liver parenchyma with use of debakey forceps crushing technique for liver parenchyma transection. the first 50 (group 1) and the last 50 (group 2) were compared to evaluate the duration of transection time, total operative time, and postoperative length of hospital stay. the design along with mechanism of functioning of debakey forceps and kelly clamps was studied and compared using photographs taken during operation. this was to ascertain the advantages of one over the other in terms of ease of usage and the versatility of the instruments. also the ergonomic differences in the wrist joint were studied, with the help of photographs while using debakey forceps and kelly clamps for liver parenchymal transection. all the patients were induced with fentanyl 2 gm / kg and propofol 2 - 3 mg / kg of body weight and intubated with atracurium 0.5 mg / kg of body weight. maintenance of anaesthesia was achieved using sevoflurane in an air - oxygen mixture with supplemental fentanyl. after induction, central venous catheterization was done uniformly in right internal jugular vein for central venous pressure (cvp) monitoring with the aim of keeping cvp less than 5 mmhg and as close to 0 mmhg as possible, during parenchymal transection. this was achieved by fluid restriction and diuretics (frusemide) in 0.51 mg / kg iv dose. in 8/100 patients we had to use nitroglycerine to reduce cvp to the desired levels. during this phase urine output and mean arterial pressures were maintained at more than 0.5 ml / kg / hr and more than 70 mmhg, respectively. this was done by 100200 ml bolus fluid challenge and norepinephrine infusion at 0.050.1 gm / kg / min. during the low cvp stage patients after the surgery all patients are reversed with neostigmine 4080 gm / kg along with glycopyrrolate 10 gm / kg. the abdomen was explored by either bilateral subcostal or triradiate incisions depending on the site and size of the lesion. the falciform ligament was then divided and the lobe to be resected was mobilized from surrounding attachments and structures like diaphragm and vena cava. only in two patients (both with hepatocellular carcinoma) undergoing right hemihepatectomy, pringle 's manoeuvre was used to facilitate removal of associated portal vein tumour thrombus in one and due to excessive bleeding in another. in all patients during parenchymal transection low central venous pressure (05 mmhg) with head - low position was maintained. the liver parenchymal transection was started with the marking of the line of resection using monopolar electrocautery followed by cutting the parenchyma for 24 mm deep. then the parenchyma was crushed using fine tip (1 mm), 8 cm long straight or 9 cm long angled debakey forceps depending on the depth of transection followed by coagulation of the small vessels of < 2 mm size using monopolar electrocautery and ligation of the biliary and larger vascular pedicles using 20/30 silk sutures. once the specimen was out the haemostasis was achieved using spray cautery and fine (30/40) prolene sutures. the roux - en - y bilioenteric anastomosis was done with the bile ducts of the remaining lobe wherever indicated. numerical variables have been compared using t - test and categorical variables using chi - square test. during the study period of january 2006 to october 2013 a total of 100 elective liver resections were performed for various indications using debakey thumb forceps for the liver parenchymal transection. there were 39 females and 61 males with a mean age of 52.4 17 years. indications and the type of liver resections performed are mentioned in tables 1 and 2. various comorbidities included hypertension (n = 11), diabetes (n = 8), and chronic obstructive pulmonary disease (copd) (n = 5), while none had coronary artery disease. group b had 8 patients, group c had 14 patients, and group d had 25 patients while normal liver parenchyma was in 53 patients. of these 14 (19%) had undergone preoperative chemotherapy (hepatoblastoma 3, gastrointestinal stromal tumor 4, and colorectal cancers 7). obstructive jaundice was in 25/100 patients (12 gallbladder cancer, 9 hilar cholangiocarcinoma, and 1 each of hydatid disease, recurrent pyogenic cholangitis and strictured hepaticojejunostomy with right lobe atrophy and recurrent cholangitis). seven of these 25 (gallbladder cancer 4, hilar cholangiocarcinoma 2, and hydatid cyst 1) had plastic stent placed in the common bile duct. all the eight patients of hepatocellular carcinoma (hcc) had cirrhosis of liver due to alcohol in two, hepatitis b virus in four, and hepatitis c virus in two patients. all of these were in child 's a status without any history of decompensation in the past. pringle 's manoeuvre was used in two patients, both with hcc (alcoholic & hepatitis c related cirrhosis). the mean age, transection time, total operative time, and postoperative length of hospital stay of 100 patients were 52.4 17.4 years, 63.4 33.4 mins, 154.11 67.6 mins, and 10.3 5.7 days. the age difference of patients in all four groups (divided on the basis of type of liver parenchyma) was not statistically significant (table 3). patients of group a (normal liver parenchyma) had significantly less transection time in comparison to group b (cirrhotic livers) and group d (cholestatic livers), while it did not reach statistical difference when compared with group c (postchemotherapy livers) patients, though there was a trend towards lesser transection time in group a. this may be due to less number of patients in group c. the total operative time was significantly less in group a patients in comparison to group b, c, and d patients. also group a patients had significantly less postoperative hospital stay in comparison with group b, c, and d patients. these results show that type of liver parenchyma affects the transection time, total operative time, and postoperative recovery as reflected by the postoperative hospital stay (table 4). also it was found that the transection time and total operative time in group 1 (first 50 patients) were significantly more than in group 2 (second 50 patients), signifying the effect of surgeon 's experience on it. though, the postoperative hospital stay was similar in both these groups (table 5). total 11/100 (11%) patients needed perioperative blood transfusions, with the range of 24 units per case. these included 1 patient of hydatid disease, 2 of secondaries liver, 1 of hilar cholangiocarcinoma, 3 of gallbladder cancer, 3 of hcc, and 1 patient of hepatoblastoma. four patients had bile leak, ten had ascites, and five had wound infections. bile leak occurred in each patient after left hemihepatectomy for hydatid, right trisectionectomy for gallbladder cancer, right hemihepatectomy with segment i resection for hilar cholangiocarcinoma, and cystopericystectomy for hydatid cyst, with daily amount of 50 ml, 100 ml, 90 ml, and 20 ml, respectively. all but cystopericystectomy patient had preoperative biliary stent placement for obstructive jaundice. in all these patients it stopped conservatively in 9, 5, 6, and 2 days, respectively. ascites was seen in 5 hcc patients, 4 gallbladder cancer patients (with jaundice), and 1 cholangiocarcinoma (with jaundice) patient, with the hospital stay ranging from 15 to 26 days. it was managed successfully by fluid restriction, diuretics, bed rest, and low - salt diet. all patients with wound infections had preoperative biliary stent placement. all of these had gram - negative organisms and were successfully managed conservatively with dressings and antibiotics based on cultures of bile taken during surgery. there were three mortalities due to hepatic encephalopathy, liver failure, and disseminated intravascular coagulation (dic) in patients with hcc, gallbladder cancer, and hepatoblastoma, respectively. kelly clamp has a hinge in the middle with two finger loops which are grasped by the thump and ring finger, while the index finger helps guide the instrument. on the other hand, debakey forceps are held between thumb and the index finger with top end resting on the first dorsal interosseous muscle at the base of the thumb and index finger. this allows one to quickly and easily grasp small tissue and to grasp and hold tissue easily with variable pressure. it is less traumatic due to its fine tip and gentle enough to fracture only the liver parenchyma without injuring the ducts or vessels. long and angled debakey forceps with fine tip facilitates crushing in the deeper planes of liver. there is a definite sensation of tissue being crushed while using debakey forceps, which thus helps in releasing the pressure timely thus preventing injury to vessels. in present study, debakey thumb forceps is found to have similar ergonomic advantages over kelly clamp during crushing of liver parenchyma, as was reported in one study when they were compared for their usage for diathermy. these advantages are that (1) a ringed handled instrument is much more difficult to pick up from a flat surface than thumb forceps like debakey forceps as like many surgeons we like to pick them ourselves due to the involved repetitive movements of this kind, (2) the grip between the thumb and the side of the index finger for picking up thumb forceps required less accurate placing of the hand than putting the two digits through the finger loops of kelly clamps which can be done without having to take focus away from the area of dissection, and (3) thumb forceps are held in the classical precision grip in which the ulnar digits help in supporting the instrument between thumb and the index finger in addition to the apex of the thumb thus increasing the accuracy of handling, whereas the hand is unsupported while using the kelly clamp. figures 18 (photographs) depict wrist joint postures during liver parenchyma transection while using kelly clamp and debakey forceps. it is clear in figures 2, 6, 7, and 8 that the wrist joint always remains in neutral posture during liver parenchyma transection with debakey forceps at various depths and angles. on the contrary figures 1, 3, 4, and 5 shows that wrist joint is always in an awkward and strainful posture while using kelly clamp for liver parenchyma transection at all the depths and angles. this study shows that use of debakey forceps crushing technique is safe and effective for liver parenchymal transection ; transection time and total operative time improve with surgeon experience and it has ergonomic advantages over kelly clamp technique. the better understanding of liver anatomy and technical developments has helped in reducing the morbidity and mortality after liver resections [1921 ]. intraoperative blood loss with subsequent need for blood transfusion is significant risk factor for increased complication rates, poor postoperative outcomes, and shorter disease - free survival [22, 23 ]. thus it is paramount to decrease the intraoperative blood loss and subsequent blood transfusions during liver resections. as most of the bleeding occurs during parenchymal transection of liver there are many methods devised from time to time to facilitate liver transection with minimal blood loss [9, 10 ]. meta - analysis of 7 rct with total 554 patients has shown that there were no clinically important benefits of an alternative transection method in terms of blood loss, parenchymal injury, transection time, and hospital stay over clamp crushing method. so clamp crushing method remains the reference technique for transection of the parenchyma in elective hepatic resections. also the 2009 cochrane review of randomized data failed to show any significant difference with regard to mortality, morbidity, and hospital stay while comparing clamp crushing technique to alternative methods. the clamp crushing avoids special equipment with similar or faster transection speed thus making it the most cost - effective technique which is 2 to 6 times cheaper than other methods depending on the number of surgeries performed each year [9, 2428 ]. our study shows that debakey clamp is an equally effective instrument for parenchymal transection in all kinds of livers in terms of transection time and safety as is shown in previous reports using kelly clamp technique. the mean transection time in our study in normal liver was 48.1 20.1 mins and it was significantly shorter than groups with patients with cirrhotic and cholestatic livers. the mean total operative time in our patients with normal liver was 110.4 35.3 mins which was significantly shorter than groups with cirrhotic, postchemotherapy, and cholestatic livers. the transfusion requirement in present study was 11% which is due to the inclusion of patients with all kinds of liver parenchyma. none of the patients with normal livers (group a) had blood transfusion which is similar to previous reports. the mean postoperative length of hospital stay in subjects with normal livers was 10.3 5.7 days which is similar to previous reports [9, 14, 28 ]. subjects with diseased livers (groups b, c, and d) had greater hospital stay signifying the role of type of liver parenchyma on overall outcomes. the morbidity rate in the present study is 14% (22 complications in 14 patients). out of these 22 complications 21 have occurred in patients having cirrhotic, cholestatic, and postchemotherapy livers, while only one occurred in a patient of hydatid cyst with normal liver. the mortality in the present study is 3% with one patient each in cirrhotic, cholestatic and postchemotherapy liver groups. all these results signify the importance of type of liver parenchyma on the transection time, total operative time, blood transfusion rates, morbidity, mortality, and postoperative hospital stay while using debakey crush technique for liver resections. the mean transection time and total operative time were found to be significantly more in the initial 50 cases of the total 100 cases suggesting the effect of surgeon 's experience, though it did not affect the postoperative length of hospital stay (table 5). technically, debakey forceps has many advantages of kelly clamp including its efficacy and safety in all kinds of livers. ergonomically, there are two aspects which make debakey forceps a preferred instrument compared to kelly clamp for liver parenchymal crushing. one is the design of the instrument and the other is the posture of the wrist joint of the surgeon while operating with these instruments as described and shown above (figures 18). these make debakey forceps more useful to the operating surgeon in terms of easy handling, precise grip, ease of usage in every depth of liver resection, being less traumatic for the tissues, and giving least strain to the wrist joint by keeping it in the neutral position. this is because in neutral posture muscles are near their resting length thus making joints comfortable. for wrist joint it is neutral when forearm, wrist, and hands are all straight and in one line. awkward postures occur when wrist is in flexion or extension [30, 31 ]. in awkward posture muscles and ligaments of joint limitations of the present study include a nonrandomised trial design. however, this study is a single surgeon experience in consecutive cases and a large sample size with careful collection of data and is therefore important. we also performed a qualitative study of comparison of debakey with kelly technique and the findings are important. in conclusion, this is the first such study showing that debakey forceps crushing technique is as safe and effective method for liver parenchymal transection in all kinds of liver parenchyma with comparable results to kelly clamp crushing method. it also shows that type of liver parenchyma has a significant effect on overall outcome while using debakey crushing technique. the technical and ergonomic differences between debakey forceps and kelly clamp, in terms of design and wrist joint posture, make debakey forceps the preferred crushing technique for liver transection although large randomised trials are needed to confirm our findings. we therefore recommend debakey forceps technique as the crushing method of choice for liver transection in elective liver resection operations in nontransplant setting. the ergonomic virtues of debakey forceps should be considered while designing newer techniques and instruments for liver transection, especially in open liver resections. | introduction and objective. bleeding is an important complication in liver transections. to determine the safety and efficacy of debakey forceps for liver parenchymal transection and its ergonomic advantages over clamp crushing method we analysed our data. methods. we used debakey crushing technique in 100 liver resections and analysed data for transection time, transfusion rate, morbidity, mortality, hospital stay, influence of different types of liver conditions, and ergonomi features of debakey forceps. results. mean age, transection time and hospital stay of 100 patients were 52.38 17.44 years, 63.36 33.4 minutes, and 10.27 5.7 days. transection time, and hospital stay in patients with cirrhotic liver (130.4 44.4 mins, 14.6 5.5 days) and cholestatic liver (75.8 19.7 mins, 16.5 5.1 days) were significantly greater than in patients with normal liver (48.1 20.1 mins, 6.7 1.8 days) (p < 0.01). transection time improved significantly with experience (first fifty versus second fifty cases70.2 31.1 mins versus 56.5 34.5 mins, p < 0.04). qualitative evaluation revealed that debakey forceps had ergonomic advantages over kelly clamp. conclusions. debakey forceps crushing technique is safe and effective for liver parenchymal transection in all kinds of liver. transection time improves with surgeon 's experience. it has ergonomic advantages over kelly clamp and is a better choice for liver transection. |
since the discovery of the fragile x mental retardation 1 (fmr1) gene in 1991, our knowledge of the molecular mechanisms underlying fragile x syndrome (fxs) has increased tremendously. the disorder is caused by dynamic mutation of a single trinucleotide sequence, cgg, on the x chromosome. in patients, cgg repeat expansion and subsequent methylation silences the fmr1 gene, preventing transcription of the fragile x mental retardation protein (fmrp). fmrp is an rna binding protein involved in mrna transport and stability and also in local protein translation. the protein is estimated to interact with 4% of all brain mrnas and with an unknown number of proteins in the mammalian brain. absence of fmrp leads to perturbation of the normal cellular function of many mrnas and proteins. the resulting fragile x phenotype is characterized by intellectual disability, specific physical abnormalities, behavioral problems and epileptic seizures. elucidating the pathophysiology of fxs is therefore not only important for the identification of potential therapeutic targets, but could also provide further insights into the general mechanisms of cognition and behavior. various techniques have been used to determine the mrna targets of fmrp, including co - immunoprecipitation followed by microarray analysis, antibody - positioned rna amplification (apra) and, most recently, high - throughput sequencing of rnas isolated by crosslinking immunoprecipitation (hits - clip). each study identified a large number of mrna targets, encoding proteins involved in synaptic signaling pathways, such as glutamate and -aminobutyric acid (gaba), in addition to proteins involved in many other intracellular pathways. proteomic analysis of murine hippocampal synapses revealed abnormalities due to loss of translational regulation of fmrp. altered expression profiles were found predominantly for proteins in specific functional groups, such as synaptic structure and cytoskeleton organization, neurotransmission and metabolism. a recent study by davidovic and colleagues, published in genome research, provides a new dimension to fragile x research by analyzing the brain metabolome of fmr1-deficient mice. by combining metabonomics data with results from previously published studies and with the knowledge of signaling and metabolic pathways gathered from databases, this work significantly increases our insight into the aberrant signaling pathways in fxs. metabonomics measures the dynamic multiparametric metabolic response of living systems to pathological stimuli or genetic modification. the collection of all metabolites (the intermediate and end products of cellular processes) in a biological sample is represented by the metabolome, which can reflect the ultimate response of biological systems to genetic or environmental changes. metabonomics is a rapidly developing field with many potential applications in cancer and neurodegenerative disease diagnostics, monitoring of disease progression, evaluation of drug toxicity and development of therapeutics. traditionally, genomics and proteomics approaches have been used to study the effect of genetic disorders. however, finding the relationship between altered gene or protein expression and the biological consequences is not always straightforward, especially given that the effect of a single genetic variation is often not limited to one biochemical pathway. metabonomics can help to identify the metabolites of seemingly unrelated biochemical pathways, and therefore might reveal potential new sites for therapeutic intervention. the metabolites can be detected by approaches based on mass spectrometry or nuclear magnetic resonance (nmr) spectroscopy. in their study, davidovic. used solid state nmr, specifically h magic - angle - spinning nmr (h hr - mas nmr) spectroscopy. this method has the advantage of being able to analyze small amounts of intact tissues, unlike other methods, which require metabolites to be in solution. the technique can thus be used even if further analysis of the tissue sample is required. the outcome of the metabonomic analysis showed 25 metabolites with altered concentrations in the fragile x brain (figure 1). these 25 metabolites could be grouped into a small number of affected pathways, involved in one of four categories : neurotransmission, osmoregulation, energy metabolism and oxidative stress response. some alterations are in line with reported anomalies in fxs, including abnormalities in the glutamatergic and the gabaergic pathways. for instance, a reduced amount of gaba in the cortex and cerebellum is compatible with earlier reports of a compromised gabaergic system in the disorder. increased glutamatergic signaling is another hallmark of the disorder. it can be hypothesized that the reduced amount of free glutamate observed by davidovic. is a protective reaction of the organism to the overstimulation of the group there have been isolated reports that the cholinergic system, a second inhibitory system in brain, is also involved in the disorder, mainly on the basis of results from fly studies. the davidovic. study clearly shows that several components in the cholinergic pathway are compromised in the mouse model. this adds further evidence to the hypothesis that fxs is a consequence of the disturbance of the delicate balance between excitatory and inhibitory signaling in the brain. the metabolites found to be downregulated (down - arrows in the left columns) or upregulated (up - arrows in the right columns) in fmr1 knockout mice compared with wild - type mice are shown for each brain region. co, cortex ; st, striatum ; ce, cerebellum ; hi, hippocampus. to integrate the abnormal metabolite concentrations with the data available from previous studies, the authors developed a novel integrated metabolome and interactome mapping (imim) approach. this systems biology approach was considered necessary because fmrp does not interact directly with the metabolites themselves and therefore can not directly influence their concentrations. all the 25 metabolites were linked to fmrp in the network by, on average, just over three intermediate proteins. the distance within the network represents the mechanistic link between fmrp and each metabolite. within the interaction network, only 12 mrnas and one protein are linked directly to fmrp. of these, some are known interactors, including the paralogous protein fxr2p and mrnas encoding subunits of the gabaa receptor. interestingly, a key interactor is the amyloid beta precursor protein (app) mrna, which is implicated in alzheimer 's disease. a previous study indicated that fmrp binds app mrna, but it is striking that app is predicted to be in the center of the metabolite network, implying a pivotal role. rhoa is a member of the rho gtpase family, and different members of this gene family have opposite roles in neurite outgrowth and retraction. although it was known that the rho gtpase rac1, which promotes neural spine formation, was involved in the disorder, this study supports the involvement of the rhoa signaling pathway, which induces spine retraction. in line with this, expression of larg, encoding a protein that activates rhoa, was reported to be compromised in fxs in an earlier study. these findings emphasize the involvement of rho gtpases in the neuro - anatomical pathophysiology of fxs, more specifically in the aberrant spine morphology associated with the disease. this story adds another layer of understanding to the complex phenotype of fxs and it contains both good and bad news for the design of drugs to treat the disorder. although it confirms a key role of inhibitory and excitatory pathways, the abnormalities in these and other affected systems seem to be specific to particular brain regions and thus may require much more sophisticated drug treatments than currently anticipated. in addition, the abnormalities were detected in very young animals and an effect of brain development on the disturbances of metabolite concentrations can not be excluded. the study from davidovic and colleagues highlights many similarities between the metabolome of fxs and that of other neurological disorders. examples include the neurodevelopmental disorder rett syndrome and the neurodegenerative disorders batten disease and huntington 's disease, which have been analyzed by other groups using metabonomics. alterations in the balance between the excitatory (glutamate) and inhibitory (gaba) neurotransmitters, osmoregulation (myo - inositol), lipid metabolism (choline) and energy metabolism (creatine) were the most commonly observed metabolic changes across all these diseases. thus, collectively these findings contribute to our understanding of the mechanisms underlying neurological disorders in general. app : amyloid beta precursor protein ; fmr1 : fragile x mental retardation 1 gene ; fmrp : fragile x mental retardation protein ; fxs : fragile x syndrome ; gaba : -aminobutyric acid ; larg : leukemia - associated rho guanine nucleotide exchange factor ; nmr : nuclear magnetic resonance ; rhoa : ras homology protein a. we are supported by the agency for innovation by science and technology in flanders (iwt) and the belgian national fund for scientific research - flanders (fwo). | fragile x syndrome is the most common cause of inherited intellectual disability, but the underlying pathophysiology is complex and effective treatments are lacking. in a recent study of fragile x mental retardation 1 (fmr1) knockout mice, the metabolic profile of the fragile x brain was determined using proton high - resolution magic angle spinning nuclear magnetic resonance spectroscopy. this analysis revealed deficiencies in four metabolic categories : neurotransmission, osmoregulation, energy metabolism and oxidative stress response. abnormalities in the metabolic phenotype were linked to the fragile x mental retardation protein using an integrated metabolome and interactome mapping approach, allowing a global picture of the disorder to emerge. |
gestational diabetes mellitus (gdm) identifies women with an increased risk of developing type 2 diabetes and cardiovascular disease. therefore, women with prior gdm (pgdm) are recommended to regularly undergo assessment of glucose tolerance in order to detect overt diabetes in time to initiate treatment to prevent complications. the current view of gdm pathophysiology is that women who develop gdm are characterized by beta - cell dysfunction based on chronic insulin resistance. consequently, disturbances in glucose metabolism are thought to be of chronic nature rather than of acute onset during pregnancy. we have previously shown that pgdm with metabolic deterioration within five years after delivery are characterized by hyperglycemia, insulin resistance, and hyperinsulinemia ; furthermore first - phase insulin secretion was impaired in these women compared to pgdm who did not experience deterioration of glucose tolerance. in addition, while those pgdm who progress to diabetes have a marked increase in glycemia and insulin resistance before diabetes onset, we observed that beta - cell function declines continuously over years. others have shown that declining beta - cell compensation for increased insulin resistance (estimated by the disposition index) characterizes pgdm who convert to overt diabetes within 12 years after delivery and that this decline was associated with an increase in body weight and c - reactive protein (crp) as well as a decrease in adiponectin. approximately 50% of pgdm develop type 2 diabetes within the first five years after delivery [8, 9 ]. at the same time, there is a group of pgdm who are able to maintain normal glucose tolerance within this vulnerable period of metabolic deterioration. although many studies describe metabolic changes in those who convert to diabetes, less is known about pgdm who are able to maintain normal glucose tolerance (ngt) over the years following delivery. it could be speculated that metabolic disturbances are cured in those pgdm and that their risk of developing diabetes equals the one of women without a history of gdm. consequently, time- and cost - consuming follow - ups would not be necessary in these women. this could help to reduce unnecessary costs. since data that would answer this question are scare, the aim of this study was to compare validated parameters of insulin sensitivity and secretion between pgdm with ngt five years after gestational diabetes and healthy controls (five years after normal pregnancy). in addition, metabolic changes in pgdm within the five - year observational period and the impact of weight loss within this time were investigated. the current study was part of the viennese post - gestational diabetes project (vpgdp), a prospective longitudinal study in women with a history of gdm. pgdm were recruited during a pregnancy complicated by gdm in the diabetes outpatient clinic of the division of endocrinology and metabolsim of the medical university of vienna, where they had been seen during pregnancy. the human ethics committee of the medical university of vienna approved the protocol and all women gave written informed consent. exclusion criteria were age 18 years, known preexisting glucose intolerance (impaired glucose tolerance, type 1 or type 2 diabetes), diagnosis of gdm before the 8th gestational week, positive diabetes - associated antibodies (assessed during and after pregnancy at our division), ethnicity other than caucasian, morbid obesity (pregestational body mass index > 40 kg / m), or evidence of chronic diseases, including kidney or liver diseases and chronic inflammatory diseases. in con, exclusion criteria additionally included the existence of any risk factors for diabetes (i.e., positive family history or chronic medication known to influence carbohydrate metabolism). for the current study, 45 pgdm with normal glucose tolerance at five - year follow - up and 18 healthy controls were matched for age and body mass index (table 1). data from the visit 5 years postpartum were compared between the groups and in pgdm also to the baseline examination (six months after delivery). furthermore, an additional subanalysis was performed, in which those pgdm with weight loss of 7% (wihtin the 5-year observational period) were compared to the healthy control group. this range was chosen in regard to the american diabetes association (ada) recommendation of 7% weight loss in patients with prediabetes. all women received dietary counseling and were recommended to regain normal body weight by intake of a healthy diet and regular physical activity. glucose tolerance tests were scheduled between day 3 and day 10 of their menstrual cycle, performed in the morning after an overnight fast of at least 8 hours. women were asked to refrain from physical activity 3 days prior to the follow - up visits. all women underwent an oral glucose tolerance test (ogtt) and the majority underwent also an intravenous glucose tolerance test (76% of pgdm and 61% of con). reasons for not undergoing an intravenous glucose tolerance test (ivgtt) were mainly problems in time scheduling (additional appointment according to menstrual cycle, more than 2 weeks after ogtt). after a venous catheter was placed into an antecubital vein, blood samples for the measurement of glucose, insulin, and c - peptide were taken at fasting as well as 10, 20, 30, 60, 90, 120, 150, and 180 minutes after ingestion of 75 g glucose in a solution. for the ivgtt, one venous catheter for blood sampling was placed in one antecubital vein and another one for intravenous administration of glucose and insulin in an antecubital vein of the other arm. blood samples (for measurement of glucose, insulin, and c - peptide) were drawn at the fasting state (10 and 0 minutes) and 3, 4, 5, 6, 8, 10, 14, 19, 22, 27, 30, 35, 40, 50, 70, 100, 140, and 180 minutes after injection of glucose (300 mg / kg body weight). at 20 minutes, normal insulin (humulin r, eli lilly, indianapolis, in, usa) was given with a concentration of 0.03 iu / kg body weight and for a duration of 5 minutes. serum samples for the assessment of insulin and c - peptide were immediately cooled down, centrifuged, stored at 20 degrees celsius, and later analyzed in the lab of the division of endocrinology and metabolism by commercially available radioimmunoassay kits : insulin (serono diagnostics, freiburg, germany) and c - peptide (cis bio international, cedex, france) with interassay coefficients of variation of < 5%. at fasting, additional blood samples were taken and the following parameters were assessed : adiponectin was measured in duplicate by an elisa system developed for the assessment of human plasma adiponectin concentrations (department of internal medicine and molecular science, osaka university, suita, osaka, japan). charles, mo, usa) and glucagon (icn biomedicals, costa mesa, ca, usa) were measured in duplicate by commercially available radioimmunoassay kits with a cv < 6% for leptin and < 8% for glucagon. hba1c (by high - performance liquid chromatography, given in %), tsh, total cholesterol, ldl - cholesterol, hdl - cholesterol, triglycerides, and c - reactive protein were measured by established methods in the central lab of the medical university of vienna. normal glucose tolerance (ngt) was defined according to the criteria of the ada : fasting plasma glucose (fpg) < 100 mg / dl and 2-hour post - ogtt glucose < 140 mg / dl. the oral glucose insulin sensitivity (ogis) index describes glucose clearance per unit change of insulin concentration. parameters of insulin secretion were described by the areas under the curve (auc) of insulin and c - peptide during ogtt and ivgtt, calculated with the trapezoidal rule. hepatic insulin extraction (hie, given in %) was quantified with a mathematical model of insulin / c - peptide interactions. from ivgtt, insulin sensitivity index (si, in 10 min/(u / ml)) describing insulin effect on glucose disappearance was computed. first - phase insulin secretion was assessed by airg calculated by averaging insulin concentrations above basal from 3 to 10 minutes and given in u / ml. the disposition index derived from ivgtt (10 min) was calculated as si airg and describes the combined effect of insulin secretion and sensitivity on glucose disposal ; it is frequently used to describe the ability of the beta cells to adapt for increased insulin resistance. between group differences were calculated by anova ; changes between baseline and follow - up visit in pgdm were calculated by a paired t - test. sas software (enterprise guide 4.3, sas institute inc., cary, nc, usa) was used for all computations. five years after the index pregnancy, pgdm despite normal glucose tolerance had significantly higher levels of plasma glucose at fasting as well as 60 minutes of the ogtt compared to con ; in line, the auc of glucose was significantly increased in pgdm compared to con (figure 1). insulin sensitivity, derived by ogis, was decreased in pgdm compared to con (figure 2(a)). furthermore, adiponectin was lower in pgdm (figure 2(b)), while blood pressure, leptin, glucagon, tsh, and lipid profile did not differ between the groups. pgdm had significantly higher waist circumference (figure 2(c)) as well as crp concentrations, (figure 2(d)). normal body weight, defined as bmi < 25 kg / m, was found in 30 out of 45 pgdm (= 66.7%) and in 14 out of 18 con (= 77.8%). insulin sensitivity (ogis) was negatively correlated with bmi and body weight in the whole study group (bmi : r = 0.3, p = 0.01 ; body weight : r = 0.3, p = 0.02). when follow - up data in pgdm were compared to their baseline examination (= six months postpartum), waist circumference (85.5 9.0 versus 81.8 9.8 cm, p = 0.002) and diastolic blood pressure (76.7 9.1 versus 72.5 9.4 mmhg, p < 0.04) were lower at five - year follow - up compared to baseline (6 months after pregnancy). in addition, pgdm had significantly lower total cholesterol (from 211.9 48.6 to 198.4 40.2 mg / dl, p < 0.006) and ldl - cholesterol (from 134.2 45.4 to 122.6 37.0 mg / dl, p = 0.002) compared to baseline. no change was observed in body weight, hba1c, glucagon, leptin, or tsh. furthermore, the disposition index (1.7 1.2 versus 2.6 2.3 10min, p < 0.004) and insulin sensitivity derived from ivgtt (from 4.3 2.5 to 6.3 3.2 10min/(u / ml), p = 0.001) were increased compared to baseline. twelve pgdm had significant weight loss (7%) within the 5-year observational period (pgdm_wl) and were compared to con, in order to assess whether weight loss was associated with an improved metabolic profile. while there was no difference in age, bmi, and waist circumference between the groups, pgdm_wl had significantly increased concentrations of glucose (auc of glucose, 1.13 0.16 versus 1.0 0.12 mol / l min, p < 0.02) and insulin during the ogtt (tis : 27.1 7.1 versus 21.8 5.7 nmol / l, p = 0.03, auc of c - peptide : 419.5 106.9 versus 347.0 81.3 nmol / l min, p = 0.04), whereas insulin sensitivity was lower compared to con (ogis : 466.9 46.4 versus 510.6 53.1 ml / min m, p < 0.03). furthermore, crp was higher in pgdm_wl compared to con (0.4 0.3 versus 0.2 0.2 mg / dl, p = 0.04). when pgdm_wl were compared to their baseline state, the significant changes in body weight (9.9 4.8 kg, bmi : from 26.8 3.5 to 23.2 2.5 kg / m, both p < 0.0001) and waist- (from 89.0 7.9 to 79.8 9.4 cm, p = 0.0002) and hip - circumference (from 107.5 5.8 to 97.8 4.6 cm, p < 0.0001) were accompanied by an improved disposition index (from 1.6 1.0 to 2.9 1.6 10 min, p = 0.01 ; in line with the whole pgdm - group), a decline in crp concentrations (from 0.6 0.2 to 0.4 0.3 mg / dl, p = 0.03), diastolic blood pressure (from 80.8 9.3 to 72.2 7.9 mmhg, p = 0.02), and leptin (from 17.7 5.5 to 14.5 7.0 ng / ml, p < 0.03). the current study aimed to assess whether disturbances in glucose metabolism can be observed in women with prior gestational diabetes (pgdm) who were able to maintain normal glucose tolerance (ngt) until five years after a gdm - pregnancy. according to our data, pgdm despite normal glucose tolerance were still characterized by decreased insulin sensitivity and increased glucose concentrations during the ogtt compared to healthy controls. furthermore, crp levels and waist circumference were higher in pgdm compared to con, despite comparable bmi, while adiponectin was decreased in pgdm. in addition, pgdm with weight loss 7% within the five - year follow - up period exhibited pronounced metabolic disturbances compared to con. insulin resistance is a frequent finding in women with prior gestational diabetes and associated with ectopic lipid accumulation in skeletal muscle and liver [20, 21 ]. it is assumed that gestational diabetes develops on the background of chronic insulin resistance, aggravated by the physiological insulin resistance of late pregnancy [2, 3 ]. according to our observations, we also assume that insulin resistance in pgdm is of chronic nature and the diagnosis of gdm during pregnancy detects a metabolic phenotype with increased insulin resistance in a young female cohort. we found a weak, but significant, inverse association between bmi and insulin sensitivity ; however, also in the subgroup of pgdm with significant weight loss (pgdm_wl) insulin sensitivity was significantly lower compared to the healthy control group. special attention has to be given to this subgroup of pgdm who had significant weight loss of 7% within the follow - up period. this group had a bmi of approximately 27 kg / m six months after delivery and weight loss in this group was accompanied by a reduction in waist- and hip - circumferences as well as leptin ; furthermore, the disposition index (in line with the whole study group) improved ; however, despite marked changes in body weight, insulin sensitivity and glucose concentrations during the ogtt and ivgtt remained unchanged compared to the baseline examination (6 months postpartum). hence, it might be speculated that obesity is not the main reason for insulin resistance in pgdm. this assumption is supported by one of our prior observations, which showed that insulin resistance is pronounced in lean subjects with gdm and persists after delivery. in addition, it is in line with prior investigations in women with a history of gdm showing that the decline in insulin sensitivity and beta - cell compensation could not be explained by changes in adiposity. it has also been shown that impaired beta - cell glucose sensitivity independent of obesity and hyperglycemia displays a risk factor in pgdm. it has to be noted that the majority of women in our study groups (66.7% in pgdm and 77.8% in con) had a bmi lower than 25 kg / m and thus fulfill the criteria of normal body weight. furthermore, mean waist circumference in pgdm was 81.8 cm, which would fulfill the who criteria for metabolic syndrome, but not those of the us national cholesterol education program adult treatment panel iii. this again leads us to conclude that obesity might not be the main trigger of glucose intolerance in this young female cohort or not in all pgdm. the observation that glucose concentrations during the ogtt were higher in pgdm compared to con clearly indicates a risk of hyperglycemia in these women. this risk would not have been detected by simply concentrating on the definition of ngt, because glucose values were within the normal range ; however, in comparison to age- and bmi - matched controls, glucose concentrations during the ogtt were significantly higher in pgdm. and even in pgdm_wl, the significant weight loss did not counteract increased glucose concentrations compared to con. the finding that adiponectin was decreased and crp increased in pgdm compared to con is of great interest. a recent study by xiang and coworkers [6, 7 ] described that declining beta - cell compensation in pgdm (described by the disposition index) is besides weight gain associated with declining levels of adiponectin and rising crp levels. to our surprise, these metabolic features were also observed in our group of pgdm who were able to maintain normal glucose tolerance within this vulnerable period of five years postpartum. hence, the question of whether these metabolic alterations can be used as markers of metabolic deterioration or simply reflect this special metabolic profile in pgdm appears. specifically a drop in adiponectin could reflect metabolic deterioration in pgdm and indicate the need for closer follow - up of these women ; however, this assumption has to be reexamined in future prospective studies. chronic inflammation and hypoadiponectinemia are frequently found in patients with diabetes ; hence it appears that these disturbances could be the cause rather than the consequences of insulin resistance and hyperglycemia. in addition, this combination of hypoadiponectinemia and increased crp might contribute to the development of atherosclerosis [26, 27 ]. hence, we could assume that pgdm despite normal glucose tolerance have an increased cardiovascular risk and should be considered as high - risk population for cardiovascular disease. several metabolic parameters, that is, ldl - cholesterol, diastolic blood pressure, and waist circumference as well as the disposition index and insulin sensitivity derived from ivgtt, were improved in pgdm at five years postpartum compared to baseline. but despite these ameliorations and the fact that the majority of our pgdm group were able to regain / maintain normal body weight and normal glucose tolerance, disturbances in glucose regulation were observed. hence, it appears that the metabolic profile and thus the risk of developing type 2 diabetes seem to be chronic and that gdm only identifies women at risk. it appears that the pivotal mechanisms that finally lead to the development of overt hyperglycemia in pgdm have not been elucidated in detail. this may include a genetic disposition in this group of gdm without obesity but still increased risk for type 2 diabetes. as shown, gdm risk is associated with genes involved in the regulation of insulin secretion. at least, more studies are needed to better understand the development of overt hyperglycemia and develop treatment strategies, which can improve prevention. while the strength of the current study lies in the investigation of a well - characterized cohort and performance of validated tests under dynamic not only fasting conditions, it also has some limitations : the study group is quite small and follow - up is limited to five years ; an extended follow - up period could allow strengthening our conclusions. consequently we can summarize that metabolic disturbances which predispose pgdm to the development of overt diabetes appear to be chronic and can be hidden but, still, remain life - long and therefore regular follow - ups should be recommended to all women with a history of gdm in order to detect diabetes in time and prevent complications, especially the onset of cardiovascualr disease. | background. the study aimed to assess whether women with prior gestational diabetes (pgdm), despite maintenance of normal glucose tolerance (ngt) five years after delivery, display metabolic disturbances compared to healthy controls. methods. 45 pgdm with ngt were compared to 18 women without a history of gdm (con), matched for age (37.0 4.1 versus 35.2 5.3, p = ns) and bmi (24.3 3.1 versus 23.3 3.3, p = ns). metabolic parameters were derived from oral and intravenous glucose tolerance tests ; furthermore lipid profile, c - reactive protein (crp), adiponectin, leptin, and glucagon were assessed. results. five years postpartum, pgdm had increased glucose concentrations during the ogtt (auc : 1.12 0.15 versus 1.0 0.12 mol / l min, p = 0.003) and insulin sensitivity was decreased compared to con (ogis : 467.2 64.1 versus 510.6 53.1 ml / min m2, p = 0.01). pgdm had lower adiponectin (8.1 2.6 versus 12.6 5.3, p < 0.008) but increased waist circumference and crp compared to con. conclusions. despite diagnosis of normal glucose tolerance, pgdm are characterized by hyperglycemia and insulin resistance compared to healthy controls, accompanied by decreased adiponectin and increased crp concentrations, thus linking metabolic disturbances to an increased cardiovascular risk in pgdm. |
a 42 year old gentleman presented with rest tremors, bradykinesia and rigidity for 10 years. he responded very well to the treatment [updrs scale (off 40, on 24) ]. after three years of onset of the disease, family members noticed that while walking he was having lateral flexion of trunk and was leaning on left side (see video). along with this he also developed striatal hand (figure 1). during supine position x - rays whole spine, mri brain and paraspinal electromyography (emg) were normal. this patient of pd was diagnosed to have pisa syndrome due to lateral flexion and axial dystonia on standing which used to completely improve in supine position. pisa syndrome is a combination of lateral deviation of spine and corresponding tendency to lean on one side.3 exact pathogenesis of ps is still not known. it is believed that cholinergic excess in patients of ad receiving cholinesterase inhibitor cause ps. in patients of pd it may be possible that striatal dopamine deficiency or imbalance in dopaminergic - cholinergic level is responsible for ps.4 this imbalance seems to be asymmetrical and is responsible for the lateral flexion on one side. our patient also had significant improvement in lateral flexion on standing during on period, however striatal hand symptoms showed no response to levodopa / carbidopa. it has been described that pd subjects in advanced stage of disease (hy iii & iv) have more postural abnormality and striatal deformities while our patient manifested in early part of disease (hy iib).3 dopa agonists and amantidine has been tried with variable response. neurosurgical interventions like pallidotomy and deep brain stimulation has also been tried in 13 subjects with good results.5 | pisa syndrome is (ps) usually seen in patients receiving antipsychotic drugs and characterised by lateral flexion of trunk and axial dystonia. it is believed that antipsychotic drugs lead to dopamine blockage causing ps. we describe a parkinson s disease patient who was doing well with levodopa / carbidopa for 3 years and developed lateral flexion of trunk. his abnormal posture used to completely improve upon lying down position. he also had striatal hand deformity suggestive of focal dystonia. |
rituximab (rituxan) is a chimeric igg1 monoclonal antibody that targets the cd20 receptor and is used in the management of lymphoproliferative disorders. rituximab is fda approved in the treatment of chronic lymphocytic leukemia (cll), non - hodgkin 's lymphoma (nhl), and rheumatoid arthritis (ra). rituximab is generally well tolerated ; however, there are risks associated with infusion - related toxicity including hypersensitivity reactions such as fever, urticaria, hypotension, and cardiovascular and respiratory compromise. the mechanism by which rituximab causes an infusion - related reaction is unclear but is thought to be due to the release of inflammatory cytokines. the likelihood of infusion - related reactions is the highest with the first infusion and decreases with subsequent infusions with time to onset ranging from 30 to 120 minutes. other risk factors for developing an infusion - related reaction include large tumor burden, pulmonary infiltrates, elderly patients, and individuals diagnosed with chronic lymphocytic leukemia or mantle cell lymphoma. infusion related reactions typically resolve with supportive care and/or a decrease in the infusion rate. the overall incidence of infusion - related reactions varies based on diagnosis but ranges from 32 to 77% with the first infusion of rituximab. the incidence of grade 3 or 4 infusion - related reactions is reported to be 9% for the first infusion of rituximab with cyclophosphamide, doxorubicin, vincristine, and prednisone (chop) or cyclophosphamide, vincristine, and prednisone (cvp) chemotherapy regimens. in an effort to prevent infusion - related reactions, the manufacturer recommends that the first infusion of rituximab be given at a slow initial rate and gradually titrated upward which may take up to 6 to 8 hours. specifically the initial rituximab infusion should be started at a rate of 50 mg / hr, and if tolerated further titrate the rate by 50 mg / hr every 30 minutes to a maximum rate of 400 mg / hr. for subsequent infusions, the starting rate should be 100 mg / hr, and if tolerated, the rate can be further increased to 100 mg / hr every 30 minutes to maximum of 400 mg / hr. additionally it is recommended to premedicate with acetaminophen and an antihistamine prior to each rituximab dose. subsequent infusions may be given at a faster rate, but they may take up to 4 hours to infuse. rituximab therapy in the maintenance setting has shown to improve progression - free survival and overall survival. maintenance rituximab can be administered according to several different schedules including one dose every 2 months, one dose every 3 months, or once weekly for 4 doses every 6 months [4, 5 ]. studies have been conducted to determine the safety and feasibility of administering rapid rituximab ; however, these studies did not include patients receiving rituximab maintenance for low - grade non - hodgkin 's lymphoma [3, 68 ]. herein, we present our experience at h. lee moffitt cancer center with rapid infusion rituximab administered as maintenance therapy for low - grade non - hodgkin 's lymphoma. h. lee moffitt cancer center has been utilizing a rapid rituximab infusion protocol since december 2007. the rapid infusion rituximab protocol indicates to infuse rituximab at a rate of 150 ml / hr for 30 minutes then increase the rate to 275 this method is used to prevent rate calculation errors and ensures all infusions are infused over 90 minutes (5 minutes) for doses between 500 mg and 1000 mg. additionally methylprednisolone, hydromorphone, diphenhydramine and epinephrine are available in the event of an infusion - related reaction. if symptoms of a reaction occur, the infusion is stopped until symptoms resolve and is resumed at 50% of the previous rate and titrated every 30 minutes as tolerated. patients must meet the following criteria in order to be eligible for rapid infusion rituximab : rituximab courses two to eight prescribed at standard doses given concurrently with chemotherapy, no grade 3/4 toxicity with previous course, subsequent rituximab monotherapy courses if no grade 3/4 toxicity with initial two courses, peripheral lymphocyte count < 4.8 k / ul, maintenance chemotherapy, following response to induction to chemotherapy.this retrospective analysis included patients diagnosed with low - grade non - hodgkin 's lymphoma who received rapid infusion rituximab for maintenance therapy from december 2007 through november 2011. patients were included if they were greater than 18 years of age, met eligibility requirements to receive rapid infusion rituximab per moffitt protocol, and diagnosed with low - grade non - hodgkin 's lymphoma. data collection included diagnosis, treatment regimen prior to maintenance therapy, rituximab maintenance schedule, administration of premedications, and any adverse events. adverse events were evaluated through documentation, vital signs, and administration of supportive care. the primary objective of this retrospective analysis was to evaluate the incidence of grade 3 and grade 4 toxicities with maintenance rapid infusion rituximab according to the common terminology criteria for adverse events version 4. secondary objectives included evaluating all infusion - related adverse events (grades 14) with maintenance rituximab and evaluating correlation of adverse events with varying schedules of rituximab maintenance therapy. rituximab courses two to eight prescribed at standard doses given concurrently with chemotherapy, no grade 3/4 toxicity with previous course, subsequent rituximab monotherapy courses if no grade 3/4 toxicity with initial two courses, peripheral lymphocyte count < 4.8 k / ul, maintenance chemotherapy, following response to induction to chemotherapy. a total of 1,070 patients received rituximab as part of their treatment regimen from december 1, 2007 through november 25, 2011. rapid rate maintenance rituximab therapy was given to 109 patients for a total of 647 doses. all of the patients received premedication with acetaminophen and an antihistamine as directed by the package insert. patients who had a prior infusion - related reaction with rituximab premedicated with corticosteroids in addition to acetaminophen and diphenhydramine. each patient received an average of six doses of maintenance rituximab therapy at a rapid infusion rate, and the average infusion time for each dose administered was approximately ninety minutes. administration of rituximab at a rapid rate saved approximately 68 minutes per patient (table 1). the majority of patients (64%) were diagnosed with follicular lymphoma and 8% had an unspecified low - grade lymphoma. the remainder of patients were diagnosed with one of the following low - grade lymphomas : mantle cell lymphoma, marginal zone lymphoma, mucosa - associated lymphoid tissue (malt), small lymphocytic leukemia, and chronic lymphocytic leukemia (figure 1). a small minority of patients received rituximab maintenance regimen every two months based on data published from the prima study (figure 2). out of the 109 patients analyzed, 3 patients developed an infusion - related reaction including one patient who developed a reaction twice. two of the patients required pharmacological management with corticosteroids, additional antihistamines, and/or inhaled beta - agonists. this analysis supports that rituximab can be safely administered as a rapid infusion during maintenance therapy. our experience resulted in a low incidence of grade 3 reactions and no grade 4 reactions. the data also reveals the incidence of developing a grade 2 reaction is quite low. out of the 109 patients analyzed, one patient experienced nausea (grade 1) with the first dose of the 3rd cycle of maintenance therapy. the other two patients experienced symptoms such as shortness of breath, facial flushing, and chest tightness (grade 3) (table 3). two of the patients were rechallenged at the rapid rate and tolerated therapy without further complications. in the patients who developed a grade 3 reaction, the incidence was too low to determine a correlation with the administration schedule of maintenance therapy. when rituximab is administered at the standard rate, the patient 's infusion time may last up to 6 hours. longer administration time is inconvenient for the patient as well as nurses. with implementation of the rapid infusion method as previously mentioned, h. lee moffitt cancer center administers rapid infusion rituximab at an initial rate of 150 ml / hr for 30 minutes followed by an increase of the rate to 275 ml / hr until completion of infusion. our method of infusion varies from the package insert recommendation ; however, the average infusion time was 90 minutes for our patients. with our method, over one hour of chair time rapid rituximab infusion improves the turn - around time for each patient and decreases resource utilization in the infusion center. with the low incidence of infusion - related reactions in combination with short administration time | rituximab is an anti - cd-20 monoclonal antibody used in the management of lymphoproliferative disorders. the use of maintenance rituximab has improved progression free survival and overall survival in follicular lymphomas. although rapid rituximab infusions have been studied extensively, there is little data on the use of rapid infusions during maintenance therapy for low grade lymphomas. the primary objective of this retrospective analysis was to evaluate the incidence of grade 3 and 4 toxicities with maintenance rapid infusion rituximab according to the common terminology criteria for adverse events version 4 (ctc v. 4). secondary objectives included evaluating all grade infusion related adverse events and correlation of adverse events with varying schedules of rituximab maintenance therapy. all patients who received rapid infusion rituximab as maintenance therapy for low grade lymphoma between december 2007 and november 2011 were included. rapid rituximab infusions were administered over 90 minutes. demographic, laboratory and clinical data were collected. a total of 109 patients received 647 rapid rituximab infusions. three patients experienced an adverse reaction which resulted in one grade 1 infusion reaction and three grade 3 infusion reactions. no patients required hospitalization. all 3 patients received pharmacological and/or supportive care to relieve symptoms associated with the reaction. |
rats were maintained in rooms with controlled temperature and light at 23 3c and 14 h light/10 h dark cycle (lights on at 0500 h). adult female rats showing at least two regular four - day estrous cycles were mated. the day of parturition was defined as day 0 (d0) of lactation and the number of pups was adjusted to eight on d1. pups were removed from their dam on d21 of lactation and mid - lactational forced weaning was performed on d10. all experiments were performed according to the guidelines for animal experiments of kitasato university and approved by the committee for laboratory animals, care and use at school of veterinary medicine, kitasato university. inguinal mammary tissues were collected from rats on d12, 21, 22, 23, or two days after forced weaning on d10. the tissues of d21 (weaning day) rats were harvested at 0 or 6 h after pup removal. the tissue samples were snap - frozen in liquid nitrogen and stored at 80c until rna extraction. total rna was extracted using trizol reagent (invitrogen, carlsbad, ca, usa) and then reverse - transcribed into cdna using a high capacity cdna reverse transcription kit (applied biosystems, foster city, ca, usa). reverse transcription - pcr (rt - pcr) for gnrh - r or ribosomal protein l19 (rpl19) was performed using premix taq (ex taq version 2.0 ; takara bio, shiga, japan) for 35 or 22 cycles, respectively, at 94c for 30 sec, 58c for 30 sec, and 72c for 60 sec, with an initial denaturing step at 94c for 2 min and a final elongation step at 72c for 7 min. primers used in the current studytargetprimer (5-3)gnrh - rforward : aatcatcttcgccctcacacreverse : agcacgggtttagaaaagcagnrh - r exon 1forward : ccgtccttggagaaatatggreverse : agcggcatgacgattagagtgnrh - r exon 2forward : tcttcaggatgatctacctagccreverse : cctgatgaaggactcgtgtggnrh - r exon 3forward : ccaagaataatatcccaagagcareverse : tcccgtatatgggtttcagcrpl19forward : ggaagcctgtgactgtccatreverse : ccatgagaatccgcttgttt. amplified products were separated by 2% agarose gel electrophoresis and detected by ethidium bromide staining. inguinal mammary tissues were also collected from rats each day from d20 to 24 for western blot analysis. the tissues of d21 (weaning day) were harvested at 0 or 6 h after pup removal. the samples containing 20 g of protein were electrophoresed on 12% polyacrylamide gels (bio - rad, hercules, ca, usa) and transferred onto polyvinylidene fluoride membranes (bio - rad). membranes were blocked with 5% skim milk (wako pure chemicals, osaka, japan) for 1 h at room temperature and then incubated with primary antibodies : anti - gnrh receptor, mouse monoclonal antibody (1:200 ; acris antibodies gmbh, herford, germany), or anti--actin mouse monoclonal antibody (1:1,000 ; c4, santa cruz biotechnology, santa cruz, ca, usa), overnight at 4c. the gnrh - r antibody was raised by immunization of the n - terminal 129 amino acid peptide of human gnrh - r, which shows 82.8% homology with rat gnrh - r and less than 40% homology with other rat proteins. after washing, the membranes were incubated with peroxidase - conjugated goat igg fraction to mouse igg (1:20,000 ; icn pharmaceuticals, aurora, oh, usa) for 2 h at room temperature. immunoreactive protein was detected with ecl plus western blotting detection reagents or ecl prime western blotting detection reagents (ge healthcare, little chalfont, uk). the signal was detected by exposure of the membrane to an x - ray film for 5, 10, or 15 min with an imagequant las 4000 digital imaging system (ge healthcare). rt - pcr examination for gnrh - r mrna showed no amplification in the mammary tissues of lactation d20, a day before weaning (fig. rt - pcr was performed with total rna isolated from mammary tissues of three rats each on day 21 before weaning and 6 h after weaning on days 21, 22, and 23. the primer sets for gnrh - r and rpl19 mrna were designed to yield 251-bp and 264-bp fragments.). gnrh - r mrna was dramatically increased over time until two days after weaning on d23 (fig. next, the expression was decreased to trace levels on d26 and 29 (fig. this indicates that the expression of gnrh - r is well - regulated and that the cessation of suckling stimuli triggers this expression. ikeda. found that gnrh mrna was expressed in the mouse mammary tissues during the lactating and involution periods, but did not detect gnrh - r mrna by pcr. did not determine the regulation of gnrh - r mrna expression during the narrow period just after weaning. rt - pcr was performed with total rna isolated from mammary tissues of three rats each on day 21 before weaning and 6 h after weaning on days 21, 22, and 23. the primer sets for gnrh - r and rpl19 mrna were designed to yield 251-bp and 264-bp fragments. we further confirmed the expression of gnrh - r in mammary tissues with different sets of primers in forced weaned rats. the gnrh - r gene consists of three exons and encodes 984 base pairs through exons 13 in rodents [33, 34 ]. mammary tissues were collected from a forced weaned rat after two days and from a lactating rat. (a) primers for exons 13 of the gnrh - r gene were used. rt - pcr was performed on dnase - treated total rna isolated from the mammary tissues on lactation day 12 (d12) and 2 days after forced weaning on day 10 (fw). amplification of gnrh - r mrna (lane 14 ; exon 1, lane 58 ; exon 2, lane 912 ; exon 3) was performed using templates with or without reverse transcription reaction (rt) to prevent the amplification of genomic dna. three primer sets specific to exons 1, 2, and 3 were designed to amplify 306-, 201-, and 271-bp amplicons, respectively. (b) rpl19 was used as an internal control for the two rats used (lane 1316).). this result again revealed that the cessation of suckling induces gnrh - r mrna expression in the mammary tissues and that full - length mrna is synthesized. (a) primers for exons 13 of the gnrh - r gene were used. rt - pcr was performed on dnase - treated total rna isolated from the mammary tissues on lactation day 12 (d12) and 2 days after forced weaning on day 10 (fw). amplification of gnrh - r mrna (lane 14 ; exon 1, lane 58 ; exon 2, lane 912 ; exon 3) was performed using templates with or without reverse transcription reaction (rt) to prevent the amplification of genomic dna. three primer sets specific to exons 1, 2, and 3 were designed to amplify 306-, 201-, and 271-bp amplicons, respectively. (b) rpl19 was used as an internal control for the two rats used (lane 1316). we previously reported that the expression of annexin a5 is dramatically increased in the epithelial cells of mammary tissues. we predicted that gnrh acts locally in the mammary gland during post - lactational involution. we also published several reports regarding the relationship between gnrh and annexin a5 in various tissues. currently, the physiological function of annexin a5 is unknown, but it is thought to be involved in apoptosis and tissue remodeling. the present data support that gnrh affects degeneration and tissue remodeling in the mammary epithelium after lactation. to confirm the translation of gnrh - r mrna, western blotting analysis using a gnrh - r antibody against the n - terminal peptide sequence was performed. the results confirmed two immunoreactive bands, approximately 60 kda and 30 kda proteins, in the lactating and post - lactating mammary tissues. (a, b) western blotting was performed with mammary tissue samples on lactation day 20 (d20) and 6 h after weaning on day 21 (d21 + 6h), day 22 (d22), day 23 (d23), and day 24 (d24). (c, d) western blotting was performed on the mammary tissues from d21 (on weaning) and d23, and the anterior pituitary tissue. -actin is used as an internal control.). a previous study that also used a gnrh - r monoclonal antibody against the n - terminal 129 amino acid residues, which differed from the antibody used in the present study, detected an approximately 60-kda protein in the rat pituitary gland. gnrh - r in mice and rats is a seven - transmembrane, g - protein - coupled receptor of 327 amino acid residues with two or three n - terminal glycosylation sites and can be detected at 5570 kda by sds - page. this band was increased after weaning and reached a peak on d23, coinciding with mrna results. interestingly, there was another 30-kda immunoreactive band, which decreased after weaning. although the sequence of the 30-kda protein is not known and was observed even during lactation while there was no detectable level of gnrh - r mrna, the gnrh - r variant may have been present alternatively, impairment of post - translational glycosylation may produce a gnrh - r with a lower apparent molecular weight on sds - page. future studies are needed to investigate the post - transcriptional or post - translational modification of gnrh - r in the mammary gland. (a, b) western blotting was performed with mammary tissue samples on lactation day 20 (d20) and 6 h after weaning on day 21 (d21 + 6h), day 22 (d22), day 23 (d23), and day 24 (d24). (c, d) western blotting was performed on the mammary tissues from d21 (on weaning) and d23, and the anterior pituitary tissue. time - specific expression of gnrh - r in the mammary gland after pup removal strongly suggests that the expression is related to changes in the endocrine milieu after lactation, specifically the cessation of massive prolactin release. we previously reported that prolactin suppresses annexin a5 expression in the corpus luteum, suggesting negative control of gnrh function by prolactin. experimental suppression of prolactin release caused luteal regression, and a gnrh antagonist was shown to inhibit the process of apoptosis. further studies are needed to clarify the role of gnrh in apoptosis and the signaling pathways involved in the post - lactational mammary gland. in summary, we detected a post - lactational increase in gnrh - r expression in rat mammary tissues. these results suggest that local gnrh is involved in the involution of mammary tissues after weaning to induce epithelial apoptosis and tissue remodeling. conflict of interests : none of the authors have any potential conflicts of interest associated with this research. | gonadotropin - releasing hormone (gnrh) is a neurohormone of the hypothalamus controlling pituitary gonadotropin secretion and hence gametogenesis. while it has also been believed that gnrh is synthesized and functions in various peripheral tissues, the expression of gnrh receptor (gnrh - r) in peripheral tissues is not well - described. we previously found that annexin a5, which is increased in the pituitary gonadotropes by gnrh, is dramatically increased in rat mammary epithelial cells after weaning, suggesting that local gnrh is responsible for this increase. annexin a5 is a member of the annexin family of proteins and is thought to be involved in various regulatory mechanisms, including apoptosis. in the present study, we examined gnrh - r expression in the mammary tissues after weaning. although gnrh - r mrna was not detected in the mammary tissues during lactation, it was dramatically increased after weaning. forced weaning at mid - lactation (day 10) also promoted the expression of gnrh - r transcripts in mammary tissues within 2 days. furthermore, western blotting analysis with anti - gnrh - r showed that the expression of an immuno - positive 60-kda protein, whose size was equivalent to that of rat gnrh - r, was confirmed to increase after weaning. these findings clarified the induction of gnrh - r in the mammary tissues after weaning and suggest that gnrh is involved in the involution and tissue remodeling of post - lactating rat mammary tissues. |
hodgkin lymphoma (hl) is a potentially curable lymphoma with distinct histology, biologic behaviour, and clinical characteristics. the reported five - year event - free survival ranges between 80 and 90% with combined modality chemotherapy and radiotherapy. despite the high cure rate with initial therapy, approximately 5% to 10% of patients have refractory disease, and 10% to 30% patients relapse after an initial complete response. autologous stem cell transplantation (asct) is the standard of care for patients with relapsed hl. about half of all patients undergoing asct are rescued and definitely cured by such an approach, but the outcome of patients relapsing or refractory to second - line chemotherapy and asct is dismal, with a median survival of less than three years. one of the most important and widely accepted prognostic factors for patients undergoing asct appears to be chemosensitivity at relapse, with patients responding to second - line chemotherapy and having a much better outcome than patients with refractory disease, whose relapse rate approaches 80% in some published series [5, 6 ]. in the functional imaging with positron emission tomography (pet) era, pet positive response at the end of induction therapy has been found to be the worst predictor of outcome [7, 8 ]. there are a few published literatures on the treatment options of patients with rr - hl after autotransplant. this paper summarizes the current available treatment modalities in these patients with emphasis on novel drugs. a diagnostic rebiopsy should be considered to confirm relapse or progressive disease if the primary diagnosis was not clear and if the relapse is late (beyond 35 years of therapy) or unusual in pattern and in pet positive lesions whenever feasible. these include radiotherapy, second asct, allogeneic stem cell transplant (allo - sct), monoclonal antibodies, chemotherapeutic drugs, and novel agents [9, 10 ]. a significant number of patients who relapse after stem cell transplant do so in previously involved sites and may present with disease that could be encompassed in a radiation field. this strategy appears most beneficial in those who present with ann arbor stage i or ii disease at relapse, without b symptoms, and no extranodal disease.. reported 5-year freedom - from - treatment failure (fftf) of 28% in patients receiving either extended - field or involved - field radiotherapy. involved - field radiation is an important option when recurrent disease extends beyond previously unirradiated lymph nodes. radiation in a prior radiation field should be considered if tissue tolerance allows ; however there is little information to support this. this option seems to be feasible for patients who relapse > 1 year after the initial transplant. a recent report from the center for international blood and marrow transplant research (cibmtr) on 40 patients undergoing second transplants included 21 patients with hl : outcomes for patients relapsing within 12 months of the first transplant were very poor, but for those with relapse > 3 years, progression - free survival (pfs) and overall survival (os) were 25% and 38%, respectively. allo - sct offers the only chance of cure for suitable patients after failed asct ; however selecting the best conditioning regimen is still controversial. myeloablative strategies achieve cure in some patients, but at the cost of high transplant - related mortality (trm), whereas reduced - intensity conditioning (ric) regimens are associated with high posttransplant relapse rates. a report from the international bone marrow transplant registry of 114 patients with lymphoma undergoing myeloablative allogeneic transplants reported a rate of disease progression at 3 years of 52% and trm of 22%. this translated to a relatively disappointing 3-year pfs of 25% and os of 33%. with further followup it was found that 5-year disease - free survival (dfs) and os were 5% and 24%, respectively. based on the assumption of allogeneic graft versus lymphoma (gvl) effect, reduced - intensity conditioning was introduced which resulted in a decreased cumulative incidence of nonrelapse mortality (nrm) ranging from 11% to 13%. nevertheless, survival outcomes were relatively unchanged, as approximately 50% of all patients undergoing allogeneic sct after ric relapsed [14, 15 ]. published the results of a retrospective multicenter study on 185 relapsed / refractory hl patients. in this study, outcomes were correlated with donor availability. the patients from the donor group experienced improved 2-year os and pfs as compared with those from the no donor group (os : 66% versus 42%, pfs : 39% versus 14%, p 6 months after asct. for patients who are not candidates for allogeneic stem cell transplant, single - agent or combination chemotherapy with gemcitabine or vinorelbine patients with early relapse (5 years from asct may be taken up for a second autologous transplant, but the long - term benefits are not known. the use of novel biological and targeted therapies is a promising approach but where to put them in treatment algorithm is still not clear. as the armamentarium for the treatment of patients with rr - hl continues to expand, | despite a high clinical success, relapse in hodgkin lymphoma occurs in 1030% of cases and 510% patients are nonresponsive to initial chemotherapy. the standard management of these patients includes high - dose chemotherapy followed by autologous stem cell transplant. however, 50% of patients ultimately relapse after autotransplant which poses a big challenge. allogeneic stem cell transplantation offers the only chance of cure in these patients. for patients who are not candidates for allogeneic stem cell transplantation, achieving cure with other possible options is highly unlikely, and thus the treatment plan becomes noncurative. various novel agents have shown promising results but the duration of response is short lived. a standard approach to deliver the most effective treatment for these patients is still lacking. this review focuses on the treatment options currently available for relapsed and refractory disease after autotransplant. |
the major problem of children with cerebral palsy is an inability to use movement control for balance as well as an inability to use the hands for reaching and manipulation in daily activities2. in daily life activities, therefore, children s physical capabilities must be evaluated in a real occupational environment. several well - established classification systems are used to characterize the functional abilities of children with cerebral palsy as they participate in their daily activities. the classification systems are divided in two types : the gross motor function classification system (gmfcs) and the manual ability classification system (macs). gmfcs scores range from level 1, which includes children with no disability and community mobility, to level 5, which includes children who are totally dependent on assistance for mobility4. the macs provides information for classifying the manual abilities of children with cerebral palsy, whether they are using their hands for manipulation or not4. both classification systems are used to evaluate children s functional status in daily routine activities. the skill of performance in daily routine activities is very important in evaluating children with cerebral palsy. in general, children s performance skill in daily activities should reflect real functions while they perform real activities such as dressing and hygiene, which are influenced by environmental, personal and contextual factors5. therefore, real performance - based evaluation is important in correctly assessing a child s degree of functional skill. nowadays, for performance - based evaluation, the assessment of motor and process skill (amps) or school amps is used. the amps is used to evaluate performance skills when children and adults perform daily tasks. it comprises two main areas : adl motor skill and adl process skill6. according to fisher, the amps adl motor skills refer to observable actions of performance used to move oneself or task objects, such as reaching, alignment and bending. process skills refer to observable actions, such as the ability to logically organize actions in time and space and the appropriate use of task tools. motor skills refer in particular to the gross and fine motor capability of an individual when performing a daily occupation7. various studies have reported on the strengths of the performance - based evaluation. to date, there has been little published evidence regarding the utility of performance - based evaluations in assessing functional ability in children with cerebral palsy. the aim of this study was to investigate the predictive utility of gmfcs and macs levels with respect to the performance - based adl motor skills of children with cerebral palsy. this study included 23 children with cerebral palsy, who attended a pediatric rehabilitation hospital in seoul. all children and their caregivers were informed about the purpose of this study in accordance with the ethical principle of the declaration of helsinki (1975, revised 1983). the study subjects were children with cerebral palsy aged 712 years old, with mild to moderate spastic cerebral palsy, who had been categorized as gmfcs and macs levels i iii. children with mixed diagnoses (e.g., severe visual and auditory problems or intellectual disability) were excluded. the gmfcs and macs were used to assess the functional capabilities of children with cerebral palsy. notably, the gmfcs uses five levels to classify those who are being evaluated., patients classified as level i handle objects easily and successfully, while those classified as level iv handle a limited selection of easily managed objects in adapted situations. those classified as level v do not handle objects4. for the amps assessment, participants were asked to complete functional tasks such as dressing, donning shoes and preparing cereal and milk. notably, motor skills represent the gross and fine motor capability of a person, while process skill is more related to cognitive and mental process capability. descriptive statistics were used to analyze the demographic characteristics of the children who participated in the study (table 1table 1.general characteristics of all children (n=23)mean sd [range]frequency (%) age (years)9.04 1.02 [811]genderboys14 (60.9)girls9 (39.1)gmfc leveli2 (8.7)ii7 (30.4)iii14 (60.9)iv0v0macs leveli2 (17.4)ii13 (56.5)iii6 (26.1)iv0v0). one - way anova and the post - hoc test in combination with scheffe s method were used to determine the mean difference in adl motor skills among gmfcs and macs levels. multiple regression analysis was used to determine the relationships among adl motor skill, gmfcs level and macs level. adl motor skills differed significantly among gmfcs (p=0.002) and macs levels (p=0.026). a post - hoc test revealed a significant difference between gmfcs levels i and iii (p=0.009) as well as between levels ii and iii (p=0.019). there was no significant mean difference in adl motor skill between gmfcs levels i and ii. there were also significant mean differences in adl motor skill between macs levels i and iii (p=0.026) (table 2table 2.mean difference of adl - motor skill among gmfc and macs levels (n=23)level ilevel iilevel iiiaveragepost - hocadl - motor skill among gmfcs level117.50 3.5399.85 11.3378.50 16.6388.39 19.50a = b, a > c, b > c adl - motor skill among macs level108.75 10.3087.76 18.1776.16 17.4788.39 19.50 a = b, a > c, b = csignificant difference at 0.002 level, significant difference at 0.026 level. a = level i, b = level ii, c = level iii). in comparison to gmfcs level i, level iii adl motor skills were 39.0 points lower (p=0.002). this regression model s explanation power was 47.6% (table 3table 3.multiple regression model to predict adl - motor skill using gmfcs level (n=23)gmfcs levelbsetrconstant117.50 10.4611.250.476level ii17.6411.861.48level iii39.0011.193.48significant level at<0.0001, significant level at 0.002. b : coefficient of regression, se : standard error, t : calculated value, r : coefficient of determination). in comparison to macs level i, level ii adl motor skills were 20.98 points lower (p<0.0001) ; level iii adl motor skills were 32.58 points lower (p=0.044). this regression model s explanation power was 30.6% (table 4table 4.multiple regression model to predict adl - motor skill using macs level (n=23)gmfcs levelbsetrconstant108.75 8.5212.760.306level ii20.989.742.15level iii32.5811.002.96significant level at<0.0001, significant level at 0.044, significant level at 0.008. b : coefficient of regression, se : standard error, t : calculated value, r : coefficient of determination). significant difference at 0.002 level, significant difference at 0.026 level. a = level i, b = level ii, c = level iii significant level at<0.0001, significant level at 0.002. b : coefficient of regression, se : standard error, t : calculated value, r : coefficient of determination significant level at<0.0001, significant level at 0.044, significant level at 0.008. b : coefficient of regression, se : standard error, t : calculated value, r : coefficient of determination children with cerebral palsy exhibit poor participation in daily activities due to motor problems such as abnormal postural tone and poor body control8. this study demonstrated the clinical utility of a performance - based assessment based on the amps motor skill test in predicting the gross and manual functions of children with cerebral palsy. first, adl motor skills in children differed significantly among gmfcs and macs levels. this result was reported previously9. according to james., amps motor skills are related to hand function in unilateral cerebral palsy9. another previous study demonstrated correlations among gmfcs, macs and wee fim scores.. showed a significant correlation among gmfcs scores, macs scores and the self - care, mobility and locomotion scores of the wee fim10. moreover, in this study, multiple regression showed that the adl motor skills of children with gmfcs level iii scored lower than those of children categorized as gmfcs level i. also, in comparison to children classified as macs level i, children labeled as macs level ii and iii had inferior adl motor skills. thus, adl motor skills differ significantly among gmfcs level iii, macs level ii and macs level iii among children with cerebral palsy. these results suggest that adl motor skills can be used to predict gmfcs or macs level in children with cerebral palsy. only spastic - type cerebral palsy patients classified as gmfcs or macs levels i iii participated. thus this study could not characterize the relationship between adl motor skills and other gmfcs or macs levels. future research is necessary to involve children with various type of cerebral palsy such as hemiplegia, diplegia and ataxic type. furthermore, future research need to investigating adl motor skills that correlation with other gmfcs or macs levels. | [purpose ] the purpose of this study was to determine effects of gross motor function classification system and manual ability classification system levels on performance - based motor skills of children with spastic cerebral palsy. [subjects and methods ] twenty - three children with cerebral palsy were included. the assessment of motor and process skills was used to evaluate performance - based motor skills in daily life. gross motor function was assessed using gross motor function classification systems, and manual function was measured using the manual ability classification system. [results ] motor skills in daily activities were significantly different on gross motor function classification system level and manual ability classification system level. according to the results of multiple regression analysis, children categorized as gross motor function classification system level iii scored lower in terms of performance based motor skills than gross motor function classification system level i children. also, when analyzed with respect to manual ability classification system level, level ii was lower than level i, and level iii was lower than level ii in terms of performance based motor skills. [conclusion ] the results of this study indicate that performance - based motor skills differ among children categorized based on gross motor function classification system and manual ability classification system levels of cerebral palsy. |
autism typically develops in childhood, and it is considered as a systemic spectrum disorder with multiple development trajectories with an incidence four times higher in males than in females (grossi and terruzzi 2014). in addition to behavioural traits, gi abnormalities such as diarrhoea, constipation, bloating and abdominal pain are common in autism and they seem to contribute to, and exacerbate, overall behaviour of children (irritability, sleeplessness, posturing) (van de sande, van buul and brouns 2014). a crosstalk exists between the gut microbiota and central nervous system (cns) mediated via a range of different chemical, immunological and signalling interactions that form part of the gut brain axis. several studies have demonstrated the role of the gut microbiota in neurodevelopment and mental health (foster and mcvey neufeld 2013), and there is increasing evidence associating gut microbial dysbiosis with gi problems that might affect autistic children. (2002) found nine unique species of clostridia in autistic children compared with controls. song, liu and finegold (2004), using qpcr analysis, found higher levels of clostridium bolteae and clostridium clusters i and xi. furthermore, parracho. (2005), using fish analysis, found greater number of species derived from the c. histolyticum group (clostridium clusters i and ii). desulfovibrio group was found to be 10 times higher in the gut microbiota of autistic children compared with controls (finegold. high - throughput sequencing has been used in more recent studies to determine bacterial composition of faecal samples from autistic children. the genera prevotella, coprococcus and unclassified veillonellaceae have been found in lower abundance in autistic individuals (kang. in addition, bifidobacterium species decreased in asd, comparing with the non - autistic control (de angelis. metabolic associations have also been identified with asd and may be attributed to gut dysbiosis in autistic individuals. abnormalities have been reported in tryptophan metabolism where higher amount of indole derivatives in the blood and higher levels of iag (indolyl - acryloyl - glycine) in the urine of autistic children have been identified. may contribute to these metabolic alterations as these organisms can metabolise tryptophan (bingham 2003). 2010) and amino acid deficiencies in autism with restricted diets, modified gut microbial population and gi symptoms being suggested as potential contributors (ming. modulation of gut microbiota is an interesting potential strategy to reduce presence of harmful microorganisms and their metabolites that might be involved in negative stimulation of cns and affect behaviour (shaw, kassen and chaves 1995 ; sandler. 2000). treating gi disorders in asd with antibiotics or pro / prebiotics has been postulated to regulate microbiota and improve gut symptoms, but the evidence is scarce, especially for prebiotics. the bifidogenic properties of b - gos (bimuno, clasado biosciences ltd, buckinghamshire, uk) have been investigated in vitro and in human intervention studies involving healthy volunteers, and conditions that have a purported microbial input such as ibs, travellers diarrhoea and obesity (tzortzis. recently, b - gos was also shown to reduce cortisol secretion and anxiety in healthy volunteers (schmidt. b - gos supplementation lowered cortisol reactivity and modulated attention to emotional stimuli compared with a placebo group, supporting the hypothesis that gut microbiota might have a role in behavioural traits (schmidt. our study aimed to assess the effects of b - gos (65% gos content) on gut microbial ecology and metabolic end products of microbial fermentation. we used in vitro, three - stage, continuous gut model systems, inoculated with faecal samples of autistic and non - autistic children, which simulated different physicochemical characteristics of the proximal, transverse and distal colons. the mixture was in syrup format consisting of 65% (w / v) gos, 10.1% (w / v) lactose, 22% (w / v) glucose and 1.8% (w / v) galactose. faecal samples were obtained from three non - autistic children and three autistic child donors (male, aged 510 years old) who were free of any metabolic and gastrointestinal diseases, were not taking probiotic or prebiotic supplements and had not taken antibiotics 6 months before faecal sample donation. all parents were then provided written informed consent for use of their children 's faeces in the study. this study was approved by the university of reading research ethics committee (urec 15/20). faecal samples were placed in an anaerobic jar (anaerojartm 2.5 l, oxoid ltd) including a gas - generating kit (anaerogentm, oxoid). an aliquot of 20 g of samples was diluted in 100 ml anaerobic pbs (0.1 mol / l phosphate buffer solution, ph 7.4, w / w) and homogenised (stomacher 400, seward, west sussex, uk) for 2 min at 240 paddle beats per minute. physicochemical conditions in the colon were replicated in a continuous culture system, comprised of a cascade of three glass fermenters of increasing working volume connected in series. a small - scale version of the validated system described by macfarlane, macfarlane and gibson (1998) was used in this study, with vessels (v) representing the proximal (v1, 80 ml, ph = 5.5), transverse (v2, 100 ml, ph = 6.2) and distal colon (v3, 120 ml, ph = 6.8). the systems were inoculated with 20% (wt : v) faecal homogenate from either non - autistic and autistic children volunteers in a growth medium (macfarlane, macfarlane and gibson 1998). following inoculation, the colonic model was run as a batch culture for 24 h in order to stabilise bacterial populations prior to the initiation of medium flow. after 24 h (t0), the medium flow was initiated and the system ran for at least 8 full volume turnovers to allow for steady state to be achieved (ss1). short - chain fatty acid (scfa) profiles (5%) were assessed before starting b - gos administration. taking into account the operating volume (300 ml) and the retention time (48 h, flow rate 6.25 ml / h) of the colonic model system, a syrup containing gos (2 g / daily, equivalent to 1 g of gos) was added daily into v1. the syrup was added to the system for at least a further 8 volume turnovers upon which steady state 2 (ss2) was achieved. the production of scfas in the fermentations was determined by hplc (merck, nj) as previously described by rodriguez - colinas. peaks were integrated using atlas lab managing software (thermo lab systems, mainz, germany). quantification of the samples was obtained through calibration curves of lactic, acetic, propionic, butyric and formic acids in concentrations 12.5, 25, 50, 75 and 100 mm, respectively. bacterial composition in the gut models was analysed for using fluorescence in situ hybridization combined with flow cytometry (fish - fcm). seven hundred and fifty microlitres of samples were centrifuged at 1136 g for 5 min. pellets were resuspended in 375 l of filtered pbs (using a 0.22-m pvdf membrane) and fixed in 1125 l of 4% (v / v) paraformaldehyde. after 4 h of incubation at 4c, samples were washed twice using 1 ml of pbs, resuspended in 600 l pbs - ethanol (1:1, v / v) and stored at 20c. permeabilisation steps were performed using 30 l of the fixed samples added to 500 l pbs and centrifuged at 1136 g for 3 min. pellets were resuspended using 100 l of filtered te - fish (tris / hcl 1 m ph 8, edta 0.5 m ph 8, distilled h2o, 0.22 m pvdf membrane) containing lysozyme (1 mg / ml of 50 000 u / mg protein) and incubated for 10 min at room temperature. solutions containing the samples were then vortexed and centrifuged at 1136 g for 3 min. pellets were washed with 500 l pbs and centrifuged (1136 g, 3 min). hybridisations were performed by resuspending the pellets in 150 l of hybridisation buffer (5 m nacl, 1 m tris / hcl ph 8, 30% formamide, ddh2o, 10% sds), vortexed and centrifuged (1136 g, 3 min). pellets were then resuspended in 1 ml of hybridisation buffer and 50 l aliquoted into eppendorf tubes. the probes used (sigma aldrich ltd, poole, dorset, uk) are reported in table 1 (devereux. 1992 ; wallner, amann and beisker 1993 ; langendijk. 1995 ; poulsen. 1995 ; manz. 1996 1999, 2000, 2002 ; hold.2003 ; lay. 2005 ; walker. 2005 ; kong. non eub338 and eub338 i - ii - iii linked at their 5 end either to alexa488 or alexa647. four microlitres of each probe and 4 l of eub338 i - ii - iii (linked to alexa488) were added to the working solution and incubated overnight at 35c in a heating block. after 12 h of incubation, an aliquot of 150 l hybridisation buffer was added to the working solution, vortexed and centrifuged (1136 g, 3 min). one hundred and fifty microlitres of supernatant were removed from each sample and the remaining volume was centrifuged (1136 g, 3 min). the pellets were washed with 200 l of washing buffer (5 m nacl, 1 m tris / hcl ph 8, 0.5 m edta ph 8, ddh2o, 10% sds), homogenised by vortexing and incubated for 20 min at 37c in a heating block. afterwards the samples were centrifuged (1136 g, 3 min) and supernatants were removed. negative control samples (no probes added) were screened by fcm to detect background before the probe samples were resuspended in an appropriate amount of pbs. numbers of specific and total bacteria were determined taking into account dilution factor, calculated from different volumes used in samples preparation steps, and events/l obtained from non eub338 and eub338 i - ii - iii probes analysed by fcm. three consecutive days of the three biological replicates for each group (autistic and non - autistic) of all time points (before and after treatment) were analysed by h - nmr (n = 27, each group). fermentation supernatants were defrosted, vortexed and centrifuged at 599 g for 5 min. the supernatants were filtered using 0.22 m low protein binding durapore polyvianylidene fluoride (pvdf) membranes (millex ; emd millipore, billerica, ma, usa) and 400 l transferred into fresh eppendorf tubes. filtered samples were combined with 200 l of phosphate buffer (0.2 m (ph 7.4) in d2o plus 0.001% tsp), mixed by vortexing, centrifuged at 1136 g for 10 min and then 550 l was transferred into 5 mm nmr tubes for analysis. all nmr spectra were acquired on a bruker avance drx 500 mhz nmr spectrometer (bruker biopsin, rheinstetten, germany) operating at 500 mhz. they were acquired using a standard 1d pulse sequence [recycle delay (rd)-90-t1 - 90-tm-90-acquire free induction decay (fid) ] with water suppression applied during rd of 2 s, a mixing time tm of 100 ms and a 90 pulse set at 7.70 s. for each spectrum, a total of 128 scans were accumulated into 64 k data points with a spectral width of 12 001 ppm. all spectra were manually phased, baseline corrected and calibrated to the chemical shift of tsp (3-(trimethylsilyl)-[2,2,3,3,h4]-propionic acid, 0.00). spectra were digitised using an in - house matlab (version r2014a, the mathworks, inc. ; the spectral region containing the water resonance was removed to minimise distortions in the baseline arising from imperfect water saturation. median fold normalization was performed for both groups : non - autistic and autistic children. before and after administration of b - gos, principal components analysis (pca) using mean - centred data was applied. orthogonal projection to latent structure discriminant analysis (opls - da) models was constructed using unit variance scaling for pairwise comparisons of the different experimental groups and time points. correlation coefficients plots were generated from the model outputs by back scaling transformation to display the contribution of each variable (metabolites) to sample classification (e.g. before and after treatment). colour represents the significance of correlation (r) for each metabolite to class membership. predictive strength (qy) of the models was obtained using a 7-fold cross - validation method and these were validated using permutation testing (number of permutations = 10 000). data from hplc and fmc - fish analyses were analysed using paired t - test in order to assess significance of results, comparing the two time points ss1 and ss2, before and after treatment, respectively. analyses were performed using graphpad prism 5.0 (graphpad software, la jolla, ca, usa). changes in bacterial compositions in gut model systems are reported in fig. 1. the data showed lower numbers of bifidobacteria in asd models compared with non - autistic ones., following the addition of b - gos to models containing both autistic and non - autistic samples, were seen. in autistic models, a significant increase of bifidobacteria occurred from 5.32 to 7.27 log10 cells / ml (p < 0.01), from 4.81 to 6.79 log10 cfu / ml (p < 0.001) and from 5.57 to 6.83 log log10 cells / ml (p < 0.05), in v1, v2 and v3, respectively. a slight but significant increase in clostridium cluster xi in v2 for autistic children was also found, as well as significant decrease in v2 in veillonellaceae group from 6.06 to 5 log10 cfu / ml (p < 0.05). in non - autistic models, there was a significant increase in numbers of bifidobacteria in v1, from 5.83 to 7.16 log log10 cells / ml (p < 0.01), and in v3, from 4.97 to 6.73 log10 cells / ml (p < 0.001) and in lactic acid bacteria (lab158) in v3 from 5.13 to 6.01 log10 cells / ml (p < 0.05). additionally, b - gos slightly increased roseburia spp. in v1 and v3 (p < 0.05) and / ml and faecalibacterium prausnitzii from 6.78 to 5.27 log10 cells / ml (p < 0.05 for both) in the second vessel, while increasing atopobium spp. from 5 to 5.92 log10 cells / ml (p < 0.05) in the third vessel of non - autistic models. in these models, numbers of clostridium coccoides eubacterium rectale were also increased from 6.76 to 7.08 log10 cells / ml (p < 0.01) in v1 and sutterella spp. significantly decreased in v1 from 7.05 to 6.49 (p < 0.01) and v2 from 7.02 to 6.37 log10 cfu / ml (p < 0.05) after b - gos administration. there was a general trend to increase all other bacterial groups analysed in all vessels but this was not significant. exceptions were seen for bacteroides (v1), clostridial cluster ix (v1), f. prausnitzii (v1), escherichia coli (v3), ruminococcus spp., clostridium leptum (v2), sutterella spp. and veillonellaceae (all vessels) in autistic models, and for clostridium coccoides eubacterium rectale (v2), atopobium spp. (v1), clostridial cluster ix (v2), clostridium cluster xi (v1, v2), e. coli (v2), sutterella spp. and veillonellaceae (all vessels) in non - autistic models that slightly decreased. bacterial groups detected by fish - fcm (log10 cfu / ml) in culture broth recovered from each vessel (v1, v2 and v3) of a colonic model before (ss1) and after (ss2) the daily administration of b - gos (2 g / d, equivalent to 1 g gos). significant difference after the treatment : p < 0.05 ; p < 0.01 ; p < 0.001. (lab158), most bacteroidaceae and prevotellaceae (bac303), clostridium coccoides eubacterium rectale group (erec482), roseburia subcluster (rrec584), f. prausnitzii (fprau655), clostridium cluster xi (clit135), sutterella spp. our data show a lower concentration of butyrate and propionate in autistic models, compared with non - autistic models, but no differences in acetate before adding b - gos into the system. after the administration of b - gos, acetate and butyrate were the main end products of microbial fermentation. supplementation of b - gos to gut models inoculated with faecal samples from autistic children led to a significant increase of acetate and butyrate in v1 and v2, simulating the proximal and transverse colons (p < 0.05), respectively, while concentration of propionate was decreased (p < 0.05) in v3 mimicking distal colon. in models simulating the colon of non - autistic children, the fermentation of b - gos mediated significant production of acetate (p < 0.05) and butyrate (p < 0.001) in v2 and v3, simulating the transverse and distal colon, respectively. there was no effect on propionate. acetate, propionate and butyrate concentrations in culture broths recovered from vessels (v1, v2 and v3) of in vitro gut model systems before (ss1) and after (ss2) administration of b - gos (1 g / daily gos). results are reported as means (mm) of the data (n = 3) : (a) autistic children and (b) non - autistic children. significant difference after the treatment : p < 0.05 ; p < 0.001. pca was performed on mean - centred data to summarise variance with the dataset. the scores plot (pc1 versus pc2) shown in fig. 3a showed separation between autistic and non - autistic models after treatment, indicating that b - gos supplementation contributed to the largest source of variance in the metabolic data. comparison of the spectra profiles from gut models before and after treatment identified that a number of metabolites changed following b - gos supplementation to characterise the metabolic variation associated with asd, b - gos supplementation and differences in microbial response to b - gos between the asd and non - asd microbiota. a significant opls - da model was obtained comparing the metabolic profiles of the autistic and non - autistic models at baseline (qy = 0.07 ; p < 0.05 ; fig. supernatants from the autistic models contained greater amounts of ethanol, glycine, tyrosine, tyramine, 5-aminopentoanate, acetate, 4-aminobutyrate and betaine, and lower amounts of butyrate compared with the non - autistic models. b - gos supplementation was found to modulate the metabolic profile of the autistic models (qy = 0.08 ; p < 0.05) increasing ethanol, lactate, acetate and butyrate and decreasing propionate and trimethylamine (fig. sb - i, supporting information). increased butyrate and acetate production was also observed in the non - autistic models following the addition of bgos (qy = 0.12 ; p < 0.01 ; fig. sb - ii, supporting information). comparing the metabolic profiles of the autistic and non - autistic models after b - gos feeding (qy = 0.17 ; p < 0.01) revealed that some of the metabolic variation was reduced (fig. there was no longer variation in 4-aminobutyrate between the models ; however, the difference in ethanol and acetate between autistic and non - autistic models was increased being higher in the autistic models. h - nmr data analysis. (a) pca score plot shows a separation between models inoculated with stool samples of non - asd and asd children after administration of b - gos. dark and light blue dots represent replicates of samples from gut models inoculated with faecal samples of autistic children, before (ss1) and after (ss2) treatment, respectively. yellow and red dots represent replicates of samples from gut models inoculated with faecal samples of non - autistic children, before (ss1) and after (ss2) treatment, respectively. (b) correlation coefficients indicating the associations of identified metabolites with autism and their alteration upon b - gos administration. recent studies have focused on the effect of pre / probiotics on the gut brain axis (liu, cao and zhang 2015). this study investigated the influence of b - gos on a small scale, in vitro, gut model system inoculated with faeces from autistic and non - autistic children. the results showed a positive modulation of bacterial populations, using an automated fish method combined with fcm. lower concentrations of scfas have previously been found in children with asd by adams. suggesting a reduced fermentation capacity by the asd microbiota. it was hypothesised that this was due to a compromised microbiota characterised by a lower number of bifidobacteria, consistent with microbial signatures observed here (adams. concomitant with these population changes, functional alterations were also observed in both autistic and non - autistic models with acetate and butyrate, the main end products of microbial fermentation, being increased. these fermentation products can cross the blood brain barrier and might influence early brain development. the synthesis of neuroactive compounds such as dopamine and serotonin can be modulated by scfa and they are able to produce reversible psychological and physiological changes in rats similar to those found in asds (wang. experimental evidence using intraventricular infusion in rats indicates that propionic acid can produce brain and behavioural changes similar to asd (macfabe. recent asd studies have shown increase in numbers of sutterella spp. and decrease in veillonellaceae group. in this study, the results did not show any significant differences between asd and non - asd group. however, a general decrease in those bacterial groups after treatment was highlighted, suggesting that b - gos administration might have an impact on the growth of these asd - associated bacteria. following b - gos feeding, the microbiota of autistic children produced greater amount of ethanol and lactate while the amount of amino acids and the scfa propionate, present in the model, was reduced. these metabolic alterations were not observed when the faecal microbiota of non - autistic individuals were fed b - gos. in a healthy colon, lactate production is generally low due to its conversion to other organic acids by many bacteria and because lactate can be used as a substrate for dissimilation of sulphate by some bacteria (e.g. desulfovibrio spp.) (fite. 2004 ; marquet. 2009 ; flint. the presence of lactate is interesting because its accumulation has been associated with neurological problems, in particular studies show the effect of lactate infusions on anxiety and panic disorders (cowley. cowley and colleagues in their findings showed that lactate infusion in patients suffering from panic disorder provokes higher panic symptoms reaction compared with controls (dillon. dillon. have showed similar results in in vivo, where panic and anxiety reaction has been measured using acute panic inventory scores. after lactate infusions, the scores were much higher in patients with panic and anxiety disorders compared with normal controls (cowley. the lysine degradation product, 5-aminopentanoic acid, was also higher in the autistic compared with the non - autistic models. it is believed to act as a methylene homologue of -aminobutyric acid (gaba) and functions as a weak gaba agonist (callery and geelhaar 1985). interestingly, gaba was also higher in the autistic models compared with the non - autistic models pre - treatment but these differences were not present following b - gos treatment. certain bacteria, such as lactobacilli, are able to produce molecules that act as neurotransmitters and directly affect the brain (wall. ethanol was found in higher amount in asd children comparing with non - asd children. the vast majority of bacteria form ethanol from acetyl - coa and the glycolytic pathway (macfarlane and macfarlane 2003). microorganisms are able to oxidase ethanol and the impact of bacterial overgrowth on ethanol production has previously been studied (baraona. metabolism of ethanol can lead to the production of toxic end products such as acetaldehyde, which may affect the gastrointestinal mucosa. the role of acetaldehyde in asd has been recently evaluated in particular for its role in oxidative stress and dna damage. under healthy conditions, ethanol is converted into acetic acid in the liver by a two - step process involving alcohol dehydrogenase and aldehyde dehydrogenase (aldh). mutation of the aldh gene has been shown to increase the accumulation of acetaldehyde and result in cancers within different regions of the gastrointestinal tract and alzheimer 's disease (jurnak 2015). the potential role of this toxic compound in neurological disorders, including autism, warrants further exploration. this in vitro study showed promising and positive results in that supplementing the microbiota of children with asd with 65%b - gos may manipulate the gut bacterial population and alter metabolic activity towards a configuration that might represent a health benefit to the host. however, further work will be required to assess such changes in an in vivo human intervention study. grg and ac were involved in designing and coordination of the study and revising the manuscript critically for important intellectual content. jv and gt are employed by clasado biosciences ltd, who provided the b - gos product, marketed as bimuno(r), used within this research. this does not alter the authors adherence to all the fems policies on sharing data and materials. the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. | abstractchildren with autism spectrum disorders (asd) often suffer gastrointestinal problems consistent with imbalances in the gut microbial population. treatment with antibiotics or pro / prebiotics has been postulated to regulate microbiota and improve gut symptoms, but there is a lack of evidence for such approaches, especially for prebiotics. this study assessed the influence of a prebiotic galactooligosaccharide (b - gos) on gut microbial ecology and metabolic function using faecal samples from autistic and non - autistic children in an in vitro gut model system. bacteriology was analysed using flow cytometry combined with fluorescence in situ hybridization and metabolic activity by hplc and 1h - nmr. consistent with previous studies, the microbiota of children with asd contained a higher number of clostridium spp. and a lower number of bifidobacteria compared with non - autistic children. b - gos administration significantly increased bifidobacterial populations in each compartment of the models, both with autistic and non - autistic - derived samples, and lactobacilli in the final vessel of non - autistic models. in addition, changes in other bacterial population have been seen in particular for clostridium, rosburia, bacteroides, atopobium, faecalibacterium prausnitzii, sutterella spp. and veillonellaceae. furthermore, the addition of b - gos to the models significantly altered short - chain fatty acid production in both groups, and increased ethanol and lactate in autistic children. |
chronic myelogenous leukemia (cml) is a malignant clonal disorder of hemopoietic stem cells characterized by abnormal proliferation and accumulation of immature granulocytes. leukostasis is one of the complications of cml and is characterized by partial or total occlusion of microcirculation by aggregation of leukemic cells and thrombi leading to respiratory, ophthalmic or neurologic symptoms. avascular necrosis of bone occurs in the areas with poor collateral circulation when the only branch supplying blood is unable to fulfill the requirements of the bone. commonly affected areas are head of the femur, head of the humerus, femoral condyles and tibial plateau. very few cases dealing with cml and avascular necrosis due to leukostasis have been reported. here, we report a rare case of 21 year - old male patient with avascular necrosis of right femoral head owing to leukostasis as the initial presentation of cml. a 21-year - old male patient was presented with pain in the right hip joint for 2 months. he was a soldier and went to the dispensary of the unit that he belonged to. he was referred to our hospital for further evaluation of abnormal complete blood cell count which was performed at the dispensary. the patient suffered from weight loss, mild dyspnea, fatigue, intermittent fever, and night sweat., it was observed that the patient was in poor general condition with pallor skin and anemic conjunctiva. laboratory investigations revealed leukocytosis with leukocyte count of 96,800/mm, increased platelets with a count of 684,000/mm, and hemoglobin level of 10.4 g / dl. bone marrow examination revealed that the entire marrow space was packed with granulocytes and megakaryocytes. the bone marrow aspiration showed 0.4% myeloblasts, 6% promyelocytes, 13.2% myelocytes, 15.2% metamyelocytes, 14.7% band neutrophils, 40% segmented neutrophils, 2.7% eosinophils, 0% basophils, 0.8% monocytes, 0.4% pronormoblasts, 0% lymphoblasts, 0% immature lymphocytes, 0.4% lymphocytes, 0.4% plasma cells, and 0% histiocytes suggesting chronic phase of cml. chromosomal analysis of bone marrow revealed philadelphia chromosome, t (9;22) and molecular cytogenetics also showed bcr - abl positive. treatment with hydroxyurea was induced, combined with immediate evaluation of right hip joint pain. radiolucency superimposing the right femoral head was seen on the pelvic x - ray (figure 1). magnetic resonance imaging (mri) of both the hips revealed inhomogenously decreased signal intensity in the both proximal femur and acetabulum on t1 weighted images. the outer areas surrounding these areas had increased signal densities on t2 weighted images (figure 2). on application of contrast medium, area with former decreased signal density on the cranial part of the right femoral head and some smaller areas of the left femoral head, showed a lack of signal enhancement. bilateral joint effusion, worse in the right than the left, was also seen. the leukocyte count decreased from 96,800/ul to 26,300/mm with the treatment of hydroxyurea (3 g / day) for 2 weeks and subsequently hydroxyurea was replaced with imatinib mesyalte. with this the size of the spleen decreased to 3 cm below the costal margin within 1 month. following normalization of the peripheral blood counts, the patient was transferred to the department of orthopedics. he underwent a bipolar hemiarthoplasty of the right hip joint, because the necrosis of the right hip joint was more severe than the left and he had no pain in the left hip joint. the postoperative course was complicated due to bleeding from the operative site possibly due to thrombocytopenia which may have been induced by imatinib mesylate. temporarily, the medication of imatinib mesylate was stopped, and the patient was transfused with platelets and packed red cells. the biopsy revealed focal subchondral necrotic areas in the right hip joint (figure 3). the patient was on a continuous follow up and showed no complications with complete cytogenetic response and has been on medication of imatinib mesylate (400 mg / day). cml is characterized by a chronic phase which lasts from months to years, and is followed by accelerated myeloproliferative phase and subsequently blast crisis. in the chronic phase, manifestations include anemia, splenomegaly, bleeding, and constitutional symptoms such as fatigue, lethargy, weight loss, or low - grade fever. as in the present case study our case of a 21-year - old male, symptoms secondary to leukostasis may also be the presenting features1). since there are no reliable clinical criteria, in practice, leukostasis is empirically diagnosed when patients present with leukemia, hyperleukocytosis and respiratoy or neurologic distress2). however, some patients have clinically suspected or pathologically proven leukostasis with leukemic blast counts significantly lower than 100,000/mm3). litchmann proposed a pathophysiological model of leukostasis, based on the rheological consequences of hyperleukocytosis. he determined that the viscosity of leukocyte suspensions in vitro increased dramatically when the fractional volume of leukocytes (also known as leukocrit) exceeded 12~15 ml / dl4). the concentration of leukocytes necessary to produce such leukocrits is a function of the mean cell volume (mcv) of the cells. since leukemic myeloblasts have larger mcv than leukemic lymphoblasts, leukostasis is more frequently observed in patients with acute or chronic myelogenous leukemia than in patients with acute or chronic lymphoblastic leukemia5). however, leukemic blast concentrations necessary to reach leukocrit of 12~15 ml / dl are rarely seen. therefore, additional factors possibly related to the adhesive or invasive properties of leukemic blasts, may be more important. there are indications that leukemic blast - endothelial cell interactions may be triggered by locally released chemoattractant factors6). several adhesion molecules (cd54, cd62e, cd62p, cd106) are up - regulated in endothelial cell of aml patients with leukocytosis6). blasts appear to have the ability to secrete cytokines such as tnf- and interleukin-1 and therefore bring about the activation of endothelium. for instance, activation of vascular endothelium by tumor necrosis factor - alpha (tnf-) has been reported to increase the adhesion of myeloid blasts8). once leukocytosis is developed, signs and symptoms are usually related to the involvement of the respiratory or central nervous system. additional but less common clinical findings include neck vein distension, gallop rhythm and electrocardiographic sign of right ventricular overload, myocardial ischemia, priapism, acute limb ischemia, bowel infarctions, renal vein thrombosis, sudden bilateral deafness and avascular necrosis of bone2). until now, in cml, avascular necrosis of femoral head due to leukostasis has not been adequately documented, probably due to the rarity of its occurrence. after reviewing the world medical literature, we encountered only few cases of cml associated with avascular necrosis of femoral head9 - 13). the current standard of care for hyperleukocytosis and leukostasis should be the immediate initiation of intravenous fluids, allopurinol, hydroxyurea and the correction of coagulopathy and thrombocytopenia2). due to paucity of data on the efficacy of leukapheresis in reducing early mortality and/or improving overall survival, unfortunately there are no evidences to prove that any of these measures have produced a reduction in the number of early deaths. a variety of traumatic and nontraumatic factors contribute to the etiology of avacular necrosis. in adult patients, corticosteroid use and excessive alcohol intake are reported to account for more than 90 percent of cases15). additionally, other causes of avascular necrosis are systemic lupus erythematus, antiphospholipid antibody, trauma, sickle cell hemoglobinopathes, gaucher 's disease, caisson disease (dysbarism), transplantation, interferon-, inherited thrombophilia, or hiv infection16 - 19). avascular necrosis usually occurs in the anterolateral femoral head, although it may also affect the humeral head, femoral condyles, proximal tibia, vertebrae, and small bones of the hand and foot20). many patients have bilateral involvement at the time of diagnosis, including disease of the hips, knees, and shoulders. the goal of therapy for avascular necrosis is to preserve the naive joint for as long as possible. | chronic myelogenous leukemia (cml) is a malignant clonal disorder of hemopoietic stem cells characterized by abnormal proliferation and accumulation of immature granulocyte. leukostasis is one of the complications of cml and is characterized by partial or total occlusion of microcirculation by aggregation of leukemic cells and thrombi leading to respiratory, ophthalmic or neurologic symptoms. we experienced a rare case of avascular necrosis of the femoral head as the initial presentation of chronic myelogenous leukemia. a 24-year - old male patient was admitted to our hospital with pain in the right hip joint. the patient was diagnosed to be suffering from chronic myelogenous leukemia by packed marrow with granulocytic and megakaryocytic hyperplasia and the presence of philadelphia chromosome. the right hip joint pain was attributed to avascular necrosis of the femoral head. and the avascular necrosis could be considered as the complication of chronic myelogenous leukemia due to microcirculatory obstruction of the femoral head. the avascular necrosis of the right femoral head was treated with bipolar hemiarthoplasty. |
copd includes chronic bronchitis and pulmonary emphysema, having as a common feature the blockage of the lung airways, thus reducing significantly the airflow at the end and trapping the air inside. the obstruction initially causes decline in lung function, leading to decreased breathing, especially after severe fatigue. moreover, everyday life of elderly is often disturbed by exacerbations of the disease, caused by frequent pulmonary infections (1,2). the main characteristic of copd is a progressive airway obstruction ; however the term copd often refers to a separate group of diseases concerning their pathophysiology, which share common causes, such as smoking and air pollution, leading to a common result of obstruction. the obstruction in copd is irreversible, but some patients have a low degree of reversibility after administration of bronchodilators and cortisone drugs, that is why often appear common characteristics with chronic asthma, where airway obstruction may be improved (3 - 5). the elderly consist a vulnerable age group, with many special needs. several times, those elderly suffering from copd apparently show complications in breathing, during sleep that characterize sleep apnea syndrome. apnea is defined as the cessation of breathing, of the airflow, in a sleeping person, at least for 10 seconds, causing electrocerebral awakening and drop of the saturation of hemoglobin in oxygen. if the cessation of airflow is accompanied by lack of respiratory movements in the chest and abdomen, and there is no aspiratory effort, then the apnea is characterized as central, where, on the contrary, if there is respiratory activity is the chest or both, and the inhalation effort is growing during apnea, then it is characterized as obstructive. the term sleep apnea is usually laboratory, in contrast with the term sleep apnea syndrome, which, in addition to apnea, requires the appearance of clinical symptoms. to show the clinical manifestations of the syndrome usually are required more than 15 apneas per one hour of sleep, and vary depending on the chronic status, the number and severity of apnea. at the beginning, clinical symptoms are of low significance or absent, but later on, as copd progresses, apneas increase, leading to more severe symptoms (6 - 8). the main clinical symptoms of the copd include (6, 9 - 11) : sleepiness during the day : it is the most important and troublesome symptome seen in an elderly patient with copd. often patients complain that they have not slept for several hours and that they fall asleep during daily activities. people who have an increased level of daytime sleepiness should be advised to avoid driving, in order to prevent accidents.snoring : it is a sound generated during sleep by the vibration of the walls of the pharynx and soft palate. the vibration is caused due to the difficulty of access to air through a clogged throat. along with sleepiness are the main symptoms of the syndrome and the type of snoring can help in diagnosis, since the instability in frequency and intensity, suggest the presence of the syndrome of obstructive sleep apnea.obesity : the syndrome occurs more often in people with increased body weight, and therefore fat tissue in the neck.frequent night urination : it forces sufferers to interrupt their sleep and occasionally to have involuntary loss of urine. this is due to release of natriuretic factor from the dilatation of the sinuses and to a pressure of the urinary bladder, caused by increased abdominal pressure during obstructive apnea.night sweats : are often due to the restless sleep and due to the intense respiratory efforts during sleep.morning headaches, irritability and memory decline : patients awake with a feeling of dizziness, headaches and dry mouth. in more severe cases, there is a memory degradation, concentration and observation. trying to overcome the drowsiness and the sense of inferiority because of the symptoms, usually leads to depressive symptoms and stress disorders.decreased sexual activity : men often face sexual problems due to loss of libido and sense of helplessness. sleepiness during the day : it is the most important and troublesome symptome seen in an elderly patient with copd. often patients complain that they have not slept for several hours and that they fall asleep during daily activities. people who have an increased level of daytime sleepiness should be advised to avoid driving, in order to prevent accidents. snoring : it is a sound generated during sleep by the vibration of the walls of the pharynx and soft palate. the vibration is caused due to the difficulty of access to air through a clogged throat. along with sleepiness are the main symptoms of the syndrome and the type of snoring can help in diagnosis, since the instability in frequency and intensity, suggest the presence of the syndrome of obstructive sleep apnea. obesity : the syndrome occurs more often in people with increased body weight, and therefore fat tissue in the neck. frequent night urination : it forces sufferers to interrupt their sleep and occasionally to have involuntary loss of urine. this is due to release of natriuretic factor from the dilatation of the sinuses and to a pressure of the urinary bladder, caused by increased abdominal pressure during obstructive apnea. night sweats : are often due to the restless sleep and due to the intense respiratory efforts during sleep. morning headaches, irritability and memory decline : patients awake with a feeling of dizziness, headaches and dry mouth. in more severe cases, there is a memory degradation, concentration and observation. trying to overcome the drowsiness and the sense of inferiority because of the symptoms, usually leads to depressive symptoms and stress disorders. decreased sexual activity : men often face sexual problems due to loss of libido and sense of helplessness. the diagnosis of obstructive sleep apnea syndrome takes place by studying the elderly patients in a special workshop during sleep, while recording parameters of sleep and the respiratory function. for the staging of sleep, eeg is recorded, along with the eye movements, with an electrocardiogram, the movements of chest and abdominal wall, the airflow in the nose and mouth, the saturation of hemoglobin and the body position during sleep. other methods that help assess the functioning of the position and the degree of airway obstruction during sleep are the imaging methods of computing and magnetic resonance imaging (mri), fluoroscopy and ultrasound. the only non - imaging method, but also the only one that directly assesses the functional competence in multiple sites of upper airways, is the measurement of the differential pressure across the palate until the rinofarynx (12 - 14). in order to choose the appropriate way of disease management, the severity of the clinical picture, the study s findings in sleep centers, as well as patients severity of copd must be taken into account. treatment of sleep apnea syndrome targets to increase the pharynx s lumen diameter during sleep. these treatments may be generic, non - invasive, and in many difficult cases, surgical. reduction of body weight, because obesity is an aggravating factor in the obstruction of the upper airway, during sleep.avoidance of alcohol consumption by patients with copd and syndrome of obstructive sleep apnea, especially before bedtime.education of the patient to take a side position rather than a supine position during sleep.continuous positive airway pressure (cpap) : it consists the continuous provision of positive air pressure in the upper airway through the nose, by a special device. the cpap device assists the normal function of the entire airway, using bigger pressure than the atmospheric one, so that at no stage and point becomes negative, creating thus a respiratory splint that keeps the upper airway free.nasopharyngeal tube : it can help directly, simply, safely and effectively all patients, bypassing the airway obstruction during sleep.surgical interventions often take place, including the permanent tracheotomy, and nose, jaws and throat surgery, aiming to keep free the upper airway. reduction of body weight, because obesity is an aggravating factor in the obstruction of the upper airway, during sleep. avoidance of alcohol consumption by patients with copd and syndrome of obstructive sleep apnea, especially before bedtime. education of the patient to take a side position rather than a supine position during sleep. continuous positive airway pressure (cpap) : it consists the continuous provision of positive air pressure in the upper airway through the nose, by a special device. the cpap device assists the normal function of the entire airway, using bigger pressure than the atmospheric one, so that at no stage and point becomes negative, creating thus a respiratory splint that keeps the upper airway free. nasopharyngeal tube : it can help directly, simply, safely and effectively all patients, bypassing the airway obstruction during sleep. surgical interventions often take place, including the permanent tracheotomy, and nose, jaws and throat surgery, aiming to keep free the upper airway. quality of life is an important factor of assessing recovery of patients suffering from copd. the recovery can take place either in specialized medical units, whose primary purpose is to address the problem, such as sleep centers, or through properly designed programs implemented at home by a group of health professionals, whose main concern is the identification, diagnosis and treatment of the problem, alongside with the application of appropriate techniques. also are required efficient teaching methods for patients and their carers, towards an active participation in the selection and application of a suitable therapeutic approach the sample of this study was composed of 500 elderly individuals (274 men and 226 women), aged from 70 years old and above. open centers for the elderly, which are located in various municipalities of greater athens. all patients completed a specialized anonymous questionnaire, with a personal interview with the investigator, after a clear explanation of the purpose of this study. during the investigation, spirometry was carried out by using a portable spirometer, mir spirobank, while the parameters examined were fev1, fevc, and particularly the ratio fev1/fevc. this ratio was also used for the classification of the disease. to investigate the occurrence of apnea in people suffering from copd were also used, specialized and standardized questionnaires, such as the berlin questionnaire and epworth sleepiness scale. contingency tables were used for the relationship between categorical variables, using fishers exact test (for tables 2x2) and person s chi square test (for all other dimensions of contingency tables), as appropriate. data processing, along with statistical analysis and visualization was performed by using the statistical package spss 17. table 1 presents the classification of the copd, regarding the sex, by using gold scale. according to table 1, there was found a statistically significant difference in the stratification of mild copd. in specific the results differ in the case of moderate copd, where men developed higher rates (61% versus 39%). there is no statistically significant difference between the genders among severe cases of copd (table 1). almost all elderly people of the sample (98%), have reported to not suffer from any sleepiness during the day (table 2). according to the berlin questionnaire, which was used to assess the existence of apnea among the population, the 28.6% of the elderly appear to suffer fro a high risk level of apnea, at a rate of 36.0% they suffer from a lower risk of apnea, while the 35.4% suffer from no apnea at all (table 3). according to table 4, a large proportion of respondents (66.4%), stated that they had not received home care for their treatment, but they believe that such an option would help them manage their health problem. on the other hand, at a rate of 12.0%, participants believe that even homecare services could not have helped them. finally, 21.6% of the sample, received home help, and found out that they have been provided with a satisfactory help. there was found statistically significant relationship between the existence of severe copd among elderly people who have low risk of apnea. as far as the daily sleepiness by gender is concerned, there was found no statistically significant difference between the two genders, both in normal and in high - risk sleepiness of the elderly during the day (table 6). it was also found that men are more likely to suffer from apnea, in comparison to women. according to the findings of the study, elderly participants consist a vulnerable group with significant health problems and with difficulties in their daily lives. no significant statistical difference between the two genders is observed, as far as the prevalence of the under investigation disease is concerned. one possible explanation have a longer life expectancy rate than men (18). as far as the classification of copd is concerned, by gender, it was found that women suffer from copd more frequently than men, and even more often they suffer from mild copd, while men show more often symptoms of moderate copd, than women. there is not any statistically significant difference in the case of severe copd between the two genders. in a similar survey, conducted in beijing by zhou ym (19), among 9.434 elderly people, coming from 7 provinces of china, it was found that 30% of the sample were patients diagnosed with copd, while from the total sample, at a rate of 12.8% copd was diagnosed in men, with only 5.4% in women. another study conducted in poland (20) in 2007, it is shown that among 603 elderly people, 22.1% suffered from mild copd, with 10.9% of them suffering of moderate to severe type of copd. based on the results of the above studies, it is evident that the gender plays an important role in the prevalence of copd, and along with smoking, exposure to inappropriate weather conditions and age, they form an unhealthy configuration that leads to a large extent to the existence of copd among the elderly. it is also observed that the vast majority of the sample (98%) does not have any symptoms of sleep apnea, as daytime sleepiness. this does not mean that they do not have sleep apnea, as 36.0% of older people suffer from sleep apnea of low risk, 28.6% of them have high risk apnea, while one out of three seniors do not suffer from sleep apnea at all. there was found statistically significant relationship between the existence of severe copd among elderly people who have low risk of apnea. two similar surveys carried out by bixler, in 1998 and 2001, in pennsylvania, found out that among 1741 adults, aged up to 99 years, obstructive sleep apnea syndrome occur at a rate of 24% among men and 9% among women (21,22). another study conducted in spain, showed sleep apnea syndrome occurs at a rate of 26.0% among men, and 28.0% among women (23). according to what has been previously been reported, it can be proved that there is no limiting factor in the occurrence of apnea between the two genders. the obstructive sleep apnea syndrome may occur with the same frequency in both elderly men and women, and occurs more frequently in people who suffer from copd and other obstructive respiratory diseases. regarding the rehabilitation and participation in a program of home care, the majority of the sample (66.4%), stated that they do not take part in a specialized program, but they do believe a program like this would help considerably. only at a rate of 21.6% did the respondents participate in a program and had already seen improvement to their health problems. it is striking though, that 12.0% of the elderly, state that they were unwilling to participate in a rehabilitation program, while they did not believe that a specialized program would be of any benefit regarding the amelioration of their symptoms. in a canadian survey comprised of a sample of two patient groups (home care rehabilitation group and inpatient rehabilitation group), admissions into hospitals and emergency care departments were decreased significantly among the home care rehabilitation group, in a time span of three months (24). this group was also found to be more satisfied with the choice of a home care program. similar are the results of another investigation that took place in australia, 2005, among 60 persons (age>60 years). the sample attended a specialized 12 week home assistance program, which included specific exercises and patients / carers education. in conclusion, it is understood that as the number of the elderly people, and their proportion in the general population increases, it is important to understand that specific changes must be made to support their efforts for well being and their active social life. furthermore, most of the elderly people need to be independent to sufficiently take care of their daily problems. on the other hand, they do ask for quality services provided by the state (26 - 30). an important step is the development of supportive social structures to monitor the elderly, in order to detect, prevent and facilitate their daily problems. it is important that the elderly can be provided with a supportive human environment, which can help them discuss about their problems and assist them in communicating their inner needs (31 - 33). institutions such as the specialized sleep centers, open care centers for the elderly and specialized pulmonary rehabilitation home care programs are vital for people with disabilities with limited access to primary health care services (32, 34 - 36). imperative is considered the staffing of these special social support organizations, who should have specialized and appropriate, up to date knowledge and skills. the above skills are required so that the health professionals can be able to plan, organize and implement strategies for the emotional and social rehabilitation of patients. it is also important the social policy of the state to move towards new directions. specialized health professionals assisted by qualified volunteers can offer their services after attending special health promotion programs. these programs are provided by community nurses, social workers and other health professionals, with expertise in the dynamics of family and social environment of the elderly (37, 38). since aging is directly related to physical illness, loss of independence and self - care, entering the golden age is an important area of study in the current health system. living conditions, social and emotional status of the elderly and their participation in society, are issues that should be further studied. therefore further research is required, not only to add extra years in the lifespan of older people, but also to improve the quality of life during their remaining years. | introduction : chronic obstructive pulmonary disease (copd) and obstructive sleep apnea syndrome are two diseases, which often coexist in one person. the sleep apnea is often caused by an interruption to breath when sleeping, due to an obstruction of the upper airway during inhalation, causing chronic snoring, morning headaches, increased body weight, blood pressure and sleepiness during the day.aim:the purpose of this study is to investigate the extend of this problem among a large group of elderly people living in the attica basin.material and methods : material of the study was 500 people aged 70 and above years old, including 274 men and 226 women. data collection was facilitated with the use of an anonymous questionnaire with the purpose to identify patients with apnea, after a personal interview with the researcher. data analysis was performed with the use of the statistical package spss 17.results:based on their responses to the questionnaires, it was found that 98.0% of the participants have abnormal sleepiness during the day. the 35.4% of them show no apnea, while 36.0% of the sample suffer from mild sleep apnea. it is important though, the fact that about one out of three seniors, 28.6%, shows sleep apnea in a high risk level.conclusions:copd is a complex disease that occurs in a large proportion of the elderly. emphasis should be given on early diagnosis and treatment of sleep apnea syndrome, in order to improve the quality of the elderly life. |
an 83-year - old woman (height : 168 cm, weight : 56 kg) was admitted to the outpatient center to receive a second transurethral resection of a bladder tumor (turbt). the patient had underlying diabetes and hypertension and was taking a calcium channel blocker (amlodipine) and an angiotensin ii receptor blocker (candelotan) for high blood pressure. when the patient visited our hospital for her second turbt the preoperative electrocardiogram (ecg) evaluation revealed atrial fibrillation with a slow ventricular response (ventricular rate : 59 beats / min) (fig. ejection fraction 63%, eccentric left ventricular hypertrophy (lvh), and left atrial enlargement (lae) findings were obtained by transthoracic echocardiography. cardiomegaly was observed in chest x - ray, and neck ct angiography showed total occlusion of the right internal carotid artery. after the patient had been taken to the operating room, her vital signs were monitored using a non - invasive blood pressure measuring device, ecg and pulse oximeter. before anesthesia, her blood pressure was 150/90 mmhg, pulse rate (ventricular rate) 90 beats / min and pulse oximetry of 100%. after use of a mask to supply 100% oxygen for 3 minutes before anesthesia, 1% lidocaine 40 mg and propofol 60 mg were injected intravenously. thereafter, blood pressure declined to 130/72 mmhg whereas ventricular rate increased slightly to 113 beats / min and then fell rapidly to 27 beats / min with blood pressure of 70/40 mmhg. ventricular rate responded with an increase to 120 beats / min but then decreased again after 8 seconds of sinus pause. afterwards, atrial fibrillation rhythm, with a ventricular rate of 100 - 130 beats / min, was observed followed by a sinus pause, sinus rhythm, with a ventricular rate of 40 - 50 beats / min (figs. an external pacemaker (lifepak 20, medtronic co., minneapolis, mn, usa) was applied and set at 60 ma, 40 counts. she was observed for 30 minutes in the operating room while the monitoring devices and use of the external pacemaker were maintained. the operation was put on hold by decision of the surgeon and the patient was transferred to the recovery room with external pacing. a 110 - 130 beats / min atrial fibrillation rhythm, 40 - 50 beats / min sinus rhythm and 40 beat / min pacing rhythm were also observed in the recovery room. there were no self - perceived symptoms. there were no abnormal findings for vascularity, and a temporary pacemaker was inserted into the right ventricle. the patient was transferred to the cardiology department and treated for 2 weeks after insertion of a permanent pacemaker. a 60 count pacing rhythm was observed in a follow - up ecg (fig. 3), and follow - up transthoracic echocardiography showed ejection fraction 65%, eccentric lvh, lae, and the pacing rhythm was maintained without complications. atrial fibrillation is the most common type of arrhythmia in the elderly and it is 10 - 20 times more common than atrial flutter. it may cause no symptoms, but is often associated with palpitations, exercise intolerance, angina, congestive heart failure (shortness of breath, edema). furthermore, as it is associated with the inability to fulfill preload caused by tachycardia, it can result in dyspnea on exertion, paroxysmal nocturnal dyspnea, presyncope and syncope. the symptoms are similar to those of atrial fibrillation : dizziness, fatigue, chest pain, angina, shortness of breath and fainting. in patients with atrial fibrillation, it is important to determine whether the fibrillation is associated with sick sinus syndrome or with chronic atrial fibrillation that have normal sinus function. in our case, although atrial fibrillation was seen in the preoperative evaluation, the medical history of the patient was not sufficiently investigated. during pre - operative checking the patient was only asked about present chest symptoms or other symptoms related to the atrial fibrillation. in a detailed interview on the patient 's medical history after she awoke in the operating room, it was discovered that the patient had experienced dizziness and felt her symptoms had been worsening over the last 2 - 3 weeks. also she had experienced dyspnea (nyha class iii) and palpitation, aggravated over the last 2 - 3 days, from one month before. if we had elicited more details of the patient 's medical history in the pre - operative evaluation, we would have obtained more information on the type and progress of atrial fibrillation, and the effects of the disease on everyday life outside the hospital. as a consequence, sick sinus syndrome would have been suspected, and additional examinations such as holter monitoring could have been carried out. atrial fibrillation is classified into 4 types according to its characteristics : initial diagnosis (first detected), self terminating episode lasting less than 7 days (paroxysmal), episode lasting more than 7 days (persistent) and sustained symptoms (permanent). this patient was not classified in terms of type of atrial fibrillation in the pre - operative evaluation. thus, access to the frequency of atrial fibrillation episodes and the level of the ordinary ventricular rate was inadequate. it is stipulated that 24 hour holter monitoring is required for the patient treatment and care before operation. our patient was classified as having persistent atrial fibrillation on her evaluation after pacemaker insertion. when an atrial fibrillation patient shows signs of hemodynamic instability or symptoms of cardiac failure, electrical cardioversion is the most effective treatment for enhancing cardiac output and reducing the risk of thromboembolism. amiodarone, diltiazem, verapamil and digoxin can be used as medications, and av nodal conduction can be slowed to adjust the ventricular response. however, treating atrial fibrillation with cardioversion or medication can be hazardous if the sinus node is impaired. as these treatments can worsen bradyarrhythmia, a pacemaker must be inserted before pharmacologic control is attempted. if the bradycardia is not the result of drug therapy (ie, digitalis, beta - blockers), an abnormal sinus node physiology should be considered. atrial fibrillation associated with tachycardia - bradycardia syndrome should be treated with a permanent pacemaker in combination with drugs. in our case, blood pressure and pulse oximetry were stable when the ventricular rate of the patient showed tachycardia. and the tachycardia was followed by severe bradycardia. since we believed that our patient had tachycardia - bradycardia syndrome we started external pacing as quickly as possible without implementing cardioversion or medication. our patient was started medication (verapamil for control rate, aspirin and warfarin for prevent thromoembolism) after insertion of pacemaker. many anesthetic agents (such as lidocaine, propofol, fentanyl, remifentanil, vecuronium and etc) have some effects on the cardiac conduction system. they could have direct effect on sinus activity and incidence of bradycardia is higher if additional vagotomic agents. choice of drug is very important in case of bradycardia and silent sick sinus syndrome. lidocaine and propofol may have played a role in the development of bradycardia in our patient. thus, it is important to conduct a detailed, accurate interview and checkup on the patient 's medical history and to evaluate the patient before operation. the existence of concealed sick sinus syndrome should be kept in mind in patients with atrial fibrillation. furthermore, a defibrillator and external pacemaker must be available at all times in the operating room. | an 83-year - old woman was scheduled for a second transurethral resection of a bladder tumor. the preoperative electrocardiogram evaluation revealed atrial fibrillation with a slow ventricular response (ventricular rate : 59 /min). after intravenous injection of 1% lidocaine 40 mg and propofol 60 mg, the ventricular rate increased to 113 beats / min and then fell rapidly to 27 beats / min. blood pressure was 70/40 mmhg. later an atrial fibrillation rhythm, with a ventricular rate of 100 - 130 beats / min, was observed together with a sinus pause and sinus rhythm with a ventricular rate of 40 - 50 beats / min. an external pacemaker was applied and set at 60 ma, 40 counts. after the patient regained consciousness, she presented an alert mental state and had no chest symptoms. she was discharged 2 weeks later without complications after insertion of a permanent pacemaker. |
omeprazole (omz), 5-methoxy-2-[[(4-methoxy-3,5-dimethyl-2-pyridinyl)methyl]sulphinyl]-1h - benzimidazole, is the first member of the proton pump inhibitors that are widely used for the prophylaxis and treatment of both gastro - duodenal ulcers and symptomatic gastrooesophageal reflux. also, it is highly effective in the treatment of zollinger - ellison syndrome [1, 2 ]. the therapeutic importance of omz was behind the development of many analytical methods for its determination in the pharmaceutical formulations and/or biological samples. these methods include spectrophotometry [313 ], electrochemical methods, hplc [1517 ], and electrophoresis. as compared to the electrochemical, electrophoretic, and chromatographic methods, despite of their higher sensitivity, the spectrophotometric methods are more versatile and easy to apply. some reported visible spectrophotometric methods, being based on the nonselective oxidation of omz, may be also influenced by the excipients resulting in inaccurate results. therefore, accurate visible spectrophotometric methods are still required for the determination of omz in quality control laboratories. the molecular interactions between the electron donating pharmaceutical compounds [1923 ] and the electron acceptors are generally associated with the formation of intensely colored charge - transfer (ct) complexes. molecular ct complexes of omz with both iodine as -acceptor and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (ddq) as -acceptor have not been studied yet. kinetic spectrophotometric methods are becoming of great interest in pharmaceutical analysis as they offered some advantages such as improved selectivity, avoiding the interference of the colored and/or turbidity background of the samples, and possible avoiding of the interference of the other active compounds present in the commercial product if they are resisting the reaction conditions established for the proposed kinetic method. therefore, the development of new kinetic spectrophotometric methods for the determination of omz in its pharmaceutical preparations was targeted. studying the kinetics, molecularities, thermodynamics, and association constants of the ct complexes as well as their examination by the spectroscopic techniques, and computational molecular modeling usually provide information about the nature and mechanism of the ct complex formation. an important application for ct complexes with iodine is that the antithyroid activity could be expected for drugs whose association constant with iodine exceeds 100 l.mol. above this value therefore, it was important to determine the association constant for omz - iodine complex beside the method development. a shimadzu model uv-1601 pc (japan) uv - vis double beam spectrophotometer with matched 1-cm quartz cells was used for recording the electronic absorption spectra and all measurements. h - nmr spectra were recorded in dmso - d6 at 500 mhz by bruker - ultra shield instrument (bruker co., usa). ir spectra were recorded by ft - ir model spectrum bx spectrometer (perkin - elmer, usa). omz (hetero drugs ltd, hyderabad, india) was used as working standard. iodine, resublimed (riedel - de - haen ag, germany), was 2 mg ml in 1,2-dichloroethane. 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (ddq ; merck, schuchardt, munich, germany) solution was 4 mg ml in acetonitrile, and it was freshly prepared daily. gastrazole capsules (riyadh pharma, saudi arabia) and losec tablets (astra zeneca, sweden) were labeled to contain 20 mg omz per capsule or tablet. all solvents and other chemicals used throughout this study were of analytical grade. into a 100 ml calibrated flask, 25 mg of omz was accurately weighed, dissolved in 20 ml 1,2-dichloroethane (for the reaction with iodine) or acetonitrile (for the reaction with ddq), and completed to volume with the same solvent to obtain a stock solution of 0.25 mg ml. these stock solutions were further diluted with the respective solvents to obtain suitable concentrations that lie in the linear range of each particular assay method. twenty tablets or the contents of twenty capsules of each formulation were weighed and finely powdered. a quantity of the powder equivalent to 25.00 mg of omz was transferred into a 100 ml calibrated flask, dissolved in 20 ml of the appropriate solvent, swirled and sonicated for 5 minutes. the flask was completed to volume with the same solvent, shaken well for 15 minutes, and filtered. the first portion of the filtrate was rejected and a measured volume of the filtrate was diluted quantitatively with the respective solvent to yield suitable concentrations that lie in the linear range of each particular assay method. accurately measured aliquots of omz solution (1.00250.00 g ml) were transferred into separate 10 ml calibrated flasks. one milliliter of iodine (2.0 mg ml) or ddq (4.0 mg ml) was added. the solution was diluted to volume with 1,2-dichloroethane (for iodine) or acetonitrile (for ddq) and mixed well. after mixing, the reaction mixture was monitored at room temperature (25 2c) and the absorbance was recorded as a function of time for 45 and 5 minutes at 362 or 418 for iodine and ddq, respectively, against reagent blank treated similarly. one milliliter of the standard or sample solution of omz (1.00250.00 g ml) was transferred into 10 ml calibrated flasks. one milliliter of iodine (2.0 mg ml) or ddq (4.0 mg ml) was added. the reaction was allowed to proceed for 45 and 5 minutes at room temperature (25 2c) for iodine and ddq, respectively. the absorbance of the resulting solutions was measured at 362 or 418 for iodine and ddq, respectively, against reagent blanks treated similarly. the concentrations of these solutions were 1 10 m (for iodine) in 1,2-dichloroethane and 1 10 m (for ddq) in acetonitrile. series of 10 ml portions of the master solutions of omz and the acceptor were made up in 10 ml calibrated flasks comprising different complementary proportions (0 : 10, 1 : 9,, 9 : 1, 10 : 0). a quantity of 0.1 mmol (~35 mg) of omz, dissolved in the appropriate solvent, was added to an equimolar amount of the acceptor in the same solvent in a round - bottom flask containing ~30 ml of the respective solvent and stirred for 30 minutes. the solvents were evaporated under reduced pressure, and the resulting residues were dried over calcium chloride. series of omz solutions (0.4 102.1 10 m) in the appropriate solvent were prepared. in addition to these solutions, iodine (3 10 m) and ddq (3.6 10 m) solutions in the appropriate solvent were equilibrated for 30 minutes in a thermostatically controlled water bath at 25 2c. five milliliters of each acceptor solution was mixed rapidly with 5 ml of omz solution in 10 ml calibrated flasks. the absorbance of the solutions was measured immediately at the corresponding maxima against reagent blanks treated similarly. the kinetic data recorded for the proposed methods was transformed to the slide write plus software, version 5.011 (advanced graphics software, inc., ca, usa) for curve fitting, regression analysis, and statistical calculations. the initial rate (v) of the reaction at different omz concentrations was obtained from the slope of the tangent of the absorbance - time curve. the calibration curve was constructed by plotting the logarithm of the initial rate of the reaction (log v) versus the logarithm of the molar concentration of omz (log c). alternatively, the calibration curve was constructed by plotting the absorbance measured after a preselected fixed time versus the concentration of omz. the limits of detection (lod) and limits of quantitation (loq) were determined using the formula : lod or loq = sda / b, where = 3.3 for lod and 10 for loq, sda is the standard deviation of the intercept, and b is the slope. the violet - colored iodine solution in 1,2-dichloroethane was changed into lemon yellow upon addition of omz. examination of the absorption spectrum of omz - iodine reaction product showed two absorption maxima at 290 and 362 nm ; the first peak is about twofold more intense than the second (figure 1). the spectrum was found to be identical with that of tri - iodide ion (i3) in 1,2-dichloroethane. this identity in both spectra proved that the color change and the appearance of the new bands at 290 and 362 nm were attributed to the formation of omz - iodine ct complex with an ionized structure omz - i this omz complex should originate from an early intermediate outer complex as explained by scheme 1. confirming the ct nature of the reaction, the violet - colored solution of iodine in 1,2-dichloroethane was restored upon extracting omz from the complex by shaking with aqueous mineral acids. measurements were carried out at 362 nm to avoid the interference from the native uv absorption of omz at 290 nm. the interaction of omz with ddq in nonpolar solvents such as chloroform led to formation of colored ct complexes with low -values. this was attributed to complete electron transfer from omz (d) to the acceptor moiety (a) accompanied by the formation of intensely colored radical ions in polar solvents, according to scheme 2. the high ionizing power of the acetonitrile is the driving force for dissociation of the (d - a) complex to form the intense colored radical ion. as shown in figure 1, omz has no considerable absorption band in the range of 400600 nm ; however, the omz - ddq ct complex has some absorption bands characterized by 4 maxima at 346, 418, 548, and 588 nm presumably due to the formation of a deep red colored ct complex. the intensity of the maximum at 346 nm is about two- to threefold the maximum at 418 nm, while the intensity of the maximum at 418 nm is about 2.7-fold the maxima at both 548 and 588 nm. investigating the electronic spectrum of omz - ddq complex as a function of time in acetonitrile (figure 2) revealed that the deep red - colored solution (the intensity of the absorption bands at the 400600 nm region) began to increase up to 5 minutes and then leveled off for about 20 minutes and finally began to disappear by increasing the time ; however, the intensity of the band at 346 nm still is increasing up to more than 3 hours. therefore, the measurements were carried out at 418 nm to obtain the higher sensitivity and to avoid the irreproducibility of the measurements at 346 nm. such spectral features were coincident with those observed for the interaction of ddq with other donors [21, 29, 30 ] and the reported values of the radical anions of ddq obtained by the reduction method. studying the effect of the reagent concentrations on ct complex formation indicated that 1 ml of iodine (2.0 mg ml) and ddq (4.0 mg ml) working solutions were the optimum concentrations (i.e., the final concentrations of both reagents in the measured solution become 0.2 and 0.4 mg ml, resp.). higher concentrations of both reagents resulted in either higher blank readings or decreased absorption intensity. in order to select the most appropriate solvent, small shifts in the position of the maximum absorption peaks were observed, and the absorption intensities were also influenced. 1,2-dichloroethane was found to be an ideal solvent in case of iodine, because it is favorable for the formation of tri - iodide ion pair. polar solvents were found to be unsuitable as their blanks with iodine gave higher readings. however, acetonitrile found to be an ideal solvent for ddq, because it offered the maximum sensitivity. this was attributed to its high dielectric constant that promotes the maximum yield of radical anions, in addition to its high solvating power for ddq. the optimum reaction temperature for the investigated acceptors was determined by following the color development at different temperatures (25, 40, 50, 60, and 70c) using fixed concentrations of omz and the acceptors. the results indicated that room temperature (25 2c) was the ideal selection and higher temperatures was found to decrease the absorption intensity. since the formation of the colored ct products increases with time, it was deemed useful to generate absorption - time curves (figure 3) in order to determine the kinetics and thermodynamics of the investigated ct reactions. complete color development was attained after 45 and 5 minutes for iodine and ddq, respectively (figure 3). the developed colors remained stable at room temperature for at least further 40 and 20 minutes for iodine and ddq, respectively. job 's method of continuous variation was used for determining the molar ratio of omz to each of the two acceptors employed in this investigation. this indicates that only one site participated in the formation of the ct complex and a univalent charged species is the possible site of the ct process. the majority of ir measurements on the complexes have been concerned with shifts in the vibration frequencies in the donor, acceptor and/or both. the ir spectra of the complexes showed differences compared with those of omz and ddq. these differences have been used to distinguish between weak ct complexes and the products of electron transfer or proton - transfer reactions. as compared to the stretching vibration frequencies of cn and c = o bands in the ir spectrum of ddq (2233, 1678 cm), a bathochromic shift was observed for these two bands in the ir spectrum of the omz - ddq complex (2227, 1654 cm). such a bathochromic shift in these two bands could be indicative for a higher charge density on their corresponding functional groups. therefore, i compared the ir spectrum of pure omz and its corresponding ct complexes with the investigated acceptors. c = n, c = c, and s = o bands (3447, 1627, 1472, 1077 cm, resp.) were bathochromic shifted to lower frequencies (3434, 1627, 1432, 1026 cm and 3422, 1618, 1452, 1018 cm for omz - iodine and omz - ddq ct complexes, resp.). in the h - nmr spectra of the complexes, generally, the protons of the donor are shifted to a lower field [19, 22, 23 ]. the h - nmr spectra of the complexes of omz with different acceptors were recorded in d6-dmso and compared with the spectrum of the free drug. the 3-methyl, 5-methyl, 4-methoxy protons and the aromatic proton in the position number 2 on the pyridine ring (= 2.2, 2.2, 3.7 and 8.2 ppm, resp.) were not affected indicating that pyridine ring might not contribute in the electron donation. the protons of ch2 attached to the sulphur atom (= 4.7) were obviously shifted (= 0.20.5 ppm) for both iodine and ddq complexes. the 5-methoxy protons of benzimidzole ring (= 3.8 ppm) were obviously downfield shifted (= 0.20.4 ppm). the aromatic protons of c4, c6, and c7 of benzimidazole ring (= 6.9, 7.1 and 7.5 ppm, resp.) were obviously downfield shifted (= 0.20.5 ppm) for both iodine and ddq complexes. these results suggested that the electron - donating site in omz is close to the aromatic protons of benzimidazole, most probably the imidzole moiety of omz. these data, together with above - mentioned ir data, confirmed the ct complex formation between omz and both acceptors. molecular modeling for the ct complexes was carried out using the mopac package in the chem 3d ultra, version 9.0 (chemoffice software, cambridgesoft corporation, cambridge, ma, usa) implemented with molecular dynamics computations software (mm2). omz and ddq were energy - minimized alone and both together to obtain the most energyminimized conformation of omz ct complexes, the minimum energy of the complex was 130.673 kcal mol. it is acceptable that certain electron density was required for achievement of a successful electron transfer. comparing the electron density on all nitrogen atoms indicated that the benzimidazole nitrogen in the para - position of och3-group has the highest density (0.323) as compared to the other benzimidazole nitrogen (0.133) and the pyridine nitrogen (0.223) making it more likely to donate its electrons to the acceptor. the spectroscopic results confirmed that only one site is possible for the formation of the ct complex. this site, taking into account the molecular modeling for the ct complexes, was postulated to be the benzimidazole moiety of omz. therefore, the plausible mechanism for the reaction of omz with the investigated acceptors, taking ddq as example, was postulated as shown in scheme 3. under the described optimum conditions, the absorbance - time curves for the reaction of omz with both iodine and ddq were generated (figure 3). the initial rates of the reactions were determined from the slope tangents of the absorption - time curves. the order of the reaction with respect to the acceptors was determined by studying the reaction at different concentrations of the acceptors with fixed concentration of omz. the plot of the initial rate, da / dt, against the initial concentrations of the acceptors was linear indicating that the initial order of the reaction with respect to the acceptors was 1. as well, the order with respect to omz was evaluated from the measurement of the rates of the ct reactions at different concentrations of omz using a fixed concentration of the acceptors, which was found to be 1, too. however, under the optimized experimental conditions, the concentration of omz was determined using relative excess amount of the acceptors. therefore, pseudo - first order conditions were obtained and the initial rates of the ct reactions were found to obey the following equation : (1)v=at = kcn, where v is the initial reaction rate, a is the absorbance, t is the measuring time, k is the pseudo - first - order rate constant, c is the molar concentration of omz, and n is the order of the reaction. the logarithmic form of the above equation is written as follows : (2)log v = log at = log k+n log c. the order of the ct reactions was obtained from the slopes (n) of log v (at different omz concentrations) versus log c. the results obtained in table 2 indicated that the value of (n) was 0.87 and 0.99 (1) for iodine and ddq reactions, respectively. the rate constants (k) were calculated from the slopes of the plots of log at versus time, where at represent the absorbance of the formed complexes at time t. table 2 indicates the values of the specific rate constants of the ct reaction of omz with both iodine and ddq. the activation energy, the minimum kinetic energy a molecule must possess in order to undergo reaction, can be determined from arrhenius equation : (3)v = k = aeea / rt, where k is the specific reaction rate, a is a constant known as arrhenius frequency factor, ea is the activation energy, t is the absolute temperature (273+c), and r is the gas constant (1.987 cal molc). the logarithmic form of the above equation is written as follows : (4)log k = log aea2.303rt. the activation energy of the kinetic reaction of omz with the investigated acceptors was determined by studying the ct reactions at different temperatures : 25, 40, 60, 70, and 80c using fixed concentrations of omz and the acceptors. the absorption - time curves at these temperatures were constructed to determine the initial rates, then plotting log k versus 1/t to determine the slope (ea/2.303r) and the intercept (log a) of the line. the negative sign indicated that the ct complex formation decreased with increase in the temperature. furthermore, the change in the entropy of activation of the transition state of the complexes was determined using the following equation : (5)a=(rtnh)es/r, where a is the arrhenius frequency factor, t is the absolute temperature, r is the gas constant, n is avogadro 's number (6.02214179 10 mol), h is planck 's constant (6.626 10 j s), and s is the change in the entropy of activation (cal mol c). the obtained large negative entropies of activation of the complexes (table 2) support the formation of more polar transition state in the polar solvent. the association constant was evaluated at the corresponding maxfor each omz acceptor complex using the benesi - hildebrand equation : (6)[a]aad=1ad+1kcadad1[d ], where [a ] is the concentration of the acceptor ; [d ] is the concentration of the donor ; a is the absorbance of the complex formed at the specific wavelength ;, the molar absorptivity of the complex formed at the specific wavelength ; k c is the association constant of the complex (l mol). on plotting the values [a ]/a versus l/[d ], straight lines were obtained, from which the association constant, correlation coefficient, and the molar absorptivity of omz acceptor complexes were calculated (table 2). the standard free energy change of the complex is related to the association constant by the following equation : (7)g0=2.303 rt log kc, where g is the standard free energy change of the complex ; r is the gas constant ; t is the absolute temperature in kelvin (273+c) ; k c is the association constant of omz - acceptor complex (l mol). as compared to omz - iodine complex, the association constant of omz - ddq complex was of much lower value (common feature for ct complexes with -acceptors) due to the dissociation of the original donor - acceptor complex to the radical anion. however, the observed high value for the association constant of the ct complex with iodine suggests that the formed omz - iodine complex is of a strong type. out of the many applications of ct complexes, the determination of the association constant of the ct complexes of drugs with iodine could determine the antithyroid activities of drugs. therefore, the determination of the association constant of omz - i2 ct complexes (21.50 10 l mol) could determine the potential iatrogenic antithyroid action of omz, and could represent a further tool for the evaluation of drug safety. (1) initial rate methodunder the above described optimum conditions, summarized in table 1, the initial rates of the ct reaction of omz with the acceptors would follow a pseudo - first - order kinetic and were found to obey the following equation : (8)log v = log at = log k+nlog c. regression analysis using the method of least square was performed to evaluate the slope, intercept, and correlation coefficient. the limits of detection (lod) were calculated and found to be 0.24 and 0.41 10 mol (0.08 and 0.14 g ml), whereas the limits of quantification (loq) were 0.73 and 1.22 10 mol (0.25 and 0.42 g ml) for both iodine and ddq methods, respectively. these low values confirmed the good sensitivity of the initial rate method and consequently its capability to determine omz in the linear range of 0.738.70 10 mol (0.253.00 g ml) and 1.4572.4 10 mol (0.525.00 g ml) for both iodine and ddq methods, respectively. under the above described optimum conditions, summarized in table 1, the initial rates of the ct reaction of omz with the acceptors would follow a pseudo - first - order kinetic and were found to obey the following equation : (8)log v = log at = log k+nlog c. regression analysis using the method of least square was performed to evaluate the slope, intercept, and correlation coefficient. the limits of detection (lod) were calculated and found to be 0.24 and 0.41 10 mol (0.08 and 0.14 g ml), whereas the limits of quantification (loq) were 0.73 and 1.22 10 mol (0.25 and 0.42 g ml) for both iodine and ddq methods, respectively. these low values confirmed the good sensitivity of the initial rate method and consequently its capability to determine omz in the linear range of 0.738.70 10 mol (0.253.00 g ml) and 1.4572.4 10 mol (0.525.00 g ml) for both iodine and ddq methods, respectively. (2) fixed time methodin this method, the absorbance of the reaction solutions containing varying amounts of omz was measured at a preselected fixed time. calibration plots of absorbance versus the concentrations of omz were established at fixed periods of time for the reactions (table 4). the regression equations, correlation coefficients, and the limits of detection and quantification are given in table 4. the lowest limits of detection and quantification were obtained with fixed times of 45 minutes (for iodine) and of 4 and 5 minutes (for ddq) methods, respectively. however, the fixed times of 5 minutes (for iodine) and of 1 minute (for ddq) methods showed wider dynamic range for quantification. therefore, on the basis of wider dynamic range and less time of analysis, the fixed time of 5 and 1 minute for iodine and ddq methods, respectively, was recommended for the determination of omz by the fixed time method, if the sensitivity is not required (otherwise, fixed time of 45 and 5 minutes is better for both methods, resp.). out of the many advantages of the kinetic spectrophotometric methods, one important advantage is that the long reaction time could be overcome by applying the initial rate method. therefore, the initial rate was preferred than the fixed time with respect to iodine method. however, the fixed time is preferred than the initial rate with respect to ddq method due to its short reaction time (5 minutes). in this method, the absorbance of the reaction solutions containing varying amounts of omz was measured at a preselected fixed time. calibration plots of absorbance versus the concentrations of omz were established at fixed periods of time for the reactions (table 4). the regression equations, correlation coefficients, and the limits of detection and quantification are given in table 4. the lowest limits of detection and quantification were obtained with fixed times of 45 minutes (for iodine) and of 4 and 5 minutes (for ddq) methods, respectively. however, the fixed times of 5 minutes (for iodine) and of 1 minute (for ddq) methods showed wider dynamic range for quantification. therefore, on the basis of wider dynamic range and less time of analysis, the fixed time of 5 and 1 minute for iodine and ddq methods, respectively, was recommended for the determination of omz by the fixed time method, if the sensitivity is not required (otherwise, fixed time of 45 and 5 minutes is better for both methods, resp.). out of the many advantages of the kinetic spectrophotometric methods, one important advantage is that the long reaction time could be overcome by applying the initial rate method. therefore, the initial rate was preferred than the fixed time with respect to iodine method. however, the fixed time is preferred than the initial rate with respect to ddq method due to its short reaction time (5 minutes). the precision and accuracy were tested by applying the proposed methods using the same experimental conditions as described under the general analytical procedure. the precision of the proposed methods was determined at three concentration levels of omz (0.5, 1.5, and 3.0 g ml for iodine and 2.0, 10.0, and 20.0 g ml for ddq methods). this was performed by determination of five replicate samples of each concentration by both the initial rate and fixed time methods. the results revealed that the relative standard deviations (rsds) of the values did not exceed 1.25%. the accuracy of the proposed methods was also evaluated using the same (above - mentioned) concentrations of omz. table 5 showed the recovery results (98.50%100.80%) with rsd not more than 1.95%, proving the high accuracy of the proposed methods. this high level of the precision and accuracy indicated the suitability of the proposed methods for the quality control analysis of omz in its pharmaceutical preparations. the proposed kinetic methods have the advantage of that all measurements are performed in the visible region, away from the near uv absorbing interfering substances that might be coextracted from omz - containing dosage forms. samples were prepared by mixing known amount (20 mg) of omz with various amounts of the common excipients such as starch, glucose, lactose, acacia, and magnesium stearate. the results (table 6) revealed that no interference was observed from any of these excipients with the proposed methods. the ruggedness was tested by applying the proposed methods to the assay of omz using the same operational conditions but using two different instruments at two different laboratories and different elapsed time. results obtained from lab - to - lab and day - to - day variations were reproducible as rsd did not exceed 3%. robustness of the proposed procedures was examined by evaluating the influence of small variation in the concentration of acceptor reagents (within 5%) or in the reaction temperature (25 2c) on the analytical performance of the proposed methods. in these experiments, one experimental variable was changed, whereas the others were kept unchanged, and the recovery percentage was calculated each time. it was found that neither the temperature (23, 25, and 27c were tested) nor the reagents concentration (1.9, 2.0, and 2.1 mg ml of iodine and 3.8, 4.0, and 4.2 mg ml of ddq reagents were tested) significantly affected the results ; the recovery percentages were 98.64%101.53% 0.921.67. depending on the obtained validation results, the proposed procedures were found to be suitable for the routine quality control analysis of omz. the proposed and the reference methods were applied to the determination of omz in its dosage forms. the results obtained by the proposed methods were statistically compared with those obtained by the reference method. the results of t- and f - tests revealed that no significant differences were found between the proposed and reference methods at 95% confidence level with respect to precision and accuracy. this proved the applicability of the proposed methods for quality control analysis of omz in its pharmaceutical preparations with comparable analytical performance. the present work utilized the colored ct complexes of omz with both iodine and ddq in the development of new simple, rapid, sensitive, and accurate kinetic spectrophotometric methods for the analysis of omz in dosage forms. the obtained ct complexes have been investigated by uv - vis spectrophotometry, ir, and h - nmr spectroscopic techniques, and by computational molecular modeling. a plausible mechanism for these ct reactions based on the spectroscopic study was postulated. the proposed kinetic methods are superior to the previously reported spectrophotometric methods in the term of sensitivity and improved selectivity because the sensitivity of the proposed methods (linear range was as low as 0.1 g ml) is higher than that of all reported spectrophotometric methods (the lowest reported linear range was 0.2 g ml) for determination of omz [313 ]. furthermore, the measurements of most of the reported spectrophotometric methods were performed at lower wavelengths (at 258320 nm) which may be influenced by the excipients of the dosage forms in contrast to the proposed kinetic methods [510 ]. moreover, some of the reported colorimetric methods are time consuming [4, 9 ], and others have been proved to be inaccurate due to matrix interference. | new rapid, sensitive, and accurate kinetic spectrophotometric methods were developed, for the first time, to determine omeprazole (omz) in its dosage forms. the methods were based on the formation of charge - transfer complexes with both iodine and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (ddq). the variables that affected the reactions were carefully studied and optimized. the formed complexes and the site of interaction were examined by uv / vis, ir, and 1h - nmr techniques, and computational molecular modeling. under optimum conditions, the stoichiometry of the reactions between omz and the acceptors was found to be 1 : 1. the order of the reactions and the specific rate constants were determined. the thermodynamics of the complexes were computed and the mechanism of the reactions was postulated. the initial rate and fixed time methods were utilized for the determination of omz concentrations. the linear ranges for the proposed methods were 0.103.00 and 0.5025.00 g ml1 with the lowest lod of 0.03 and 0.14 g ml1 for iodine and ddq, respectively. analytical performance of the methods was statistically validated ; rsd was < 1.25% for the precision and < 1.95% for the accuracy. the proposed methods were successfully applied to the analysis of omz in its dosage forms ; the recovery was 98.91100.32% 0.941.84, and was found to be comparable with that of reference method. |
lingual orthodontics, a more esthetic orthodontic technique than labial orthodontics, has developed rapidly in recent years.1 - 3 many case reports and papers have documented the treatment effects, and a variety of bracket designs have been produced.4,5 the disadvantages of lingual orthodontics include the excessive chair time, complicated biomechanics, patient discomfort, expensive lab procedures, and high material prices.6,7 however, several innovations have improved the use of lingual orthodontics, such as customized lingual brackets and 2-dimensional lingual brackets that can be bonded directly.8,9 nonetheless, the efficient control of anterior torque and intrusion during retraction continues to be a limiting challenge. mini - screws and mini - implants (the osseointegrating type) have been successfully applied to lingual orthodontics.9,10 mini - implants placed on each side of the palate have been used to avoid uncontrolled tipping and the deepening of the anterior bite during en masse retraction. typically, the treatment protocol involves the soldering of a lever arm to the main lingual arch wire.10,11 the lever arm moves the force vector apically and closer to the center of resistance, thereby allowing better control of torque during retraction. one disadvantage of this mechanics is that play within the slot allows some of the torque to be lost during retraction. in addition, if bilateral mini - implants are not in the same horizontal plane, which is sometimes required by the anatomy of the maxilla, the clinician may see unwanted canting of the occlusal plane due to different force vectors generated during retraction. moreover, the sliding mechanics in a full - arch appliance using mini - implant - assisted anterior retraction may be adversely affected by friction within bracket slots and tubes, causing unwanted distalization of posterior teeth. this new treatment system allows en masse retraction of the anterior teeth independently of the posterior teeth by using a c - retractor and palatal miniplate (figures 1 and 2). the c - retractor is constructed by soldering a 0.9-mm stainless steel wire onto mesh - bonding pads and is subsequently bonded to the lingual surfaces of the 6 or 8 anterior teeth.14 unlike typical bracket / arch wire setups, slot play is not an issue in this type of setup. furthermore, the c - retractor is adequately rigid to resist deformation under a normal retraction force. this particular feature facilitates control of the axes of the anterior teeth during retraction of the anterior segment. also, selection of the appropriate vertical height of the lingual anterior retraction hooks (larhs) allows the clinician to produce controlled tipping, bodily movement, and lingual root movement during retraction (figure 3). patient compliance is unnecessary, and patient comfort is improved when compared to lingual brackets. for cases in which the upper second premolars are affected by certain conditions (e.g., dilacerated roots, short roots, compromised teeth, or dens invaginatus), extraction of the second premolars is usually indicated, even though the goal of lingual biocreative therapy is maximum anterior retraction. in a previously cited clinical study,12 miniplates in the palate (c - plates ; jin biomed co., bucheon, korea) were the only source of anchorage for the en masse retraction of the 6 or 8 maxillary anterior teeth. the c - plates were designed to have adjustable extension wings to allow the clinician to alter the force vectors. further, the c - plate is fixed to the cortical bone of the maxillary palate, and a flap does not need to be laid. hence, damage to the roots of adjacent teeth or anatomical structures is not a concern. since the applied orthodontic forces during anterior retraction are against the c - plate and not against orthodontic appliances fixed to the posterior teeth, no change in the posterior occlusion is expected during retraction.12,13,15 to date, however, no studies have analyzed the force systems involved in the control of anterior torque and intrusion by this technique, with the exception of studies in clinical literature and case reports. the aim of this study was to use finite element analysis (fea) to evaluate the effectiveness of anterior segment retraction using the c - plate while varying the vertical height and location of the c - retractor hook. we obtained tooth outlines by performing three - dimensional (3d) laser scanning of a right maxillary tooth from a dental study model of the normal adult dentition (nissin dental products inc., we aligned and leveled the dental arches using a broad arch form (ormco, glendora, ca, usa) and referred to previous studies to assign inclination and angulation.16,17 neither a curve of spee nor a curve of wilson was added (figure 4). the thickness of the periodontal ligament was assumed to be uniform (0.25 mm).18 the alveolar bone crest was constructed to follow the cemento - enamel junction (cej) curvature 1 mm apical to the cej. the 3d - finite element model included 12 teeth, an open space to correspond to the missing first premolars or second premolars, periodontal space and alveolar bone. the model was also bilaterally symmetrical. in the finite element model, the teeth, alveolar bone, and periodontal spaces were constructed with fine tetrahedron solid elements, and node - to - node contact elements were installed between adjacent teeth to represent tooth interactions. in this study, the teeth, alveolar bone, and periodontal spaces were assumed to be isotropic and homogeneous linear elastic bodies, and the material properties of the elements were based on values for young 's modulus and poisson 's ratio, according to previous studies (table 1).19 - 21 in the system studies, we assignedd the x - axis to the median - lateral direction, the y - axis to the anterior - posterior direction, and the z - axis to the coronal - apical direction. furthermore, we defined + x as the left central incisor direction, + y as the labial (anterior) direction, + z as the apical direction, and the x - y plane as the occlusal plane of the teeth. in all cases, we assumed no movement of the posterior teeth, since they received no force application. to fabricate the c - retractor, a 0.9-mm stainless - steel round wire (this round wire is a 2-noded, 3d beam element that has 3 transitional and 3 rotational degrees of freedom and can represent the bending characteristics of wires) afterwards, an additional wire was used to construct the lever arm hook, which was connected to the c - retractor by node sharing. the wire system was connected to stainless steel pads (tetrahedron solid element) by node sharing as well to complete the appliance (figure 4). the c - retractor was adjoined to the lingual surfaces of the upper anterior teeth at 5.5 mm apical to the incisal edge of the maxillary central incisor by node sharing. four experimental conditions were used in this study, and were based on the teeth extracted and the placement of the larhs. the maxillary first premolar extraction cases were conditions 1 and 2, while the second premolar extraction cases were conditions 3 and 4. the larh position between the maxillary central and lateral incisors comprised conditions 1 and 3, while larh placement between the lateral incisors and canines made up conditions 2 and 4 (figure 5). the larhs were constructed close to the surface of the palatal rugae, and the element analysis was implemented for each case using different vertical heights (1, 4, 7, 10, and 13 mm) for the larhs. the vertical height was measured from the plane of the mesh - plate to the end of the hook perpendicular to the occlusal plane. in clinical studies, the retraction force was applied from the c - plate ; however, in this fea study, the c - plate model was not included in the analysis, and was therefore not fabricated. this reduced complications in the analysis. using the usual position and dimensions of the c - plate as a reference, the hooks extending laterally from the c - plate were laterally 8.2 mm from the mid - palatal suture, sagitally located between the upper first and second molar, and 12 mm apical to the common lingual bracket position. a retraction force of 200 g was applied to each side (figures 4 and 5). the tooth displacement was marked by applying the x, y, z coordinates at the midpoint of the incisal edges of # 11 and # 12, the cusp tip of # 13, and the corresponding root tips. the fea was performed using ansys 11 (swanson analysis system, canonsburg, pa, usa), the universal finite element program, on an hp - xw6400 workstation (hewlett - packard co., palo alto, ca, usa). we obtained tooth outlines by performing three - dimensional (3d) laser scanning of a right maxillary tooth from a dental study model of the normal adult dentition (nissin dental products inc., we aligned and leveled the dental arches using a broad arch form (ormco, glendora, ca, usa) and referred to previous studies to assign inclination and angulation.16,17 neither a curve of spee nor a curve of wilson was added (figure 4). the thickness of the periodontal ligament was assumed to be uniform (0.25 mm).18 the alveolar bone crest was constructed to follow the cemento - enamel junction (cej) curvature 1 mm apical to the cej. the 3d - finite element model included 12 teeth, an open space to correspond to the missing first premolars or second premolars, periodontal space and alveolar bone. the model was also bilaterally symmetrical. in the finite element model, the teeth, alveolar bone, and periodontal spaces were constructed with fine tetrahedron solid elements, and node - to - node contact elements were installed between adjacent teeth to represent tooth interactions. in this study, the teeth, alveolar bone, and periodontal spaces were assumed to be isotropic and homogeneous linear elastic bodies, and the material properties of the elements were based on values for young 's modulus and poisson 's ratio, according to previous studies (table 1).19 - 21 in the system studies, we assignedd the x - axis to the median - lateral direction, the y - axis to the anterior - posterior direction, and the z - axis to the coronal - apical direction. furthermore, we defined + x as the left central incisor direction, + y as the labial (anterior) direction, + z as the apical direction, and the x - y plane as the occlusal plane of the teeth. in all cases, we assumed no movement of the posterior teeth, since they received no force application. to fabricate the c - retractor, a 0.9-mm stainless - steel round wire (this round wire is a 2-noded, 3d beam element that has 3 transitional and 3 rotational degrees of freedom and can represent the bending characteristics of wires) was formed passively along the lingual surfaces of the upper anterior teeth. afterwards, an additional wire was used to construct the lever arm hook, which was connected to the c - retractor by node sharing. the wire system was connected to stainless steel pads (tetrahedron solid element) by node sharing as well to complete the appliance (figure 4). the c - retractor was adjoined to the lingual surfaces of the upper anterior teeth at 5.5 mm apical to the incisal edge of the maxillary central incisor by node sharing. four experimental conditions were used in this study, and were based on the teeth extracted and the placement of the larhs. the maxillary first premolar extraction cases were conditions 1 and 2, while the second premolar extraction cases were conditions 3 and 4. the larh position between the maxillary central and lateral incisors comprised conditions 1 and 3, while larh placement between the lateral incisors and canines made up conditions 2 and 4 (figure 5). the larhs were constructed close to the surface of the palatal rugae, and the element analysis was implemented for each case using different vertical heights (1, 4, 7, 10, and 13 mm) for the larhs. the vertical height was measured from the plane of the mesh - plate to the end of the hook perpendicular to the occlusal plane. in clinical studies, the retraction force was applied from the c - plate ; however, in this fea study, the c - plate model was not included in the analysis, and was therefore not fabricated. this reduced complications in the analysis. using the usual position and dimensions of the c - plate as a reference, the hooks extending laterally from the c - plate were laterally 8.2 mm from the mid - palatal suture, sagitally located between the upper first and second molar, and 12 mm apical to the common lingual bracket position. a retraction force of 200 g was applied to each side (figures 4 and 5). the tooth displacement was marked by applying the x, y, z coordinates at the midpoint of the incisal edges of # 11 and # 12, the cusp tip of # 13, and the corresponding root tips. the fea was performed using ansys 11 (swanson analysis system, canonsburg, pa, usa), the universal finite element program, on an hp - xw6400 workstation (hewlett - packard co., palo alto, ca, usa). two hundred grams of retraction force was applied to the c - retractor hook under the 4 conditions described in the materials and methods section. the results of the relationship between the tooth displacement pattern on the z - axis (the plus [+ ] and minus [- ] symbols refer to intrusion and extrusion, respectively) and the vertical height of the larh are shown in table 2 and figures 6 and 7. for condition 1, the incisal edge of # 11 and the cusp tip of # 13 were intruded using the larh vertical heights of 10 and 4 mm, respectively. the degree of extrusion was greater for condition 2 than for condition 1 at the same larh height. for condition 2, the incisal edge of # 11 and the cusp tip of # 13 were intruded at the larh vertical heights of 13 and 10 mm, respectively. the results for conditions 3 and 4 were similar to those for conditions 1 and 2, respectively ; however, the amount of tooth displacement under conditions 3 and 4 were reduced relative to conditions 1 and 2. for condition 1, a retraction force of 200 g resulted in lingual and uncontrolled tipping of the maxillary central incisor crown when the larh vertical height was 1 mm (table 3, figures 6 and 8). controlled tipping was observed at the larh vertical heights of 4 and 7 mm, while bodily displacement and the occurrence of root retraction was noted at the larh vertical heights of 10 and 13 mm. for condition 2, the degree of the lingual tipping of # 11, # 12, and # 13 increased in comparison to condition 1 at the same larh vertical height. for condition 2, the maxillary central incisors at the larh vertical heights of 7 and 10 mm showed controlled tipping, while actual root retraction was observed with the larh vertical height of 13 mm. the pattern of tooth movement was similar between conditions 1 and 3, but bodily displacement for condition 3 was observed at a lower vertical height of 7 mm. meanwhile, a similar pattern of tooth displacement was found between conditions 1 and 4, but bodily movement in condition 4 was observed only when the vertical height was more than 10 mm. two hundred grams of retraction force was applied to the c - retractor hook under the 4 conditions described in the materials and methods section. the results of the relationship between the tooth displacement pattern on the z - axis (the plus [+ ] and minus [- ] symbols refer to intrusion and extrusion, respectively) and the vertical height of the larh are shown in table 2 and figures 6 and 7. for condition 1, the incisal edge of # 11 and the cusp tip of # 13 were intruded using the larh vertical heights of 10 and 4 mm, respectively. the degree of extrusion was greater for condition 2 than for condition 1 at the same larh height. for condition 2, the incisal edge of # 11 and the cusp tip of # 13 were intruded at the larh vertical heights of 13 and 10 mm, respectively. the results for conditions 3 and 4 were similar to those for conditions 1 and 2, respectively ; however, the amount of tooth displacement under conditions 3 and 4 were reduced relative to conditions 1 and 2. for condition 1, a retraction force of 200 g resulted in lingual and uncontrolled tipping of the maxillary central incisor crown when the larh vertical height was 1 mm (table 3, figures 6 and 8). controlled tipping was observed at the larh vertical heights of 4 and 7 mm, while bodily displacement and the occurrence of root retraction was noted at the larh vertical heights of 10 and 13 mm. for condition 2, the degree of the lingual tipping of # 11, # 12, and # 13 increased in comparison to condition 1 at the same larh vertical height. for condition 2, the maxillary central incisors at the larh vertical heights of 7 and 10 mm showed controlled tipping, while actual root retraction was observed with the larh vertical height of 13 mm. the pattern of tooth movement was similar between conditions 1 and 3, but bodily displacement for condition 3 was observed at a lower vertical height of 7 mm. meanwhile, a similar pattern of tooth displacement was found between conditions 1 and 4, but bodily movement in condition 4 was observed only when the vertical height was more than 10 mm. in lingual orthodontic treatment, the attachment and removal of lingual brackets are technique sensitive, and thus challenging and time consuming. because of these issues, these proceudres may involve a complex and expensive set - up process. moreover, routine adjustments and archwire fabrication require expertise, experience, and technical skill. as a result of these challenges, for instance, in cases in which the treatment of anterior protrusion requires maximum anchorage, complicated overlay archwires and/or mini - implant anchorage have been recommended to achieve controlled 3d tooth movement.22 the lingual biocreative therapy applied in this study is a method to retract the anterior dental segment using forces between the c - retractor and the c - plate. the biomechanical premise underlying segmental orthodontics is adapted from one of burstone 's protocols,23 but differs in that the force is applied to a segment from a skeletal anchor with no connection to the posterior teeth. extended lever arms have been used in conventional lingual orthodontics for retraction against mini - screw - anchors, but torque loss is a common side effect due to slot play within the appliance as well as flexibility of the archwire. biocreative therapy with the c - retractor eliminates these side effects, because the anterior segment is bonded as a unit with a rigidly constructed device. furthermore, retraction control is in the hands of the clinician, since controlled bodily displacement, tipping, and root retraction is possible through altering the vertical height of the larh.24,25 the results of the current study are similar to those of the fea study of jang.,26 which used a modified c - retractor and various miniscrew positions. in that study, the optimal choice for vertical height of the larh was found to be related to the goals for retraction (i.e., controlled tipping, bodily displacement, root retraction). the device was bonded to the lingual surfaces of the upper 6 or 8 anterior teeth, and retraction was implemented by applying a closed niti coil spring between the extension hook of the c - plate and the larh of the c - retractor. in the current study, 3d tooth displacement was controlled by varying the vertical height of the larh. our results were different from those of a previous clinical study to control torque,27,28 as well as the study by mo.,29 which attempted 3d tooth movement through the control of intrusion and retraction in a labial treatment method. the latter study showed differences between the control of the incisors and canines, but found that only variation of the vertical height of the larh provided the desired 3d control during retraction of the anterior teeth.29 one of the potential reasons for this difference between the previous report and the current one may be the rigidity afforded by the c - retractor. the 0.9-mm wire is much stiffer than a standard archwire placed in conventional lingual brackets. future studies may show this rigid c - retractor to be valuable when applying heavy retraction forces, as in the case of perisegmental corticotomy for inducing rapid tooth movement.13,30 in the current study, the retraction pattern depended on the position of the larh. although both positions met the requirements of controlling the upper incisor axes and preventing deepening of the bite, the position for conditions 1 and 3 (between the maxillary central and lateral incisors) had more significant treatment effects for the same vertical height than that of conditions 2 and 4 (between the lateral incisors and the canines). one advantage of using the larh position in conditions 1 or 3 is that the canine can be segmented from the c - retractor, allowing detailing of the canine while still retaining incisor retraction with the c - plate (figure 9). therefore, we recommend that as a rule of thumb, the larh should be placed distal to the central incisors rather than distal to the lateral incisors. this study examined the initial displacement due to orthodontic forces, using the fe method. hence, further studies on the clinical long - term effects, the retraction pattern, and the risk of root resorption for lingual biocreative therapy using the c - retractor and c - plate will be needed. in addition, we anticipate further studies on the design and treatment effects of c - retractors in asymmetrical premolar extraction cases. the following conclusions can be made on the basis of the findings in this study : 1. fe studies have demonstrated that variations in the vertical height of the larh affect the vector of the retraction force and produce measurable effects on the inclination and vertical position of the anterior teeth during anterior retraction. the larh can be placed between the central and lateral incisors or between the lateral incisors and canines. placement distal to the central incisors was considered preferable because the treatment effects were better. if the larh is distal to the lateral incisors, a vertically higher hook is necessary to achieve bodily displacement. | objectiveto evaluate the factors that affect torque control during anterior retraction when utilizing the c - retractor with a palatal miniplate as an exclusive source of anchorage without posterior appliances.methodsthe c - retractor was modeled using a 3-dimensional beam element (0.9-mm - diameter stainless - steel wire) attached to mesh bonding pads. various vertical heights and 2 attachment positions for the lingual anterior retraction hooks (larhs) were evaluated. a force of 200 g was applied from each side hook of the miniplate to the splinted segment of 6 or 8 anterior teeth.resultsduring anterior retraction, an increase in the larh vertical height increased the amount of lingual root torque and intrusion of the incisors. in particular, with increasing vertical height, the tooth displacement pattern changed from controlled tipping to bodily displacement and then to lingual root displacement. the effects were enhanced when the larh was located between the central and lateral incisors, as compared to when the larh was located between the lateral incisors and canines.conclusionsthree-dimensional lingual anterior retraction of the 6 or 8 anterior teeth can be accomplished using the palatal miniplate as the only anchorage source. using larhs at different heights or positions affects the quality of torque and intrusion. |
low back pain is a highly prevalent complaint, reported to affect 26.4% of us adults at some point in the last three months, although fewer than 1% of patients proceed to undergo surgical intervention. in spite of methodical advances in surgical techniques, incidental durotomy (iatrogenic dural tears) during posterior spinal surgery incidental durotomy or iatrogenic dural tears remain a relatively commonplace and potentially serious complication of lumbar spine surgery [35 ]. delineating anatomical variants in the posterior lumbar spine may be crucial in reducing future rates of incidental durotomy. the aim of this study was to investigate variations in the anatomical and histological features of the posterior epidural ligaments, passing between the dura mater and the ligamentum flavum in the lumbar spine. seventeen lumbar spines were dissected from randomly selected soft - fixed cadavers with no known history of spinal disease or spinal surgery. cross - sectional anatomical dissection at the levels l1-s1 via a dorsal approach was performed. anterior retraction of the dura mater identified any connection between the dorsal surface of the dura mater and the ligamentum flavum. any attachments between the dura and the ligamentum flavum were dissected out in their entirety, fixed in 10% formalin and stained with hematoxylin and eosin (h&e) and elastic van gieson (evg). a number of randomly selected control specimens from levels l1-s1, without connection, were also stained with h&e and evg. staining with evg differentiated collagen from elastin in the tissue structure excised. posterior epidural ligaments were identified as a connection between the dorsal surface of the dura mater and the ligamentum flavum. nine (9) separate posterior epidural ligaments were identified in these cadavers, with 3 (33.3%) at l3/l4, 5 (55.6%) at l4/l5, and 1 (11.1%) at l5/s1 (table 1). figures 1 and 2 demonstrate the variable presentations of the ligament and its identification at different vertebral levels. histology confirmed the presence of poorly differentiated collagen - based connective tissue, distinct from the normal anatomy (figure 3). histological analysis highlighted variants in the presentations of the ligament (figures 4 and 5). figure 4 shows a ligament with a substantial coalition and fusion of fibres, which are distinct from the standard tissue plane. on other occasions, the ligament was observed as a discrete stranded structure with ventral - dorsal interdigitation of fibres (figure 5). despite methodical approaches and improved surgical technique, the incidence of incidental durotomy in posterior lumbar spine surgery has not significantly decreased. unidentified incidental durotomy has a clear postoperative risk, and the existence of variants in the lumbar spine anatomy has been hypothesised as a possible cause for the stagnant incidence rates of incidental durotomy. previous anatomical studies have delineated ventral lumbar dural adhesions, which authors have reported them to be an adjuvant source of discogenic back pain. in the posterior spine, lateral (hoffman 's ligaments), dorsolateral, and dorsomedial epidural attachments have been described (reviewed by kimmel.). the attention to terminal attachment (ata) has been described originating from the dorsal surface of the dura mater at the level of s1 projecting towards the ligamentum flavum. this intraoperative finding had similar histological features to those identified in this anatomical study and was also described as a possible causative factor in dural tears. however, the dorsal ligaments described here are clearly different from the ata as they were found at levels l3 to s1 and present a clear extension from the dorsal dura mater to the ligamentum flavum. posterior decompression of lumbar neural structures often involves removal or part removal of the lumbar lamina and ligamentum flavum. in patients with severe stenosis, surgeons regularly encounter adherent ligamentum flavum to the dura mater, where no epidural fat is present to define the surgical plane. we observed variable ligamentous attachments between the dorsal surface of the dura mater and ligamentum flavum at levels l3-s1. these ventral - dorsal ligaments tethered the dura mater to the ligamentum flavum forming a substantial communication (figures 1 and 2). the presence of such ligaments, in the anatomical plane where lumbar spine surgery is most commonly undertaken, may have a role in the aetiology of intraoperative incidental durotomy. histology staining confirmed the presence of collagen - rich tissue that was distinct from the normal elastic structure of the ligamentum flavum (figure 3). the variable histological presentations of the ligaments were outlined (figures 4 and 5). the histological union of the dura mater and ligamentum flavum makes the intraoperative identification of separate tissue structures difficult, and care should be taken during surgery [11, 12 ]. with the knowledge of the existence of the posterior epidural ligament at levels l3-s1, the authors advocate surgical caution in the region with the employment of complication avoidance techniques when approaching the ligamentum flavum. the authors support the previous literature [10, 11 ] that recommends that two - step flavotomies, using semisharp dissectors, during a posterior approach to the lumbar spine should be standard surgical practice. furthermore, with knowledge of the features of the posterior epidural ligaments, it is recommended that in cases where movement of the dura is visualized, areas of adherent ligamentum flavum should be left in situ. the presence of ligamentous attachments between the dorsal surface of the dura mater and the ligamentum flavum is of surgical and anatomical importance. in this paper, we show these ligaments arising at vertebral levels l3-s1 in over half of the cadavers investigated and describe their varied anatomical and histological presentation. the delineation of the posterior epidural ligaments in the lumbar spine is of clear clinical importance in posterior lumbar spine surgery. | purpose. incidental durotomy is a relatively common complication for patients undergoing posterior spinal surgery. delineating anatomical variants in the posterior lumbar spinal canal is crucial in reducing future rates of incidental durotomy. materials and methods. the ligamentous attachments between the dura mater and ligamentum flavum in the lumbar region of 17 soft - fixed cadavers were investigated. the lumbar vertebral columns were removed, and cross - sectional dissection was performed at levels l1-s1. anterior retraction of the dorsal dura mater identified attachments between the dorsal surface of the dura mater and the ligamentum flavum. histological staining of the ligamentous attachments was carried out with hematoxylin and eosin (h&e) and elastic van gieson (evg). results. posterior epidural ligaments were present in 9 (52.9%) cadavers. nine (9) separate ligaments were identified in these cadavers, with 3 (33.3%) at l3/l4, 5 (55.5%) at l4/l5, and 1 (11.1%) at l5/s1. histology confirmed the presence of poorly differentiated collagen - based connective tissue, distinct from the normal anatomy. conclusions. this study confirms the presence of multiple dorsomedial posterior epidural ligaments at the main sites for posterior spinal surgery (l3-s1). an intraoperative awareness of the variability of such connections may be an important step in reducing static rates of incidental durotomy. |
undifferentiated pleomorphic sarcoma (ups), formerly known as malignant fibrous histiocytoma (mfh), is a common soft tissue sarcoma in which any attempt to describe the line of differentiation fails. ups was first described by ozzello. in 1963, and feldman and norman first described primary malignant tumor of bone in 1972 (quoted from). it most commonly involves the extremities and retro - peritoneum. in the head and neck region, the most commonly affected sites are the nasal cavity and the paranasal sinuses (54.3%). the mandible is a rare location, accounting for only 3% of tumors within bones. ups of head and neck that extend into bony structures are associated with a much more aggressive clinical course than those that are restricted to soft tissues. in this article, we report a rare case of ups of the mandible in a 44-year - old korean man. a 44-year - old man was referred to dankook dental hospital from a local neurosurgery clinic. the patient presented with the chief complaint of a rapidly growing mass in the left mandible area within the last two months. extra - oral examination revealed a tender, 4 to 5 cm sized solid mass in the left mandible area. no specific lesion such as ulcer or infection was found in intra - oral examination. the patient s medical history was non - contributory and there was no history of trauma or dental treatment recently. nothing remarkable was found through laboratory data including complete blood count, urine analysis, or chest x - ray in the preoperative exam. preoperative panoramic radiographs showed an ill - defined radiolucent, radiopaque mixed lesion on the left mandible area (fig. 1). a neck computed tomography (ct) with contrast revealed a 4 cm heterogeneously enhancing mass lesion in the left mandible body extending to adjacent soft tissue with bony destruction. 2). positron emission tomography - computed tomography (pet - ct) revealed a large lobulating hypermetabolic lesion in and around the left submandibular area with possible bone invasion. in both lungs, a few tiny nodular opacities were seen, but these were too small to be characterized (fig. 3). with a clinical diagnosis of malignant tumor of the mandible, surgery was performed. under general anesthesia, a mandibulectomy from the symphysis to the left ascending ramus was performed, and reconstructed with vascularized osteocutaneous fibula free flap. to stabilize the flap, inter - maxillary fixation with arch - bar was applied in right half maxilla and mandible (fig. the main tumor mass, with hematoxylin and eosin staining, was highly vascular and hypercellular with an apparent whitish slit - like hemorrhagic space at 100 magnification. at 200 magnification, tissue presented generalized myxoid pattern with a loose matrix. the tumor consisted of fibroblast - like spindle - shaped cells, specifically osteoclast - like giant cells with remarkably active mitosis. at high - power field (400 magnification), nuclear pleomorphism with prominent nuclei that varied in size, figure, and postoperatively, the patient was referred to the department of hemato - oncology for adjuvant chemotherapy. upon examination at two months, metastasis to the lung and local recurrence on primary mandible area was detected in pec - ct. thus, metastatectomy was performed for metastasis of lung, and the patient is receiving continuous palliative chemotherapy. the older term for ups was mfh, based on the resemblance of the cells to histiocytes and fibroblasts (due to their elongated shapes). the name was modified to ups because the morphologic pattern of mfh is similar to many other sarcomas. the usual presentation in the literature is a painful mass that expands over a few weeks or months. in our case, the patient was a male in his forties, complaining of a rapidly growing painful mass. non - specific symptoms such as paresthesia make diagnosis of the tumor by clinical symptoms difficult. this implies that some of these tumors may represent an initial proliferative response to trauma. ups is either primary or secondary, and secondary tumors are more aggressive and less common than primary tumors. park. reported in a review that ct and magnetic resonance imaging (mri) features of ups of head and neck are nonspecific. on ct scans, ups appears as a non - specific, large, lobulated, soft tissue mass iso - attenuated to muscle. central areas of low attenuation may be due to necrosis, hemorrhage or myxoid material. in 5% to 20% of the cases, calcification or ossification can be detected. on mri, ups shows a heterogeneous hyper - intense pattern on t2-weighted images and isointensity that is almost same as muscles on t1-weighted images. non - immunoreactivity for pan - ck, ck8, ck18 allows sarcomatoid or anaplastic hepatocellular carcinoma and cholangiocarcinoma to be ruled out. angiosarcomas are excluded due to no reactivity for the usual vascular antigens, such as cd31, cd34. liposarcoma or malignant peripheral nerve sheath tumor (s-100), rhabdomyosarcoma (desmin), malignant melanoma (s-100 and hmb-45), malignant lymphoma (lca) are also excluded. s100, desmin, s-100, hmb-45, lca in parenthesises are antibody of liposarcoma, rhabdomyosarcoma, malignant melanoma, malignant lymphoma respectively. fine needle aspiration biopsy can be used for obtaining of biopsy samples, but sensitivity is poor, ranging from 60% to 80%. this is due to the inability to obtain immunohistochemical stains on most samples from fine needle aspiration biopsy. the management of ups of the mandible requires early radical surgery including removal of adjacent normal tissue, with a minimum of 3 cm tumor - free margins. elective neck dissection is performed only when there is an evidence of cervical lymph node metastasis. the patient should be re - examined often to rapidly detect local recurrence and distant metastasis. the efficacy of chemotherapy and radiotherapy treatment for ups of mandible is not well established. this is based on randomized trials comparing surgery with or without radiotherapy that demonstrate the efficacy of radiotherapy as an adjuvant treatment in decreasing local recurrence rates. adjuvant radiotherapy may be indicated for patients with probable insufficient surgical margin when re - resection is not possible. one observer noted that cyvadic did not improve the prognosis, and a more intensive regimen could have been more effective. therefore, if adjuvant chemotherapy is needed, more intensive chemotherapy might be employed only for the high - risk group such as tumors with history of local recurrence and histologically high - grade because of possible subclinical or microscopic metastasis. perhaps 25% to 35% of patients with ups of head and neck region will develop metastases, with the lungs as the most common metastatic site from ups in the head and neck, followed by bone and liver. in this case, we detected no lymph node metastasis at the pre - operative examination so we did not perform neck dissection. this infiltrative growth is an adverse prognostic factor not only for local control, but also for disease - free and meta - stasis - free survival. tumor infiltration into adjacent soft tissue including muscles occurred in this case, accompanied by local recurrence. background (primary or recurrent), histologic grade, tumor size, and surgical margin are the significant prognostic factors for the outcome after surgical treatment. in conclusion, the diagnosis of ups should be reserved only for those cases in which any attempt to identify a line of differentiation has failed. due to the low numbers of patient with ups of the head and neck region, a consistent therapeutic strategy for this disease is unlikely to be established. | undifferentiated pleomorphic sarcoma (ups), previously known as malignant fibrous histiocytoma, occurs commonly in the soft tissues in adult, but is rare in the maxillofacial region. it consists of undifferentiated mesenchymal tumor cells resembling histiocytes and fibroblasts. the purpose of this article is to report a case of ups in the mandible. a 44-year - old patient presented with a painful growing mass in the mandible of two months duration. computed tomography and positron emission tomography - computed tomography revealed an ill - defined heterogenous, hypermetabolic mass about 4 cm in size in the left mandible invading adjacent soft tissues. a left mandiblulectomy and reconstruction with a fibular free flap were performed. immunohistochemical study gave a diagnosis of ups. the patient was referred for adjuvant chemotherapy after surgical removal of the tumor. |
ramadan is a holy month observed by muslims all over the world, during which fasting is rigorously observed from dawn until sunset. the duration of fasting varies by geographical location and season. during summer and northern latitudes, fasting begins after a meal before dawn (sahur) and is completed after sunset by usually consuming sweetened water or dates followed by a meal (iftar). individuals are exempted from ramadan fasting in circumstances such as illness or menstruation that could adversely affect health outcomes leading to interruption of fasting during such periods. in such cases is generally assumed that the calorie consumption is low and there is a greater tendency for hypoglycemia and irregular glycemic control in subjects with type 2 diabetes mellitus.(t2 dm). the effect of experimental short - term fasting on carbohydrate metabolism has been published in the literature. it has been uniformly found that a slight decrease in serum glucose from 3.3 mmol to 3.9 mmol (60 mg / dl to 70 mg / dl) occurs in normal adults a few hours after fasting has begun. however, this reduction in serum glucose tends to cease due to increased gluconeogenesis in the liver during fasting, and related decrease in insulin concentration and a rise in glucagon and sympathetic activity.[24 ] glycogen stores, along with some degree of gluconeogenesis, may maintain normal serum glucose when a fast follows a large pre - dawn meal. the epidar study that recruited 12,914 muslim diabetic subjects from thirteen countries reported prevalence estimates of fasting during ramadan as 43% for type 1 diabetes mellitus and 86% for t2 dm. although this study was the largest epidemiological report that described diabetes - related practices during ramadan, the study failed to include a detailed dietary assessment during the fasting period although the authors report unaltered life style practices in about 50% of the individuals during ramadan. it is generally perceived that calorie consumption is restricted during the fasting period, but there exists some degree of dietary indiscretion during the non - fasting period with excessive compensatory eating, which may contribute to hyperglycemia and weight gain. there is paucity of information available on dietary practices and diet components during ramadan fasting. since calorie restriction, adherence to dietary counseling and maintenance of balanced diet forms an integral component of general diabetes management and especially during critical periods such as fasting, the current study aimed to evaluate dietary patterns and diet - adherence at different time points during 30-day fasting period of ramadan amongst muslims living in south india. diabetes management during fasting is challenging since physical activity is restricted and pharmacological therapy is usually lowered in fear of hypoglycemia. therefore appropriate glycemic control largely relies on appropriate diet modulation of diet that efficiently maintains the blood glucose and counter - balances the fasting effects, especially hypoglycemia, without compromising religious fasting. the importance of dietary management have been strongly underscored by the diabetes control and complications trial (dcct) and uk prospective diabetes study (ukpds).[810 ] although, during ramadan, the benefits of fasting appear only in patients who maintain their appropriate diets. most patients with diabetes find it increasingly difficult to understand and adhere to the nutritional component of their treatment.[1114 ] in the current study, we therefore describe the general dietary pattern followed by muslims during religious fasting and the degree of compliance to the prescribed diet during ramadan all subjects in this dietary survey were muslims with t2 dm who intended to undertake fasting for the entire holy month of ramadan (30 days). the participants were recruited from an original multicenter, randomized double blind, placebo controlled trial where pioglitazone was used along with conventional ohas to assess the status of glycemic control and incidence of hypoglycemia during ramadan described elsewhere. table 1 shows the baseline characteristics of subjects who participated in the study prior to any type of dietary assessment. baseline characteristics of subjects who took part in the dietary survey all subjects were subjected to 24-hour dietary recall and diabetic diet counseling at screening. follow - up dietary assessment was carried out at three stages, i.e., before initiation of ramadan fasting, mid - ramadan (15 days after initiating fasting) and post - ramadan (1 week after completing the fasting period) by a trained dietician. diet was assessed using 24-hour dietary recall method and through standard questionnaires based on food frequencies as detailed below. study design ; flow of patients regular dietary pattern was obtained by a 24-hour recall method, and quantitative food assessment was done using a food frequency questionnaire. food frequency questionnaires (ffq) based on self - reported intake of daily food have been shown to validly measure long - term dietary patterns in epidemiological studies. the food frequency questionnaire used for the assessment of nutrient intake in the north indian population was easy to administer, showed moderate to good correlation with the 5-day diet record, and was reproducible. a set of standardized cups, spoons, and glasses were used to aid the subject to recall the quantity of food prepared and consumed. the amount of food intake and raw ingredients used for each food preparation was obtained individually from each subject using reference standardized cups. participants were asked to recall their usual intake of foods and beverages over the past 6 months. both the frequency of intake and the serving size were ascertained. the subject 's intake in terms of raw equivalent was calculated as the product of the total raw amount of each ingredient and the amount of cooked food intake divided by the total food cooked per day. this method is useful in obtaining qualitative details of diet and the pattern of food consumption at household level and also includes assessment of the frequency of consumption of different foods on daily, weekly, fortnight or occasional basis. participants were also queried with regard to the special dietary practices they followed during the fasting period, the use of dietary supplements, and food preparation methods. energy, carbohydrates, proteins, fat, and fiber were calculated using the food composition table reference from national institute of nutrition and indian council for medical research. based on the dietary intake pattern, along with anthropometric measurements (height, weight), cultural preferences, beliefs, lifestyle and customary intake, a standard diabetic diet sheet was given to each individual. the dietary recommendations were adapted to the specific needs of the individual based on nutrition assessment, desired treatment outcomes, and modification of usual food intake. all subjects were advised to strictly adhere to a two meal, two snack (one meal and one snack before onset of fasting and one meal and one snack after breaking of fast) pattern during the fasting period of ramadan. a typical day 's meal and snacks aimed at providing 1500 to 2000 calories with 65 - 70% calories from carbohydrate, 15- 20% from protein and 10 - -15% from fat. prototype dietary recommendation based on body mass index the food exchange system for planning diabetic diets for the current study was based on the concept of food equivalents. seven food groups listed in the food exchange system (cereals, pulses, meat, milk, vegetables, fruits, and fat) were included in the diet prescribed to the participants.. the description of food exchanges according to their general composition and characteristics are included in table 3. food exchanges recommended during fasting dietary counseling was given individually and food portions were instructed using standardized cups and spoons. a - days dietary pattern with traditional foods was displayed in portions to ease understanding of dietary recommendations. in this visual method, a plate served as a pie chart to show the proportions of the plate that have to be covered by various food groups. overall glycemic control during 30 days of fasting was assessed using serum fructosamaine, measured by boehringer mannheim automated analysis (bm / hitachi system 912). values for optimal glycemic control was based on comparison of serum fructosamaine to hba1c values performed on non - diabetic controls recruited in the original study. glycemic control was classified as good, suboptimal and poor if the corresponding serum fructosamine were 185.285 um / l, 286 - 485 um / l and > carbohydrate, protein, and fat intake were expressed as percent contribution to daily energy intake. repeated measure anova was used to compare the difference in proportions in calorie consumption during different time points of fasting. all subjects in this dietary survey were muslims with t2 dm who intended to undertake fasting for the entire holy month of ramadan (30 days). the participants were recruited from an original multicenter, randomized double blind, placebo controlled trial where pioglitazone was used along with conventional ohas to assess the status of glycemic control and incidence of hypoglycemia during ramadan described elsewhere. table 1 shows the baseline characteristics of subjects who participated in the study prior to any type of dietary assessment. follow - up dietary assessment was carried out at three stages, i.e., before initiation of ramadan fasting, mid - ramadan (15 days after initiating fasting) and post - ramadan (1 week after completing the fasting period) by a trained dietician. diet was assessed using 24-hour dietary recall method and through standard questionnaires based on food frequencies as detailed below. regular dietary pattern was obtained by a 24-hour recall method, and quantitative food assessment was done using a food frequency questionnaire. food frequency questionnaires (ffq) based on self - reported intake of daily food have been shown to validly measure long - term dietary patterns in epidemiological studies. the food frequency questionnaire used for the assessment of nutrient intake in the north indian population was easy to administer, showed moderate to good correlation with the 5-day diet record, and was reproducible. a set of standardized cups, spoons, and glasses were used to aid the subject to recall the quantity of food prepared and consumed. the amount of food intake and raw ingredients used for each food preparation was obtained individually from each subject using reference standardized cups. participants were asked to recall their usual intake of foods and beverages over the past 6 months. both the frequency of intake and the serving size were ascertained. the subject 's intake in terms of raw equivalent was calculated as the product of the total raw amount of each ingredient and the amount of cooked food intake divided by the total food cooked per day. this method is useful in obtaining qualitative details of diet and the pattern of food consumption at household level and also includes assessment of the frequency of consumption of different foods on daily, weekly, fortnight or occasional basis. participants were also queried with regard to the special dietary practices they followed during the fasting period, the use of dietary supplements, and food preparation methods. energy, carbohydrates, proteins, fat, and fiber were calculated using the food composition table reference from national institute of nutrition and indian council for medical research. based on the dietary intake pattern, along with anthropometric measurements (height, weight), cultural preferences, beliefs, lifestyle and customary intake, the dietary recommendations were adapted to the specific needs of the individual based on nutrition assessment, desired treatment outcomes, and modification of usual food intake. all subjects were advised to strictly adhere to a two meal, two snack (one meal and one snack before onset of fasting and one meal and one snack after breaking of fast) pattern during the fasting period of ramadan. a typical day 's meal and snacks aimed at providing 1500 to 2000 calories with 65 - 70% calories from carbohydrate, 15- 20% from protein and 10 - -15% from fat. the food exchange system for planning diabetic diets for the current study was based on the concept of food equivalents. seven food groups listed in the food exchange system (cereals, pulses, meat, milk, vegetables, fruits, and fat) were included in the diet prescribed to the participants.. the description of food exchanges according to their general composition and characteristics are included in table 3. dietary counseling was given individually and food portions were instructed using standardized cups and spoons. a - days dietary pattern with traditional foods was displayed in portions to ease understanding of dietary recommendations. in this visual method, a plate served as a pie chart to show the proportions of the plate that have to be covered by various food groups. overall glycemic control during 30 days of fasting was assessed using serum fructosamaine, measured by boehringer mannheim automated analysis (bm / hitachi system 912). values for optimal glycemic control was based on comparison of serum fructosamaine to hba1c values performed on non - diabetic controls recruited in the original study. glycemic control was classified as good, suboptimal and poor if the corresponding serum fructosamine were 185.285 um / l, 286 - 485 um / l and > carbohydrate, protein, and fat intake were expressed as percent contribution to daily energy intake. repeated measure anova was used to compare the difference in proportions in calorie consumption during different time points of fasting. a the current study examined dietary patterns and adherence to diet prescribed during fasting days of ramadan in 72 muslim subjects with t2 dm. the participants were middle aged (45 9 years) with a shorter duration of diabetes (8.0 2 years) and mean bmi was 29.8 5 kg / m. all subjects included in the study were on anti - diabetic medications either in the form of monotherapy (n = 12), or combination therapy (n = 38). the overall calorie consumption during ramadan varied significantly prior to initiation of fasting, 15 days after initiation of fasting and end of the fasting period (p=0.001). the percentage of energy consumption from dietary carbohydrate prior to fasting (64.11 6.73), during fasting (68.41 4.41), and at the end of fasting (70.46 5.45) remained almost unchanged with marginal statistical significance (p=0.041). analysis of individual components of diet consumption showed a substantial increase in dietary components during mid - ramadan fasting [table 4 ]. dietary intake and glycemic control in patients compared from before and after during the fasting period of ramadan a significant reduction in fasting and post prandial glucose was observed during mid - ramadan, but the glycemic patterns resumed to baseline values when subjects resumed their normal dietary pattern after ramadan fasting. overall glycemic control as assessed by serum fructosamine at the end of the fasting period was optimal (p=0.005). nearly one - fourth of the world 's population constitutes muslims. over 50 countries across the globe the 2011 population census of india (http://censusindia.gov.in) report an approximate 13.4% (138 million) muslims living in india clustered in minority communities, which is more than islamic community in the arab countries. however, it is evident from clinical observations an health surveys that most of the muslims in india practice religious fasting during the holy month of ramadan. the major changes in life style pattern during ramadan fasting are chrono - biological and behavioral. although there is considerable evidence on physiological alterations and diabetes management with oral hypoglycemic drugs or insulin, little emphasis is laid upon dietary management during fasting. in the current study we aimed to investigate the dietary patterns during the ramadan fasting and also assess adherence to prescribed diets to optimize blood sugar control among south indian muslims who undertook 30 day fasting during ramadan. results from our study demonstrates that there is an overall increase in calorie consumption during ramadan fasting despite sufficient dietary counseling in our participants. although religious fasting permits intake of food only twice a day (before and after sunrise and sunset), there appears to be compensatory overeating in adults as observed by the increase in total calorie intake during fasting. there was a significant increase in the percentage carbohydrates, protein and fats consumed during fasting, most of the later are a part of the traditional foods that are specifically consumed during ramadan in this population. contrary to our findings, a study among 22 diabetic muslims from malaysia have shown low calorie consumption during ramadan fasting, however detailed dietary description was not reported among these individuals. the calorie consumption during the entire period of fasting was also not reported among these individuals. we identified a distinct dietary behavioral pattern during the fasting period among our particpants, with regard to type of food groups consumed with a mean increase in consumption of all components of diet during 30-day fasting. this specific pattern of high calorie food intake was independent of the socio - economic strata and educational status. it was evident from the patient - dietician interview that most patients perceived that intake of large quantities of food that yield sufficient energy could help them sustain fasting and prevent hypoglycemia. observed an increase consumption of dietary fat (35.8%), especially saturated fats (43.3%) among muslims during fasting periods similar to our observations (44.2%) during mid - ramadan. although this dietary practice of high calorie compensation was contrary to actual religious preaching (of non - compensatory eating), the main concern among participants was interruption of continuous 30-day fasting due to hypoglycemia. most dietary guidelines for diabetes management are targeted towards intake of specific macronutrients. in an indian setting, many individuals find it difficult to make dietary changes based on such numerical criteria. therefore, our recommendations were based on overall pattern of dietary intake focusing on appropriate food choices. we focused mainly on high - carbohydrate and high - fiber (hchf) diet during the nonfasting hours, as it is noted that diets which were high in rich complex carbohydrates and dietary fiber, and relatively low in fat, benefited individuals with diabetes mellitus. despite repeititive counseling on dietary patterns and reinforcing prescribed diet according to patients choices and traditional practices, the compliance to prescribed diet was poor. these conclusions were not supported by questionnaire based assessments but, from overall assessment of the diet diary where the patient recorded foods consumed during fasting. we learn that although targeted counseling is implemented, it is difficult to break the traditional pattern of high - fat and high - protein diet during the period of ramadan fasting. glycemic assessment by serum fructosamine at the end of the study was optimal (p = 0.005) among our participants. several studies have reported no change in hba1c or fructosamine in subjects who undertook fasting during ramadan. it is important to note that the variation in diet pattern during fasting is crucial leading to hypo- and hyperglycemia. hypoglycemia is one of the major concerns both among patients and treating physicians who undertake short - term fasting due to religious practices. hypoglycemia assessment (based on self - reports) was not seen among our participants similar to previous observations in fasting diabetic patients treated with anti - diabetic drugs or insulin. however, a large epidemiological study demonstrated that the risk of severe hypoglycemia increased by 4.7 and 7.5 folds in subjects with type 1 and type 2 diabetes who undertook fasting during ramadan. in another muticentric observational study, symptomatic hypoglycemia we did not observe significant difference in weight or bmi change before, during or after fasting similar to observations by mguil. a power calculation to determine the sample size was not used due to the lack of prior similar studies evaluating the dietary habits during ramadan. the 24-hour dietary recalls can mostly give an estimate of the average nutrient intake, and this possibly explains the difference in food intake when comparing the two dietary assessment methods. there is some element of bias in the recall of food intake as most subjects volunteered participation. however the present study gives us an insight into the dietary habits, food adherence patterns, and traditional beliefs in a unique group of muslim subjects who undertook fasting during ramadan. the large proportion of muslim subjects who undertake fasting during ramadan represents a challenge to the treating physicians. the present study is one of the first studies that have attempted to present the overall picture of the food pattern in fasting muslim subjects during ramadan, and our prototype diet based on regional food practices and a culturally acceptable diet pattern is a recommendation for patients with diabetes who fast during ramadan in india. there is a overwhelming need for additional studies that could provide assessment and dietary recommendations that are ethnic / culture specific and form mainstay of diabetes management in all religious groups that practice short - term fasting. | aims : the aim was to assess the dietary pattern during ramadan season among type 2 diabetic muslim subjects who underwent fasting and intensive dietary counseling.materials and methods : the study was conducted among 70 muslim subjects with type 2 diabetes mellitus who undertook fasting during ramadan and was part of a randomized control trial using pioglitazone published previously. all subjects were subjected to a dietary assessment and counseling at three stages, i.e., initiation of the study, mid - ramadan and post - ramadan, by a trained dietician. dietary assessment was done by the 24-hour dietary recall method and the food frequency questionnaire. diabetic diet sheets were dispensed to subjects based on their body mass index (bmi), daily activity, and needs.results:the mean caloric intake between pre - ramadan (before fasting) and mid - ramadan (15 days after fasting) were 1506.80 kcal and 1614.29 (p = 0.001) respectively. the distribution of active components pre and during ramadan were : carbohydrates (g) 260.76 and 265.35 g (p = 0.001), proteins (g) 43.64 and 46.19 (p = 0.001) and fat (g) was 32.88 and 44.16 (p = 0.0001) respectively. the percentage of energy from dietary carbohydrate prior to fasting (64.11 6.73) and during fasting (68.41 4.41) remained almost unchanged but statistically significant when compared at different intervals before and during fasting. fat intake increased significantly during fasting (p = < 0.001).conclusions : the dietary composition in a type 2 diabetic muslim population who undertook fasting during ramadan showed a mean increase in consumption of all components of diet during the period of fasting. nutritional compliance during such a time seems to be difficult and warrants repeated counseling and regular follow - up to achieve targets. |
renal diseases contribute a major component to morbidity and mortality ; with a prevalence of 17.2%. it has become a global problem necessitating early detection, evaluation, and preventive management to delay progression and to prevent adverse outcomes. the incidence of renal failure has doubled in the last 15 years. with progressive renal failure, glomerular filtration rate reduces below 15 ml / min leading to accumulation of metabolic byproducts such as urea and creatinine along with imbalance of electrolytes in serum. this necessitates renal replacement therapy (rrt) to avoid the serious complications leading to death. alternate to rrt, constant timely hemodialysis at regular intervals can be life - sustaining tool for these chronic renal failure patients. frequency of dialysis or time to initiate dialysis remains the key factor for maintaining homeostasis and to improve the quality of life of these patients. constant monitoring of serum levels of metabolic byproducts such as creatinine, urea, and potassium is needed. repeated venipuncture increases patient 's infection risks. saliva is considered as a filtrate of the blood where various molecules pass through transcellular (passive intracellular diffusion and active transport) or paracellular routes (extracellular ultrafiltration) into saliva. as a result, saliva is equivalent to serum, thereby reflecting the physiological state of the body. studies have shown variations in salivary levels of urea, creatinine, sodium, and potassium in renal failure patients. based on the availability of improved salivary diagnostic systems, this study was designed to test the diagnostic accuracy of salivary levels of creatinine, urea, sodium, potassium, and calcium using diagnostic validity test, receiver operator characteristic (roc) curve, and we also aimed to determine cutoff values for salivary creatinine, urea, sodium, potassium, and calcium as indicators of dialysis need in patients with renal failure. the study group consisted of 41 recently diagnosed renal failure patients undergoing dialysis for the first time while 41 healthy age- and sex - matched individuals constituted the control group. written informed consent individuals with other diseases, medications, and habits that affect water and electrolyte balance were excluded from the study. under aseptic conditions, the samples were centrifuged at 2000 revolutions / min (rpm) for 23 min to obtain serum. all participants were instructed to avoid eating or drinking for 2 h before collection of saliva. the samples were immediately transferred to a vaccine carrier with ice pack to avoid biochemical changes and carried to the laboratory. the samples were centrifuged at 4000 rpm for 10 min to obtain supernatant saliva. in renal failure patients, blood and saliva urea, creatinine, sodium, potassium, calcium, and phosphorus levels were determined in serum and supernatant saliva using semi - autoanalyzer. comparison of levels of serum and salivary urea, creatinine, sodium, potassium, calcium, and phosphorus between renal failure cases and age- and sex - matched healthy controls was done using unpaired t - test. pearson 's correlation coefficient was used to measure the degree of relationship between salivary and serum parameters. pearson 's correlation coefficient has been represented as r value, which signifies the extent of linear relationship between two variables (serum and salivary parameters). any value between 1 and 0 indicates negative correlation and between 0 and + 1 indicates positive correlation. 1 indicates perfect negative linear relationship, + 1 indicates perfect positive linear relationship, and 0 indicates two variables are independent of each other. diagnostic values of salivary parameters were assessed using diagnostic validity tests and were confirmed using roc curve. comparison of levels of serum and salivary urea, creatinine, sodium, potassium, calcium, and phosphorus between renal failure cases and age- and sex - matched healthy controls was done using unpaired t - test. pearson 's correlation coefficient was used to measure the degree of relationship between salivary and serum parameters. pearson 's correlation coefficient has been represented as r value, which signifies the extent of linear relationship between two variables (serum and salivary parameters). any value between 1 and 0 indicates negative correlation and between 0 and + 1 indicates positive correlation. 1 indicates perfect negative linear relationship, + 1 indicates perfect positive linear relationship, and 0 indicates two variables are independent of each other. diagnostic values of salivary parameters were assessed using diagnostic validity tests and were confirmed using roc curve. the values of urea and creatinine were significantly high in serum and saliva of cases when compared to controls. a statistically significant positive correlation was detected between serum and salivary urea concentration [r = + 0.81, graph 1, p = 0.00 ] and between serum and salivary creatinine concentration [r = + 0.65, graph 2, p = 0.00, table 1 ]. area under the curve in roc for salivary urea [graph 3a ] and creatinine [graph 3b ] was 0.9, suggestive of excellent diagnostic accuracy [table 2 ]. correlation of serum and salivary parameters in cases and controls (a) receiver operating characteristic curve for salivary urea, (b) receiver operating characteristic curve for salivary creatinine. combined table of diagnostic validity tests for all the variables the sodium levels were increased significantly with cases both in serum and saliva compared to controls. a statistically significant positive correlation was detected between serum and salivary sodium concentration [r = + 0.74, p = 0.00, table 1 ]. diagnostic accuracy and area under curve in roc for salivary sodium were 73% and 0.7, respectively, suggestive of good diagnostic accuracy [table 2 ]. a slightly negative correlation was detected between serum and salivary potassium concentration [r = 0.03, p = 0.88, table 1 ]. diagnostic accuracy of salivary potassium was 89% and area under the curve in roc was 0.9, suggestive of excellent diagnostic accuracy [table 2 ]. the values of serum and salivary calcium showed slight reduction in cases when compared to controls. the values of serum and salivary phosphorus showed slight increase in cases compared to controls. correlation between serum and salivary calcium (r = + 0.29, p = 0.06) and between serum and salivary phosphorus [r = + 0.271, p = 0.09, table 1 ] was not statistically significant. hence, cutoff value and diagnostic validity tests were not applicable. the achieved diagnostic accuracy of salivary urea, creatinine, sodium, and potassium in this study proved that saliva can be used as noninvasive diagnostic fluid in renal failure patients to monitor the levels of above - mentioned parameters. salivary levels of urea, creatinine, sodium, and potassium were proportional with their serum counterparts, and the mean values of the same are discussed in table 3. mean values and cutoff values of serum and salivary urea, creatinine, sodium, potassium, calcium, and phosphorus cutoff values of the salivary levels of individual parameters were also evaluated. the cutoff values were evaluated in comparison with serum levels, which means any value of a parameter above the cutoff value would be considered as abnormal. the values of urea and creatinine were significantly high in serum and saliva of cases when compared to controls. a statistically significant positive correlation was detected between serum and salivary urea concentration [r = + 0.81, graph 1, p = 0.00 ] and between serum and salivary creatinine concentration [r = + 0.65, graph 2, p = 0.00, table 1 ]. area under the curve in roc for salivary urea [graph 3a ] and creatinine [graph 3b ] was 0.9, suggestive of excellent diagnostic accuracy [table 2 ]. correlation of serum and salivary parameters in cases and controls (a) receiver operating characteristic curve for salivary urea, (b) receiver operating characteristic curve for salivary creatinine. the sodium levels were increased significantly with cases both in serum and saliva compared to controls. a statistically significant positive correlation was detected between serum and salivary sodium concentration [r = + 0.74, p = 0.00, table 1 ]. diagnostic accuracy and area under curve in roc for salivary sodium were 73% and 0.7, respectively, suggestive of good diagnostic accuracy [table 2 ]. a slightly negative correlation was detected between serum and salivary potassium concentration [r = 0.03, p = 0.88, table 1 ]. diagnostic accuracy of salivary potassium was 89% and area under the curve in roc was 0.9, suggestive of excellent diagnostic accuracy [table 2 ]. the values of serum and salivary calcium showed slight reduction in cases when compared to controls. the values of serum and salivary phosphorus showed slight increase in cases compared to controls. correlation between serum and salivary calcium (r = + 0.29, p = 0.06) and between serum and salivary phosphorus [r = + 0.271, p = 0.09, table 1 ] was not statistically significant. the achieved diagnostic accuracy of salivary urea, creatinine, sodium, and potassium in this study proved that saliva can be used as noninvasive diagnostic fluid in renal failure patients to monitor the levels of above - mentioned parameters. salivary levels of urea, creatinine, sodium, and potassium were proportional with their serum counterparts, and the mean values of the same are discussed in table 3. mean values and cutoff values of serum and salivary urea, creatinine, sodium, potassium, calcium, and phosphorus cutoff values of the salivary levels of individual parameters were also evaluated. the cutoff values were evaluated in comparison with serum levels, which means any value of a parameter above the cutoff value would be considered as abnormal. kidneys regulate the volume and composition of the extracellular fluid to maintain homeostasis by constantly processing the plasma by filtration, reabsorption, and secretion of substances, thereby help in preserving the internal environment of the body. renal damage reduces glomerular filtration capacity of kidneys and leads to increased serum levels of metabolic byproducts. among the byproducts, urea and creatinine dialysis is used to remove excess metabolic byproducts in cases of renal failure. during renal failure, continuous monitoring of serum levels of metabolic byproducts decides the need for dialysis. among all the metabolic byproducts, urea, creatinine, and potassium levels have been considered to be decisive indicators for initiation of dialysis. considering the disadvantages of invasive serum collection method and ease of saliva collection, saliva is a filtrate of serum and has been explored as an alternative to serum. in this study, we examined the use of saliva as an alternative to monitor the metabolic byproducts of kidney failure. sialometric parameters vary with age and sex. after reaching maximum development at the age of 15 years, salivary gland parenchyma will be gradually replaced by adipose and fibrovascular tissue leading to reduction in volume of acini in turn leading to sialometric alterations. in addition, women present smaller salivary glands in comparison with men ; this along with female hormonal pattern may contribute to variation in salivary parameters among the sexes. considering the above factors, age- and sex - matched controls salivary urea and creatinine levels showed positive correlation with serum levels both in cases and controls. the correlation of salivary urea and creatinine level so with serum further saliva as an ultra - filtrate of serum. in this study, the correlation coefficient of salivary and serum urea level was 0.8 while that of salivary and serum creatinine was 0.69. although no previous explanation is available for this finding, the cause may be the lower molecular weight and size of urea at 60.03 d and 0.26 nm, respectively, in comparison with that of creatinine at 113 d and 0.3 nm, leading to a greater filtration of urea in comparison to creatinine. a significant positive correlation was found between salivary sodium level and serum sodium level, whereas slight negative correlation was obtained when salivary and serum potassium levels were compared and correlated. the salivary concentration of these ions (sodium and potassium) does not depend entirely on their serum concentration, and instead depend on differing, reabsorption of sodium and secretion of potassium in the striated ducts of salivary glands, thus explaining the increase potassium ion concentration in saliva than in serum. very high correlation coefficient of r = + 0.5 was found between serum and salivary potassium in patients undergoing dialysis by nagler. the reduction in serum and salivary calcium level is the consequence of a fall in 1,25 dihydroxycholecalciferol, an active metabolite of vitamin d synthesized in the kidney which plays a main role in calcium absorption from intestine, thereby causing dip in the calcium levels in serum and saliva. serum and salivary phosphorus values showed statistically nonsignificant increase in the study group and positive correlation was obtained between serum and salivary phosphorus but was statistically nonsignificant. our finding of increased level of salivary phosphorus was in agreement with a study done by savica. the increase in serum and salivary phosphorus levels can be explained by diminished phosphate load in the filtrate. the amount of the phosphate filtered is completely reabsorbed in the tubules, thus increasing plasma level of phosphorus. hence, hyperphosphatemia is dangerous due to increased risk of precipitation of calcium phosphate in soft tissue and in walls of blood vessels, contributing to cardiovascular calcification in renal failure patients. hence, increase in the level of salivary phosphorus is due to renal function deterioration. the salivary urea showed sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv) of 93% and overall diagnostic accuracy of salivary urea was found to be 93% in this study. this suggested that salivary urea has excellent diagnostic accuracy which was also confirmed by its score of 0.9 of area under the curve in roc. similar findings were obtained by other authors. however, comparatively, lesser values of sensitivity of 80%, specificity of 71%, ppv of 80%, and npv of 71% were demonstrated by zuniga., whereas sensitivity of 80%, specificity of 90%, and area under the curve of 0.898 in roc were demonstrated by xai. the salivary creatinine showed sensitivity of 93%, specificity of 90%, ppv of 90%, npv of 93%, and overall diagnostic accuracy of 91% which suggested that salivary creatinine has excellent diagnostic accuracy, which was also confirmed by its score of 0.9 of area under the curve in roc. parallel findings were obtained by previous studies. however, comparatively, less diagnostic accuracy was demonstrated by xai., in which sensitivity was 77%, specificity was 98%, and area under the curve was 0.897 in roc. minor disparity in the diagnostic accuracy of urea and creatinine between studies could be due to difference in sample size, method of estimation, time and method of sample collection. the salivary sodium showed sensitivity, specificity, ppv, npv of 73% and overall diagnostic accuracy of 73%, which suggested that salivary sodium had good diagnostic accuracy and was confirmed by its score of 0.7 of area under the curve in roc. the salivary potassium showed sensitivity of 83%, specificity of 78%, ppv of 79%, npv of 82% with overall diagnostic accuracy of 89% which suggested that salivary potassium had excellent diagnostic accuracy confirmed by its score of 0.9 of area under the curve in roc. based on the findings of this study, we concluded that salivary diagnostics is a simple, quick, noninvasive, inexpensive, highly accurate, and reliable technique to assess the serum levels of metabolic byproducts and electrolytes in patients with renal failure. the salivary urea, creatinine, sodium, and potassium are diagnostically accurate and can be used to monitor serum levels of metabolic byproducts such as urea and creatinine and for screening of high - risk patients to assess the need for dialysis. the authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or non - financial in this article. the authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or non - financial in this article. | background : the prevalence of chronic renal failure is increasing because of increase in chronic debilitating diseases and progressing age of population. these patients experience accumulation of metabolic byproducts and electrolyte imbalance, which has harmful effects on their health. timely hemodialysis at regular intervals is a life - saving procedure for these patients. salivary diagnostics is increasingly used as an alternative to the traditional methods. thus, the aim of the present study was to determine the diagnostic efficacy of saliva in chronic renal failure patients.materials and methods : this case control study included 82 individuals, of which 41 were chronic renal failure patients and 41 were age- and sex - matched controls. blood and saliva were collected and centrifuged. serum and supernatant saliva were used for biochemical analysis. serum and salivary urea, creatinine, sodium, potassium, calcium, and phosphorus were evaluated and correlated in chronic renal failure patients using unpaired t - test, pearson 's correlation coefficient, diagnostic validity tests, and receiver operative curve.results:when compared to serum ; salivary urea, creatinine, sodium, and potassium showed diagnostic accuracy of 93%, 91%, 73%, and 89%, respectively, based on the findings of study.conclusion:it can be concluded that salivary investigation is a dependable, noninvasive, noninfectious, simple, and quick method for screening the mineral and metabolite values of high - risk patients and monitoring the renal failure patients. |
the online version of this article (doi:10.1007/s00134 - 007 - 0545-y) contains supplementary material, which is available to authorized users high - frequency ventilation (hfv) has been compared with conventional mechanical ventilation (cmv) since the 1980s. in hfv, patients are ventilated with small tidal volumes, even smaller than the dead space of their airways, at high frequencies, normally between 5 and 10 hz. because hfv combines high mean airway pressures with small tidal volumes, this technique of ventilation has been regarded by some to be the most optimal form in patients with infant respiratory distress syndrome (irds), adult respiratory distress syndrome (ards), and other forms of severe lung disease. the hfv has been extensively investigated in premature neonates with irds, a population specifically at risk for chronic lung disease (cld). unfortunately, the results of these studies were equivocal [2, 3 ] ; thus, the question remains whether or not hfv better prevents cld than conventional mechanical ventilation (cmv) in patients with severe lung disease. a significant number of meta - analyses have been performed to answer this question [4, 5, 6, 7, 8 ]. pooled estimates of pulmonary outcomes failed to show clinically relevant differences among hfv and cmv ; however, significant heterogeneity existed between studies included in these meta - analyses. in a recent cumulative meta - analysis we identified improvements of the conventional treatment of irds and ventilation strategies applied in both hfv and cmv as important sources of heterogeneity. although a meta - analysis may pool results from randomized trials, differences among trials will not be randomly or independently distributed. a meta - analysis constitutes an observational study of trials, subjected to bias inherent to observational research. in a meta - regression analysis it is possible to adjust for confounding covariates. a number of alternative hypotheses have been formulated to explain heterogeneity between trials : the observed regression of the cumulative relative risks to the level of unity was due to publication bias.use of the sensormedics ventilator resulted in better results in hfv treated patients.a prolonged ventilation on cmv before initiating hfv treatment could reduce the benefits of hfv.in subgroups of more premature neonates with lower birth weight with a higher susceptibility for cld, hfv could result in better pulmonary outcome.with outcome rates increasingly representing more severe disease, hfv could have an increasing advantage over cmv [9, 10 ] ; therefore, we used meta - regression analysis to better estimate relative treatment effects through adjustments for factors that could explain trial heterogeneity. the observed regression of the cumulative relative risks to the level of unity was due to publication bias. use of the sensormedics ventilator resulted in better results in hfv treated patients. a prolonged ventilation on cmv before initiating hfv treatment could reduce the benefits of hfv. in subgroups of more premature neonates with lower birth weight with a higher susceptibility for cld, hfv could result in better pulmonary outcome. with outcome rates increasingly representing more severe disease, hfv could have an increasing advantage over cmv [9, 10 ] ; therefore, we used meta - regression analysis to better estimate relative treatment effects through adjustments for factors that could explain trial heterogeneity. trials were included based on a previous meta - analysis that we had conducted. the same search strategy, as well as the same inclusion and exclusion criteria as in our previous meta - analysis, were used for an update, yielding two more studies that could be included for this meta - regression analysis. the following outcome measures were used : mortality, chronic lung disease (cld) as defined by supplemental oxygen need or ventilator dependency at the age of 3036 weeks post - menstrual. a number of explanatory variables were extracted as well : year of publication ; type of ventilator used for hfv (sensormedics 3100a ventilator versus other) ; ventilation strategies applied in the hfv and cmv treatment groups were obtained as previously described ; time on cmv before study initiation ; gestational age and birth weight ; and outcome rates in the control population were taken as proxy for baseline disease severity in the source population. the sensormedics ventilator was singled out because previous research suggested better performance compared with other oscillator ventilators [2, 4 ]. the number of patients surviving without chronic lung disease was subtracted from the total number of randomized patients in each treatment arm to calculate the composite outcome of death or cld. to calculate the risk of cld publication bias was assessed by visual appraisal of symmetry of funnel plots and performing rank tests. smaller studies could show different results than larger studies which could suggest publication bias, but in fact was caused by systematic differences among studies ; therefore, an analysis of publication bias stratified for ventilation strategies was performed to determine whether the observed association between the inverse of the standard error with the risk ratio was confounded by ventilation strategies. the dependent variables, rr of cld and rr of cld or death, were natural log transformed to linearize the regression models. individual studies were weighted by inverse variances of relative risks of outcomes of interest so that the more precise studies had more influence in the analysis. secondly, linear regression analyses with continuous covariates were conducted stratified by hlvs, lpvs, and use of surfactant. finally, multivariable linear regression analyses were performed to calculate adjusted contributions of different explanatory variables of rivalling hypotheses to changes in rr. a relative risk ratio quantifies the relative change in rr that is associated with a specified change of a covariate. for continuous variables the rrr was calculated for the ranges of minimum and maximum values of covariates that were reported in trials. for example, the rrr for year of publication was calculated by using the range between the publication year of the first year and the publication year of the last included trial. the rrr for year of publication thus estimates the relative change in rr due to the difference in years of publication between the first and last trials. all analyses were conducted using spss 12.0.1 for windows software (spss, chicago, ill.) and excel (microsoft, redmond, wash.). all data were extracted according to the intention - to - treat principle. the number of patients surviving without chronic lung disease was subtracted from the total number of randomized patients in each treatment arm to calculate the composite outcome of death or cld. to calculate the risk of cld, the number of surviving patients was put in the denominator. publication bias was assessed by visual appraisal of symmetry of funnel plots and performing rank tests. smaller studies could show different results than larger studies which could suggest publication bias, but in fact was caused by systematic differences among studies ; therefore, an analysis of publication bias stratified for ventilation strategies was performed to determine whether the observed association between the inverse of the standard error with the risk ratio was confounded by ventilation strategies. the dependent variables, rr of cld and rr of cld or death, were natural log transformed to linearize the regression models. individual studies were weighted by inverse variances of relative risks of outcomes of interest so that the more precise studies had more influence in the analysis. secondly, linear regression analyses with continuous covariates were conducted stratified by hlvs, lpvs, and use of surfactant. finally, multivariable linear regression analyses were performed to calculate adjusted contributions of different explanatory variables of rivalling hypotheses to changes in rr. a relative risk ratio quantifies the relative change in rr that is associated with a specified change of a covariate. for continuous variables the rrr was calculated for the ranges of minimum and maximum values of covariates that were reported in trials. for example, the rrr for year of publication was calculated by using the range between the publication year of the first year and the publication year of the last included trial. the rrr for year of publication thus estimates the relative change in rr due to the difference in years of publication between the first and last trials. all analyses were conducted using spss 12.0.1 for windows software (spss, chicago, ill.) and excel (microsoft, redmond, wash.). for the analyses 15 studies were available that specified either cld in survivors or death or cld as outcome measures [2, 3, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 ]. in 11 trials a high frequency oscillatory ventilator was used [2, 3, 12, 13, 16, 17, 19, 20, 22, 23, 24 ], in 7 of these trials this was the sensormedics ventilator [2, 12, 13, 17, 22, 23 ]. two studies used a high - frequency jet ventilator [14, 15 ] and in two studies a high - frequency flow interrupter ventilator was used [18, 21 ]. in the hfv group a total of 1141 patients were included for the outcome of cld with 373 events and a total of 1457 patients with 671 events for the outcome death or cld. in the cmv group a total 1159 patients were reported for the outcome of cld with 428 events and a total of 1473 patients with 730 events for the outcome death or cld. time on cmv to start of the study was not reported by plavka. and craft.. in only one study was surfactant not used as concomitant treatment. a high lung volume strategy (hlvs) was used in all but two studies [14, 16 ]. a ventilation strategy in the cmv - treated patients that could qualify as lung protective (lpvs) was reported in the most recent 9 studies [2, 3, 18, 19, 20, 21, 22, 23, 24 ]. other reported ranges of covariates were 8.7 h average time on cmv before start of study, 5 weeks average gestational age, and 0.65 kg average birth weight. two studies dominated the analyses by virtue of the weight they received in the analyses : johnson. and courtney. (together 69% for cld and 73% for death or cld as outcome). table 1study characteristicsreferenceyeartime on cmvagebirth weightsensormhlvslpvssurfcld lnrrweightdeath or cld lnrrweight 1992 9.0281.100yynn-1.290.01 - 0.580.01 1996 3.0311.500yyny-0.670.04 - 0.550.02 1996 7.2270.950nnny 0.020.01 - 0.230.10 1997 8.0271.020nyny-0.700.03 0.480.03 1998 1.0281.100nnny 0.000.00 0.310.00 1999260.850yyny-1.030.01 - 0.740.01 1999 0.5270.870nyyy 0.090.06 0.010.04 2001 2.6260.840yyyy-0.980.02 - 0.590.02 2001 0.3280.990nyyy-0.200.05 - 0.060.05 2002 2.7260.850yyyy-0.060.16 - 0.220.13 2002 1.0260.850nyyy-0.010.54 - 0.020.60 2003 1.0291.200yyyy 0.320.03 0.270.04 200314.0270.980yyyy-0.040.05 2003260.726nyyy 0.100.05 0.090.03 2005 0.3270.880nyyy-1.440.01 - 1.200.00year : year of publication. hlvs : high lung volume strategy in the hfv group, lpvs : lung protective ventilation strategy in the cmv group, surf : use of surfactant in the study. cld : chronic lung disease, defined as on oxygen at 3036 weeks postmenstrual age, lnrr : natural log of the relative risk study characteristics year : year of publication. hlvs : high lung volume strategy in the hfv group, lpvs : lung protective ventilation strategy in the cmv group, surf : use of surfactant in the study. cld : chronic lung disease, defined as on oxygen at 3036 weeks postmenstrual age, lnrr : natural log of the relative risk a funnel plot of the inverse of the standard error against the natural logarithm of the rr for cld was indicative of publication bias because of asymmetry round the line of the pooled effect (fig. 1). a stratified analysis of publication bias is indicated by different colors in fig. 1. to visually evaluate publication bias within subgroups of ventilation strategy, the distribution of trials round the corresponding colored lines (mean effect size within subgroup) was assessed. stratification by ventilation strategy (hlvs and lpvs vs either no hlvs and/or no lpvs) showed p - values of 0.456 and 0.851, respectively, indicating less evidence of publication bias. the distribution of stratified studies round the lines of pooled estimates showed less asymmetry (fig. 1). publication bias for the composite outcome of death or cld was less likely with a p - value of 0.329. selection bias in reporting rr of chronic lung disease (cld) as suggested by asymmetry of the distribution of studies. x - axis : inverse of the standard error of the rr ; y - axis : natural logarithm of the rr. blue diamonds : studies with either no high lung volume strategy (hlvs) or no lung protective volume strategy (lpvs) ; pink diamonds : studies with both hlvs and lpvs ; dotted line : estimated pooled rr including all studies ; dashed colored lines : pooled rr of subgroups of studies. publication bias was visually appraised by assessing symmetry of distribution of studies around the lines of pooled estimates. selection bias in reporting rr of chronic lung disease (cld) as suggested by asymmetry of the distribution of studies. x - axis : inverse of the standard error of the rr ; y - axis : natural logarithm of the rr. blue diamonds : studies with either no high lung volume strategy (hlvs) or no lung protective volume strategy (lpvs) ; pink diamonds : studies with both hlvs and lpvs ; dotted line : estimated pooled rr including all studies ; dashed colored lines : pooled rr of subgroups of studies. publication bias was visually appraised by assessing symmetry of distribution of studies around the lines of pooled estimates. cmv conventional mechanical ventilation figures 2, 3, 4 show the results of the linear meta - regression analyses for continuous explanatory variables with relative risk of cld as dependent variable. two studies dominate these figures, designated by the weight they received in the analyses [2, 3 ]. over the years the reported benefit of hfv over cmv seemed to diminish (fig. 2). a longer time on cmv prior to study initiation and a higher gestational age and increase of birth weight (data not shown) seemed to be positively associated with a relatively better outcome in hfv (figs. 3, 4). table 2 shows the results of linear meta - regression analyses, showing significant associations with year of publication (3.1 times higher rr with change of publication year from 1992 to 2005) and whether or not a protective ventilation strategy was applied (1.9 times higher rr with change of protective ventilation from no to yes ; table 2). in the linear regression analyses with death or cld as composite outcome no significant associations were detected. whether or not a sensormedics high - frequency oscillatory ventilator was used and baseline incidence in cmv (0.75 vs 0.08) treated patients displayed the smallest effects on trial outcome (rrr = 0.84 and 0.90 for cld and rrr = 0.85 and 0.99 for death or cld, respectively). crude and subgroup linear regression analyses of the effect of year of publication, prior time on cmv and gestational age with natural logarithm of rr of cld as dependent variable. y - axis : natural logarithm of the rr ; x - axis : explanatory variables. blue diamonds : studies with either no hlvs or no lpvs ; pink diamonds : studies with both hlvs and lpvs. the size of the diamonds reflects the weights the individual trials contribute to the analyses. thin blue line : regression line including all studies ; thick pink line : regression line including only studies with both hlvs and lpvs. 2table 2univariable linear regression analysis95% confidence interval95% confidence intervalcrude bsig.lowerupperrrrlowerupperboundaryboundaryboundaryboundaryall studiescld year 0.090.025 0.010.163.131.18 8.27 sensorm-0.170.351 - 0.550.210.840.58 1.24 (no to yes) timecmv-0.090.055 - 0.190.000.440.19 1.02 age-0.080.237 - 0.230.060.660.32 1.36 weight-0.760.163 - 1.870.350.540.22 1.33 hlvs-0.110.883 - 1.741.520.890.17 4.57 lpvs 0.640.009 0.191.101.911.21 3.00 surf 1.210.168 - 0.593.003.340.5620.03 cmv-0.180.774 - 1.531.170.900.42 1.92death or cld year 0.050.096 - 0.010.122.010.86 4.65 sensorm-0.170.132 - 0.390.060.850.67 1.06 timecmv-0.010.590 - 0.050.030.920.65 1.29 age-0.020.733 - 0.130.100.910.52 1.61 weight-0.220.611 - 1.160.710.840.40 1.77 hlvs-0.370.698 - 2.441.690.690.09 5.45 lpvs 0.190.275 - 0.180.561.210.84 1.76 surf 0.520.289 - 0.511.561.690.60 4.75 cmv-0.020.963 - 0.910.870.990.60 1.63studies with surfactant, hlvs, and lpvscld year 0.000.971 - 0.230.220.960.0517.34 timecmv-0.050.698 - 0.340.250.660.05 8.75 age 0.040.727 - 0.220.301.220.33 4.49 weight 0.410.693 - 1.992.811.380.20 9.44death or cld year 0.010.846 - 0.150.171.200.15 9.72 timecmv 0.000.819 - 0.050.040.960.65 1.43 age 0.060.406 - 0.100.211.340.61 2.92 weight 0.550.396 - 0.891.991.550.49 4.90simple linear regression analyses were calculated for chronic lung disease (cld), defined as on oxygen at 3036 weeks postgestational age, and death or cld. the following co - variates were evaluated : year : number of years after the first included study ; sensorm : whether or not a sensormedics type of hfv was used ; timecmv : mean time on cmv before start of the study in hours ; age : mean gestational age (weeks) ; weight : mean birth weight (kg) ; hlvs : high lung volume strategy in the hfv group ; lpvs : lung protective ventilation strategy in the cmv group ; surf : use of surfactant in the study ; b was the estimated crude coefficient ; rrr : relative risk ratio = rrcovariate=1/rrcovariate=0, for binary variables (sensorm, hlvs and lpvs), for continues variables the extreme values reported in the studies were used to calculate the ranges, 13 for years, 8.7 for time on cmv, 5 for age, 0.8 for weight and 0.65 for cmv (rrryears = rryear=2005/rryear=1992, rrrtime on cmv = rrtime=9 h / rrtime=0.3 h, rrrage = rrage=31 weeks / rryear=26 weeks, rrrweight = rrweight=1.5 kg / rryear=0.7 kg, rrrincidence of cld in cmv=0.75/rrincidence=0.08) linear regression analyses. crude and subgroup linear regression analyses of the effect of year of publication, prior time on cmv and gestational age with natural logarithm of rr of cld as dependent variable. y - axis : natural logarithm of the rr ; x - axis : explanatory variables. blue diamonds : studies with either no hlvs or no lpvs ; pink diamonds : studies with both hlvs and lpvs. the size of the diamonds reflects the weights the individual trials contribute to the analyses. thin blue line : regression line including all studies ; thick pink line : regression line including only studies with both hlvs and lpvs. cmv conventional mechanical ventilation univariable linear regression analysis simple linear regression analyses were calculated for chronic lung disease (cld), defined as on oxygen at 3036 weeks postgestational age, and death or cld. the following co - variates were evaluated : year : number of years after the first included study ; sensorm : whether or not a sensormedics type of hfv was used ; timecmv : mean time on cmv before start of the study in hours ; age : mean gestational age (weeks) ; weight : mean birth weight (kg) ; hlvs : high lung volume strategy in the hfv group ; lpvs : lung protective ventilation strategy in the cmv group ; surf : use of surfactant in the study ; b was the estimated crude coefficient ; rrr : relative risk ratio = rrcovariate=1/rrcovariate=0, for binary variables (sensorm, hlvs and lpvs), for continues variables the extreme values reported in the studies were used to calculate the ranges, 13 for years, 8.7 for time on cmv, 5 for age, 0.8 for weight and 0.65 for cmv (rrryears = rryear=2005/rryear=1992, rrrtime on cmv = rrtime=9 h / rrtime=0.3 h, rrrage = rrage=31 weeks / rryear=26 weeks, rrrweight = rrweight=1.5 kg / rryear=0.7 kg, rrrincidence of cld in cmv=0.75/rrincidence=0.08) figure 5 shows how the incidence of cld in the cmv treated patients was related to the incidence in hfv treated patients for each of the studies. a trend line was fitted by weighted linear regression, showing a small effect of change in incidence in cmv on incidence in hfv - treated patients. 5linear regression analysis of incidence of cld in cmv on incidence of cld in hfv. y - axis : incidence of cld in hfv ; x - axis : incidence of cld in cmv. thin pink line : regression line including all studies linear regression analysis of incidence of cld in cmv on incidence of cld in hfv. y - axis : incidence of cld in hfv ; x - axis : incidence of cld in cmv. thin pink line : regression line including all studies year of publication was not related to change in relative risk of cld in the subgroup of studies with hlvs, lpvs and concomitant use of surfactant (rrr = 0.96). there was only a small increase in relative risk for death or cld (rrr = 1.20 ; fig. 2 ; table 2). opposite effects of gestational age (rrr = 1.22 for cld and 1.38 for death or cld vs rrr = 0.66 for cld and 0.91 for death or cld, respectively) and birth weight were detected in the subgroup analysis (fig. 4 ; table 2). prior time on cmv exerted less effect on outcome compared with the crude analysis, rrr = 0.66 for cld and 0.96 for death or cld and rrr = 0.44 for cld and 0.92 for death or cld in the adjusted and crude analyses, respectively (fig. 3 ; table 2). multivariable linear regression analyses were conducted to assess the independent contributions to change in rr by explanatory variables (table 3). the rrrs in table 3 have the same meaning as in table 2, only they represented adjusted rrrs. year of publication was not considered as an independent explanatory variable but rather as proxy for changes in treatment and patient population. gestational age and birth weight were collinearly related by nature ; only gestational age was fitted in the model. one study contributed to the fact that surfactant was not used ; therefore, surfactant was not used in the multiple linear regression analyses. model a used sensormedics, time on cmv, gestational age, hlvs, and lpvs as covariates. the largest estimated effects were caused by ventilation strategies, hlvs, and lpvs, adjusted for use of sensormedics ventilator, prior time on cmv, and gestational age. these estimations were consistent for the outcomes cld (rrr = 0.42 and rrr = 2.02 for hlvs and lpvs, respectively) and death or cld (rrr = 0.42 and rrr = 1.98 fro hlvs and lpvs, respectively). use of a sensormedics ventilator seemed to have a much smaller effect on rr for outcome. the rrr of gestational age, comparing 26 weeks with 31 weeks, for cld and death or cld were larger (rrr = 1.17 and rrr = 1.47). the effect of a difference in prior time on cmv of 8.7 h on cld vs death or cld was not consistent (rrr = 0.85 and rrr = 1.07, respectively). table 3multivariable linear regression analysisadjusted95% confidence interval95% confidence intervalbsig.lower boundaryupper boundaryrrrlower boundaryupper boundarymodel acld (constant)-0.660.900 - 13.0311.70 sensorm-0.040.884 -0.75 0.660.960.47 1.94 timecmv-0.020.903 -0.38 0.340.850.0419.22 age 0.030.850 -0.36 0.421.170.16 8.32 hlvs-0.880.306 -2.80 1.040.420.06 2.84 lpvs 0.700.506 -1.73 3.142.020.1823.12death or cld (constant)-1.860.412 -7.22 3.49 sensorm-0.170.309 -0.55 0.210.850.58 1.24 timecmv 0.010.722 -0.05 0.061.070.68 1.69 age 0.080.299 -0.09 0.251.470.62 3.47 hlvs-0.880.407 -3.38 1.620.420.03 5.06 lpvs 0.680.127 -0.28 1.651.980.76 5.19model bcld (constant) 0.070.904 -1.21 1.35 sensorm-0.060.698 -0.38 0.260.940.69 1.30 hlvs-0.810.203 -2.14 0.520.440.12 1.68 lpvs 0.720.011 0.21 1.232.061.23 3.43death or cld (constant) sensorm-0.110.318 -0.33 0.120.900.72 1.13 hlvs-0.790.363 -2.66 1.080.450.07 2.93 lpvs 0.460.089 -0.09 1.011.590.92 2.74multiple linear regression analyses were calculated for chronic lung disease (cld), defined as on oxygen at 30 - 36 weeks postgestational age, and death or cld. the following co - variates were evaluated : sensorm whether or not a sensormedics type of hfv was used ; timecmv mean time on cmv before start of the study (in hours). hlvs high lung volume strategy in the hfv group, lpvs lung protective ventilation strategy in the cmv group. rrr relative risk ratio = rrcovariate=1/rrcovariate=0, for binary variables (sensorm, hlvs and lpvs), for continues variables the extreme values reported in the studies were used, 8.7 for time on cmv (rrrage = rrage=31 weeks / rryear=26 weeks) multivariable linear regression analysis multiple linear regression analyses were calculated for chronic lung disease (cld), defined as on oxygen at 30 - 36 weeks postgestational age, and death or cld. the following co - variates were evaluated : sensorm whether or not a sensormedics type of hfv was used ; timecmv mean time on cmv before start of the study (in hours). hlvs high lung volume strategy in the hfv group, lpvs lung protective ventilation strategy in the cmv group. rrr relative risk ratio = rrcovariate=1/rrcovariate=0, for binary variables (sensorm, hlvs and lpvs), for continues variables the extreme values reported in the studies were used, 8.7 for time on cmv (rrrage = rrage=31 weeks / rryear=26 weeks) a sensitivity analysis was conducted by fitting a second model (model b) with the most important variables, hlvs and lpvs, combined with whether or not a sensormedics ventilator was used. type of ventilator did not have a large effect compared with ventilation strategies (rrr = 0.94 and rrr = 0.90). the hlvs was associated with a decrease of the rrs comparing hfv with cmv (rrr = 0.44 and rrr = 0.45), while lpvs increased the rrs to the line of no effect (rrr = 2.06 and rrr = 1.59). our meta - regression analysis showed a clear trend of decreasing differences in pulmonary outcome between hfv and cmv in randomized trials conducted in premature neonates with irds over the years. the most likely hypothesis for this trend was the application of a lpvs in the most recent studies. use of surfactant could also have a significant contribution, but only one study did not use surfactant. in previous meta - analyses, subgroup analyses or cumulative methods subgroup analysis is equivalent to meta - regression with a categorical trial - level covariate. considering subgroup analysis formally as a meta - regression has advantages, since it focuses on differences between subgroups as is appropriate, rather than the effects in each subgroup separately. furthermore, it is appropriate to use meta - regression to explore sources of heterogeneity, even if an initial overall test for heterogeneity is non - significant. this test often has low power and therefore a non - significant result does not reliably identify lack of heterogeneity. in this meta - regression analysis we evaluated in a quantitative way a number of hypotheses that were raised to account for different results between randomized trials. a relatively large proportion of well - conducted trials were available for the analyses. the effects of the two most important covariates, hlvs and lpvs, were consistent in the different models and were even increased in effect size by adjusting for other covariates. none of the competing hypotheses were more likely to influence results as shown by calculating the rrrs. common pitfalls in meta - regression analysis can occur, such as multiple or post - hoc analyses, and lead to data dredging and a high probability of false - positive conclusions. we, therefore, restricted our analyses to a limited number of pre - specified explanatory covariates. publication bias was considered unlikely as an explanation of the apparent diminishing relative effect of hfv. if trials are selectively published either because of their size or because of significant results, this would result in an association between trial size and/or precision and the trial outcome. strictly speaking, funnel plots probe whether studies with little precision (small studies) give different results from studies with greater precision (larger studies). asymmetry in the funnel plot may therefore result not from a systematic under - reporting of negative trials but from an essential difference between smaller and larger studies that arises from inherent between - study heterogeneity ; thus, if larger studies were also associated with changes in ventilation strategies and these strategies resulted in changes in reported rrs, the assumed publication bias would be, in fact, a real association between ventilation strategy and study outcome ; therefore, we conditioned the association between precision and effect size, presumably caused by publication bias, on ventilation strategies. this resulted in a lower p - value for publication bias and more symmetrical distribution of studies in subgroups in the funnel plots ; therefore, what appeared to be publication bias could also be explained by differences in ventilation strategies related to both study size and observed relative risks. however, it should be pointed out that the strength of this evidence is difficult to assess because fewer studies in the subgroups automatically resulted in less power to detect publication bias. other alternative hypotheses that have been formulated to explain differences among studies were also less compatible with the evidence. the type of ventilator, sensormedics vs other types of high - frequency ventilators, displayed rrr close to one. in the crude analyses, prior time on cmv before study initiation showed contradictory effects to what was hypothesized. gestational age and birth weight could also influence the magnitude of the effect of hfv compared with cmv. in the adjusted analysis gestational age did not change the rr for cld but showed an increase of the rr for less premature neonates. finally, an increased risk of cld was not accompanied by a greater relative benefit of hfv as compared with cmv. the observed effects of continuous variables, such as time on cmv or gestational age, could be exaggerated by small studies with outlying results. for the covariate, time on cmv, the two largest studies showed results that were compatible with the hypothesis that this had no important impact on the results of these trials [2, 3 ]. the same fact applied to the effect of baseline incidence of cld or death or cld. gestational age and weight were comparable between the two largest trials, which made it more difficult to ascertain the relevance of the hypothesis that in smaller and more premature infants hfv performed better than cmv treatment. the observed direction of the effect of gestational age and birth weight, however, was opposite to what the hypothesis predicted. if gestational age was to be interpreted as a higher risk of acquiring cld, one would expect that an increase in the incidence of cld was associated with a relatively lower incidence of cld in hfv treated patients ; however, linear regression analysis showed perfectly equal increase in both treatment groups. still, the possibility remains that the relationship with patient averages, such as gestational age and birth weight, across trials was not the same as the relationship for patients within trials, and therefore an effect of these patient characteristics can not be excluded but only considered in relation to other covariates. similar findings of the effects of ventilation strategies have been reported by us and other authors as well [4, 5 ] ; however, meta - analyses are subject to bias when differences among trials are used to explain differences in reported rrs. in this meta - regression analysis we were able to estimate adjusted association measures, thereby diminishing the effects of possible confounders / effect - modifiers. by calculating less biased estimates of the effects of ventilation strategies and the effect of using a sensormedics ventilator instead of other ventilators on the outcome in the different hfv trials we were able to reinforce the hypothesis that ventilation strategies are more important than type of ventilator to prevent cld. the results of this meta - analysis stresses the importance of using appropriate ventilation strategies to prevent ventilator - induced lung damage in a highly vulnerable group of patients ; therefore, in clinical practice the question of how to use the ventilator is more important than the question of which ventilator should be used. the major theoretical advantage of hfv to cmv is delivery of smaller tidal volumes to an optimally recruited lung. as this meta - regression analysis did not confirm that subgroups of more premature neonates, avoidance of cmv prior to initiating hfv, or neonates with higher risk of cld were more likely to benefit form elective hfv in irds, future research should be directed at identifying patients in whom hfv does have a benefit over cmv. to improve the robustness of these conclusions and to avoid the limitations of meta - analysis of trials, an individual - patient - data - based meta - regression analysis should be conducted. in conclusion, confining randomized trails to smaller or more premature children with irds did not seem to result in better pulmonary outcomes of hfv compared with cmv. a generally held opinion that a prolonged ventilation time on cmv prior to initiating hfv diminished the benefits of hfv was not in agreement with the current evidence. the most important effects resulting in differences among trials were probably caused by ventilation strategies applied in hfv- and cmv - treated patients. | objectivethere is considerable heterogeneity among randomized trials comparing high - frequency ventilation (hfv) with conventional mechanical ventilation (cmv) in premature neonates with respiratory distress syndrome. we investigated what factors explained differences in outcome among these trials.designmeta-regression analysis of 15 randomized trials.measurements and resultsvariables were extracted to explain heterogeneity : year of publication ; use of sensormedics 3100a ventilator for hfv ; time on cmv prior to start of study ; gestational age ; use of surfactant ; high lung volume strategy in hfv ; and lung protective ventilation strategy in cmv and baseline risk. chronic lung disease (cld) and death or cld were outcome measures. relative risk ratios were calculated to estimate effect sizes of explanatory variables on reported relative risks. adjusted estimates of relative risk ratios of high lung volume strategy and lung protective ventilation strategy were 0.42 (95% ci 0.062.48) and 2.02 (95% ci 0.1823.12) for cld, respectively. the effect of gestational age was less pronounced (rrr = 1.17 (95% ci 0.168.32) for cld, respectively). use of sensormedics and prior time on cmv had the smallest effects [rrr = 0.96 (95% ci 0.471.94) and rrr = 0.85 (95% ci 0.581.24) for cld, respectively) ]. the same results applied to cld or death as outcome.conclusionsvariation in ventilation strategies that were used in trials comparing hfv with cmv in premature neonates offered the most likely explanation for the observed differences in the outcome of these trials compared with other explanatory factors.electronic supplementary materialthe online version of this article (doi:10.1007/s00134 - 007 - 0545-y) contains supplementary material, which is available to authorized users |
a 12-year - old male, whose father was a ldl receptor (ldl - r) mutation carrier, developed corneal arcus and multiple skin lesions with a 10-year history of xanthomas. both his parents had elevated levels of total serum cholesterol and ldl cholesterol. his elder brother died of myocardial infarction secondary to fh at the age of 7 years. physical examination showed the presence of subcutaneous yellow nodules at the knuckles of his fingers [fig. some other yellow nodules of varying sizes under the skin erupted over the buttocks [fig. the ocular exam revealed a partial circumferential white - grey deposit corresponding to corneal arcus [fig. b - scan revealed extensive plaques and enhanced intima - media thickness of common carotid arterial wall. laboratory studies disclosed the following values : total serum cholesterol, 752.1 mg / dl (normal range, 110 - 220 mg / dl) ; triglyceride, 96.6 mg / dl (normal range, 50 - 150 mg / dl) ; ldl cholesterol, 661.3 mg / dl (normal range, 80 - 140 mg / dl). findings were consistent with type iia hyperlipoproteinemia. as is shown in the figure [fig. 3 ], there are cytosine (c)>thymine (t) heterozygous double peaks at 97 in the second exon of ldl - r gene, which is in genbank as a known mutation of nm_001195798.1:c. this mutation resulted in the change from c to t 33 codon in e2 of ldl - r gene and, thus, glutamine became the stop codon in the corresponding amino acid (np_001182732.1:p.gln33x). findings at presentation (a) xanthomas over the fingers (b) xanthomas over both elbows(c) xanthomas over both knees tending to coalesce (d) xanthomas over the achilles tendons (e) xanthomas of varying sizes under the skin erupted over the buttocks findings at presentation. the ocular exam showed a partial circumferential (from 2 oclock to 4 oclock) white - grey deposit corresponding to corneal arcus. (a) right eye the ocular exam showed a partial circumferential (from 2 oclock to 4 oclock) white - grey deposit corresponding to corneal arcus. there are c > t heterozygous double peaks at 97 in the second exon of ldl - r gene homozygous fh is clinically characterized by cutaneous xanthomas, enlarged achilles tendons, atherosclerosis, and corneal arcus, usually developing from early childhood. although no significant correlations were obtained between corneal arcus and patterns of hyperlipoproteinaemia in previous observations, a recent study of homozygous familial hypercholesterolemia series indicated that patients with corneal arcus had higher cholesterol - year score and was correlated with calcific atherosclerosis. one explanation is the proximity to limbal vasculature that may increase endothelial permeability to lipids via active scavenging mechanisms. another is the temperature gradient that can alter lipid deposition as the infiltrating particles pass from limbal blood vessel into the cornea. early combination therapy with ldl apheresis, statins, and cholesterol absorption inhibitors are advised in children with homozygous fh at the highest risk. | we report the case of a 12-year - old male who developed corneal arcus and multiple skin lesions with a 10-year history of xanthomas. the lesions appeared over his fingers, hands, elbows, knees, buttocks and feet. laboratory studies showed a total serum cholesterol level of 752.1 mg / dl ; a triglyceride level of 96.6 mg / dl ; a low - density lipoprotein cholesterol level of 661.3 mg / dl. findings were consistent with homozygous familial hypercholesterolemia. to our knowledge, this is the first such case to be reported from china. |
during pregnancy in larger mammals, the maternal immune system must tolerate the fetus for months. although in mice tolerance is accomplished by suppression of maternal immune cells, species with longer pregnancies probably can not suppress their immune systems to the same extent because this would make them prone to infections. to examine how the immunological challenge of a long gestation period is met, we chose three well - studied species with pregnancies that last nine months or longer : humans, cattle, and horses. in these species, tolerance is mediated by major histocompatibility complex proteins, by leukocytes, and by the cytokines secreted by the leukocytes. if the mother misunderstands the signal sent by the fetus during pregnancy, the fetus will be miscarried or delivered preterm. interestingly, although it might be assumed that tolerance would be accomplished in all these species by hiding the fetus from the maternal immune system, paternally inherited antigens are expressed during early pregnancy by trophoblast cells in cattle and horses. at parturition, paternal antigens are known to be expressed in cows, possibly so that the maternal immune system will reject the placenta and help to expel it [4, 5 ]. this review compares fetal - maternal crosstalk that is mediated by the immune system in humans, cattle, and horses. it examines physiological pregnancy (in which gestation is not shortened and the fetus is not miscarried or delivered preterm), pathological pregnancy, and parturition. it suggests the hypothesis that, in horses and cows, the expression of paternal antigens by invading trophoblast cells may educate the maternal immune system and prepare it for rapid rejection of fetal membranes at parturition. there are two classes of mhc i : classical and nonclassical (table 1). classical mhc are highly polymorphic, which means they have the ability to present many antigens including foreign antigens. if cells express these foreign antigens they are attacked by cytotoxic t lymphocytes (ctl). nonclassical mhc i are not polymorphic and present a so - called zero antigen. antigen fills a groove in nonclassical mhc i proteins and is recognized by leukocytes as a maternal self antigen ; however, it is not of maternal origin. the cells that express zero antigen are protected because cells that do not express any antigen are attacked by uterine natural killer cells (unk) [8, 9 ]. communication between mhc i and leukocytes (uterine natural killer cells, macrophages, and t lymphocytes) induces and maintains maternal tolerance during physiological pregnancy. in humans, cattle, and horses, expression of mhc i is increased by trophoblast cells that invade the endometrium as they become more exposed to the maternal immune system. the pattern of expression of mhc i differs according to the species. in humans, zero antigen that protects the cells by binding with unk [8, 9 ]. in cattle and horses, the invasive trophoblast expresses classical mhc i with paternal antigens [10, 11 ], and this pattern of expression stimulates a response from cytotoxic t lymphocytes (ctl) [6, 7 ]. the reasons for different patterns of expression of mhc i on invasive trophoblast are not clear, although this might be associated with the structure of the placenta in different species. in humans, nonclassical mhc i enters the maternal circulation, which is probably facilitated by the invasive placenta structure (hemochorial placenta) that has been found in this species, as well as in apes, monkeys, and rodents. in these species with invasive placentas, a part of the trophoblast that is called the extravillous trophoblast destroys 3 layers of endometrial tissue so that it can be in direct contact with maternal blood. the blood passes through a disk - shaped zone, which maternal arteries and veins access from the endometrium. nourishment is passed to the fetus through 3 layers of cells in highly vascularized villi that sink into this disk and are washed by maternal blood. to ensure that enough blood can circulate through this disk, blood pressure in the maternal arteries is increased by a process called spiral artery remodeling [8, 9, 14 ]. the structure and expression of the mhc i that mediate tolerance and support of pregnancy have been best defined in humans, in which mhc i are referred to as human leukocyte antigen (hla) (table 1). human classical mhc i are polymorphic and are known to exist in several classes. of these classes, only hla - c bind zero antigens. hla - c is expressed on the entire surface of the trophoblast villi (figure 1). when hla - c is bound by the kir2d receptor on uterine natural killer cells (unk), this leads to optimum blood supply to the trophoblast, thus supporting the fetus. zero antigens are expressed by all 3 known classes : hla - e, hla - f, and hla - g [1719 ]. these hla are soluble and are expressed on the whole surface of the trophoblast villi (the villous and extravillous trophoblast) (figure 1). tolerance is induced by hla - g, which is known to enter the maternal circulation and bind with the leukocytes immunoglobulin - like receptors (lir-1 and lir-2) on unk, macrophages, and t lymphocytes. after binding, the leukocytes are inactivated and express more lir-1 receptors [1, 16, 1823 ] (table 3). even in pathological pregnancies, only hla that induce tolerance have been found, that is, hla with zero antigens. yang. took samples from the trophoblast during pregnancy and cultured them with inf - gamma, a strong proinflammatory cytokine. normally, when tissues are treated with this cytokine, they respond by expressing hla - a and hla - b. these are classical mhc i that induce inflammation and their expression of immune rejection might lead to recognition by cytotoxic t lymphocytes and immune rejection. however, the cultured trophoblast cells in this experiment continued to express only tolerance - inducing hla - g. this suggests that the mechanisms that lead to expression of only hla with zero ruminants are known to have noninvasive placentas ; of these species, cows have been studied the most. in noninvasive placentas, instead, nourishment is passed from the mother to the fetus through structures called placentomes. in cows, there are 70120 placentomes scattered throughout the entire placenta. placentomes consist of vascularized villi that originate in the trophoblast and the corresponding endometrial crypts into which the villi fit. nourishment passes from the maternal to the fetal blood through six layers of cells, three in the endometrium and three in the trophoblast villi. in cows, mhc i are referred to as bovine leukocyte antigen (bola) (table 1). unlike in humans, classical bola with paternal antigens are known to be expressed during physiological pregnancy, in addition to nonclassical bola with the paternal antigens are expressed on binuclear cells, which have a role in supporting pregnancy. binuclear cells originate in the trophoblast, although the exact details of their origin are unknown. the cells migrate from the trophoblast and invade the endometrium, where they fuse with endometrial cells to create giant trinuclear cells. these giant cells lose the paternal antigens and express no bola at all (figure 2) [10, 36 ]. giant cells help to stabilize pregnancy by secreting bovine placenta lactogen, which influences ovarian and placental steroidogenesis and alters maternal metabolism to support fetal growth and development. in cows, zero antigens, and they may have a role in inducing tolerance. unlike humans, these nonclassical bola have not been found on the entire surface of the trophoblast but only on the regions between the placentomes (interplacentomal region) and between the villi (arcade region). moreover, these bola have only been found during the last trimester of pregnancy, not throughout the entire pregnancy as in humans [2, 10 ]. nonclassical bola are produced in both nonsoluble and soluble forms, so it can be speculated that the soluble bola also bind lir-1 receptors on leukocytes in cows, which could inhibit the leukocytes, similar to as in humans. during clone pregnancies in cows, classical bola with paternal antigens have been found on the trophoblast surface during the first month of pregnancy. it is speculated that this presentation of paternal antigens is connected with the high number of clone pregnancies that are lost due to attack by activated cytotoxic t lymphocytes (ctl) [37, 40 ]. it is not known why paternal antigens are only presented on the trophoblast in these pregnancies, but it is likely that this is due to altered gene expression caused by the nuclear transfer process. although little is known about immunological activity at the time of parturition, research in cows suggests that expulsion of fetal membranes is promoted when the maternal immune system rejects paternal antigens that are presented by the fetal membranes [4, 5 ]. classical bola with paternal antigens have been found to be expressed by cows at parturition [4143 ]. the trophoblast villi are the contact zone in the placentomes, and, up to one month before parturition, the endometrial epithelium undergoes a thinning process and then disappears completely. this histological change leads to loosening of the contact area, so that the trophoblast epithelium contacts the connective tissue of the endometrium (the placenta changes from epitheliochorial to synepitheliochorial) [44, 45 ]. in addition, when paternal antigens are presented by classical bola protein on the surface of chorion cells, the antigens are recognized by t lymphocytes (cd 8 +). this recognition can be seen as an increased migration of these lymphocytes to the placenta surface. the increased chemotactic activity of the lymphocytes has been well investigated in cows and this activity decreases when cows retain fetal membranes. in a study by benedictus., classical bola compatibility from the point of view of the immune systems of both the calf and the dam gave a significantly higher risk of retention of fetal membranes with an odds ratio of 16.25. in a study by streyl. that compared mrna expression 6 to 26 days before parturition with expression during physiological parturition, upregulation of certain genes during parturition suggested that increased numbers of leukocytes were present in the fetal - maternal contact zones in the placentomes. nothing further is known about the immunological mechanisms that lead to retention of fetal membranes in cows or in other species. however, it is possible that immunological communication during pregnancy may prepare the cow for rejection and expulsion of fetal membranes at parturition. horses have an epitheliochorial placenta, as do species that are classified as having a noninvasive placenta. however, because the horse placenta has a subpopulation of highly invasive trophoblast cells (called the chorionic girdle), the authors here will refer to this kind of placenta as semi - invasive. this subpopulation of invasive cells forms a chorionic girdle that encircles the fetus. by day 35 of pregnancy, cells of the chorionic girdle adhere to the endometrial epithelium and begin to invade the endometrium [4952 ] (figure 3). the aggressive invasive behavior of these cells is similar to the behavior of cells in the human extravillous trophoblast and to metastatic tumor cells. the chorionic girdle disappears at about days 3638 of pregnancy [50, 54, 55 ]. chorionic girdle cells have been found to express mhc i, which is referred to in horses as equine leukocyte antigen (ela) [49, 56 ]. this expression quickly diminishes after invasion and is not found in mature endometrial cups [57, 58 ]. the expression of ela by other cells during horse pregnancy and at any other time during pregnancy has not been investigated. moreover, it has not been established whether nonclassical or classical ela are expressed, nor whether the ela bind zero or paternal antigens. evidence for the binding of paternal antigens by what would probably be classical ela is the fact that cd8 + t leukocytes have been found to be attracted to the cells that express ela. these cd8 + t leukocytes have been found around the chorionic girdle on the same days that mhc i was expressed [11, 30, 51, 56, 57, 59, 60 ]. antibodies to paternal antigens that were produced by b lymphocytes have been found at stable levels in the peripheral blood throughout the rest of pregnancy [54, 6163 ]. however, cd8 + t leukocytes were not found to attack trophoblast cells that were expressing ela. this may be because the paternal antigens are expressed for too short a time for the immune system to prepare itself to attack the paternal - antigen presenting cells (the chorionic girdle disappears on days 3638) [50, 51, 54, 55 ]. when the chorionic girdle invades the endometrium, it forms distinct nodules in the endometrial stroma. these nodules are called endometrial cups ; mature endometrial cups do not express any ela [57, 58 ] (figure 3). the endometrial cups remain and continue to secrete ecg until about days 90120, by which time they have degenerated. the chorionic girdle and the endometrial cups are important for maintaining pregnancy. in donkey - in - horse pregnancies there is no chorionic girdle or endometrial cups, and it has been speculated that this is related to the high rate of abortions in these pregnancies [67, 68 ]. cells in the endometrial cups have il-22r1 receptors which bind il-22, which is secreted by the chorionic girdle. binding of il-22 helps to maintain mucosal immunity, by facilitating endometrial reepithelization and upregulating antimicrobial proteins. during pregnancy in all three species described here, maternal leukocytes behave differently in the uterus than they do in the rest of the mother 's body. when unk from humans, macrophages from humans and cows [69, 70 ], and t lymphocytes from horses have been taken from pregnant uteruses and compared to leukocytes taken from the peripheral blood, the uterine leukocytes were found to be inhibited from engaging in normal immune responses, although the mother is able to resist general infection. this phenomenon is known as split immune tolerance. in humans, uterine natural killer cells unk cells are the most abundant leukocytes in the placenta (table 2), and their number remains constant throughout pregnancy. unk and their receptors are a type of nk cells that are unique to the uterus and they differ structurally from peripheral nk [71, 72 ]. the phenotype of unk (cd56, cd16, and cd3) distinguishes them from nk in peripheral blood (cd56, cd16, and cd3). unk do not attack the trophoblast ; this is mediated by nonclassical mhc i (hla - g), as mentioned before (section 2.2). unk cells change their structure as pregnancy progresses, and these changes are related to the roles that unk play in inducing tolerance and support of the fetus and placenta. in the first trimester unk the granules contain angiogenic growth factor and vascular endothelial factor c. angiogenic growth factor is released when hla - g binds to the unk lir-1 receptor. this growth factor promotes spiral artery remodeling and may increase vascularization in the syncytial villi. this seems to be a feedback loop that helps to stabilize immunological tolerance of the fetus. in the second trimester unk undergo a degranulation process and in the third trimester only degranulated unk cells are present in the endometrium. when degranulation starts, unk stop secreting the above factors and begin to secrete ifn - gamma. this cytokine inhibits the migration of trophoblast cells, protecting the uterus from too much destruction by these invasive cells [7, 31, 75 ]. they induce optimal blood supply for the fetus by participating in spiral artery remodeling [8, 9, 14, 16 ] when their kir2d receptors bind hla - c that is present on the surface of the trophoblast. unk secrete the matrix metalloproteinases mmp2 and mmp9 during implantation on the 8th to 10th day after ovulation. these enzymes break down fibrous proteins that are known as the extracellular matrix. by breaking down this matrix, these metalloproteinases reduce the intercellular gap between the trophoblast and the endometrium. the mechanisms that induce unk to secrete these metalloproteinases are unknown. unk are inhibited from attacking the fetal unit when their lir-1 receptors bind soluble hla - g that has entered the maternal circulation (as part of the extravillous trophoblast figure 1). in addition, the number of inhibitory lir-1 receptors increases both on unk and on macrophages and t leukocytes when the kir2dl4 receptors on unk are bound by hla - g [7880 ]. it is not known if and how unk communicate with the other leukocytes to effect this change in the number of their inhibitory receptors.. altered numbers of unk or decreased numbers of kir2d receptors on unk have been associated with fetal growth restriction and insufficient trophoblast invasion, miscarriage [81, 82 ], implantation failure, and preeclampsia. some nk cells have been found in the horse placenta, but none in cows ; therefore all the information that we have about the activity of unk comes from studies with humans. macrophages protect pregnancy in humans throughout all of gestation by inhibiting the immune response by unk, t lymphocytes, and other macrophages. after this happens, macrophages do not secrete the proinflammatory cytokines tnf - alpha and inf - gamma. instead they synthesize more lir-1 receptors and secrete prostaglandin pge2, which suppresses the activity of other macrophages, t lymphocytes, and unk [7880 ]. cd9 protein is expressed by uterine macrophages ; it is bound by pregnancy specific protein (psg), which is secreted by the trophoblast. after these receptors are bound, the macrophages secrete il-10, which inhibits secretion of tnf - alpha by unk and t lymphocytes [85, 86 ]. to prevent activation of maternal t lymphocytes, macrophages reduce expression of the costimulatory molecules cd80 and cd86 and express indoleamine-2,3-dioxygenase (ido) [7880, 86 ]. ido is an immunomodulatory enzyme that catalyses degradation of essential l - tryptophan, which inhibits proliferation of t lymphocytes and prevents their activation., there is a bias toward cytokines production by cd4 + t - helper (th2) cells and against cytokines production by cd4 + th1 cells. nave cd4 + t lymphocytes recognize hla - g zero antigens (presented on antigen presenting cells) when the antigens bind with their lir-1 receptors. this recognition inhibits proliferation of cd4 + t - cells, induces their long - term unresponsiveness, and causes differentiation of the cd4 + t - cells into suppressive th2 cells. also, secretion of proinflammatory tnf - alpha by cd4 + th1 cells is inhibited by il-10 secreted by macrophages. by avoiding the th1 response, ctl cells are also inhibited when their lir-1 receptors bind with hla - g. moreover, suppressor cd4 + cd25 cells inhibit activation of ctl cells by the same ido mechanism as macrophages (mentioned in the macrophages section). however, cd8 + t lymphocytes are present at the site where placenta implantation takes place. these lymphocytes are thought to protect the pregnancy against external antigens and to support trophoblast growth by secreting il-8, which promotes trophoblast invasion. horses. in mares, cd4 + and cd8 + t lymphocytes have been found to cluster around the cells of the invading chorionic girdle, and around the early, mature, and dying endometrial cups (which are formed by the fusion of the girdle cells with endometrial cells) in greater numbers than are found in the rest of the endometrium. as detailed in the section on horse mhc i (ela), the invading cells express ela with paternal antigens, but the mature and dying cups do not, and it is unclear how t cells recognize these cells that do not express ela [57, 58 ]. it is also unclear why ctl do not destroy the ela - expressing cells of the invading chorionic girdle and of the early endometrial cup. it appears that both a systemic increase in t - cell tolerance during pregnancy and an unknown inhibitory factor that is produced by trophoblast cells help protect the ela - expressing cells [55, 9092 ]. to mediate tolerance by the maternal immune system during pregnancies in humans, cows, and horses, which normally last for nine months or longer, mhc i present zero antigens, which are recognized by leukocytes. these leukocytes secrete cytokines that increase expression of zero antigens, further induce tolerance in other leukocytes, and support pregnancy. although the details of this process are best known in humans, there are findings of immune activity in other species that have not yet been investigated in humans. much remains to be done both to clarify the mechanisms of maternal immunological tolerance in individual species and to find out which mechanisms are common to all placental mammals and which species are specific. as bainbridge points out, the differences between immunological fetal - maternal crosstalk in humans, cattle, and horses may exist because their common ancestor may have had a short gestation period. thus, these species may have independently evolved different mechanisms to protect the fetus from longer exposure to the maternal immune system. however, it is interesting to note that, in all three species examined in this review, invasive trophoblast cells increase their expression of mhc i as they become more exposed to the maternal immune system. bainbridge has advanced three hypotheses to explain this phenomenon : (1) mhc on trophoblast cells may help them adhere to and invade maternal tissue, (2) mhc expression may protect the invading cell from the maternal immune system, although it is difficult to understand how the paternal antigens on cattle and horse cells would pacify the maternal immune system, and (3) this expression of mhc may protect the entire fetoplacental unit from the maternal immune system, at least in humans, where hla - g has been found to be able to suppress the proliferation of peripheral blood lymphocytes. we suggest a fourth hypothesis be added to the three above : in horses and cows, the expression of paternal antigens by invading trophoblast cells may educate the maternal immune system and prepare it for rapid rejection of fetal membranes at parturition (figure 4). in horses, when trophoblast cells that display paternal antigens invade the endometrium, cd8 + t lymphocytes are attracted to those cells [11, 30, 51, 56, 57, 59 ]. this persistence could prepare the immune system for a rapid response to paternal antigens that are presented at or just before parturition, if the unknown factor that inhibited the t - cells is not present in the uterus at parturition. after invasion by chorionic girdle cells, antibodies to paternal antigens that were produced by b lymphocytes have been found to remain at stable levels in the mare 's peripheral blood throughout the rest of pregnancy [52, 6163 ]. at parturition, the mare 's immune response to paternal antigens may be similar to what is known to occur in humans when macrophages recognize foreign antigens : the macrophages secrete proinflammatory tnf - alpha and inf - gamma, which activate both other macrophages and t lymphocytes. similar mechanisms in cows would help explain why classical mhc i compatibility from the point of view of the immune systems of both the calf and the dam gave a significantly higher risk of retention of fetal membranes. in mammalian species in which pregnancy lasts for months, the maternal immune system must be able to resist infection while tolerating paternal antigens that are expressed by the fetus. because of the length of pregnancy in these species, simply relying on extensive suppression of the mother 's immune response is probably too risky. humans, cows, and horses all have gestation periods of nine months or longer, and they have evolved similar mechanisms for meeting this immunological challenge. when mhc i present zero antigens, these antigens are recognized by leukocytes, and these leukocytes secrete cytokines which induce tolerance in other leukocytes, stimulate the expression of more zero antigens, and help support pregnancy. the details of this process differ between the three species, and much needs to be done to determine which mechanisms are common to all three species, and which are different. in addition, there are reports that suggest that immunological communication may prepare for and promote rapid rejection of fetal membranes during parturition in cows and horses, but whether or how this is done also needs to be determined. | during pregnancy in larger mammals, the maternal immune system must tolerate the fetus for months while resisting external infection. this tolerance is facilitated by immunological communication between the fetus and the mother, which is mediated by major histocompatibility complex i (mhc i) proteins, by leukocytes, and by the cytokines secreted by the leukocytes. fetal - maternal immunological communication also supports pregnancy by inducing physiological changes in the mother. if the mother misunderstands the signal sent by the fetus during pregnancy, the fetus will be miscarried or delivered preterm. unlike any other maternal organ, the placenta can express paternal antigens. at parturition, paternal antigens are known to be expressed in cows and may be expressed in horses, possibly so that the maternal immune system will reject the placenta and help to expel it. this review compares fetal - maternal crosstalk that is mediated by the immune system in three species with pregnancies that last for nine months or longer : humans, cattle, and horses. it raises the possibility that immunological communication early in pregnancy may prepare the mother for successful expulsion of fetal membranes at parturition. |
patients with eos usually present in the fourth and fifth decades of life unlike skeletal osteosarcoma patients who present in the fifth and sixth decades of life. there are also reports of eos involving the face, breast, abdominal wall, soft tissues of the back and retroperitoneum, and site of a vaccination scar. found that 10% of the patients had previous irradiation and 13% of the patients had history of trauma related to the site of eos. the genetic change most commonly associated with osteosarcoma is the loss of the p53 tumor suppressor gene on chromosome 17 and loss of retinoblastoma gene rb1 on chromosome 13. a genetic association between paget 's disease and osteosarcoma has also been identified on chromosome 18q. other genomic changes, such as loss on 13q and 15q and gain on 1q and 8q, have been detected in patients with eos. the contribution of the major susceptibility genes on chromosome 10q (ret proto - oncogene) is well established in hd. in addition, potential modifying associations exist with chromosomes 2, 9, 13, 20, 21, and 22. variations of main ret proto - oncogene account for as much as 50% of familial and 2030% of sporadic cases. the etiology of the majority of sporadic cases is not clear, appearing to arise from combined cumulative effects of susceptibility loci on other chromosomes controlling the mechanism of cell proliferation, differentiation, and maturation. presence of genitourinary abnormalities in the form of horse - shaped kidney and azoospermia, and sporadic hd pointed toward a genetic abnormality in our patient. hence, we performed genetic screening at the common sites for both hd and osteosarcoma on chromosome 10 (ret proto - oncogene) and chromosome 17 (p53 tumor suppressor gene). however, we found no abnormalities at both these loci, suggesting a role of combined cumulative effects of susceptibility genes on other chromosomes. inactivation or alteration of a gene located at this susceptibility loci could have been an early event in the development of eos in our patient. the cornerstone of treatment consists of radical surgery and polychemotherapy. due to the rare nature of this disease, no data from randomized studies concerning the type of chemotherapeutic combinations are available. in a small retrospective study, goldstein - jackson. reported favorable results when eoss were treated as conventional osteosarcomas. active chemotherapeutic agents for skeletal osteosarcoma consist of a combination of doxorubicin and cisplatin, with or without the addition of other drugs such as methothrexate, ifosfamide, and etoposide. the use of multi - agent chemotherapy has extensively improved the outcome for skeletal osteosarcoma. however, these treatments are very intensive, and therefore acute and delayed toxicities can be expected. doxorubicin can cause bone marrow toxicity and cardiotoxicity, whereas cisplatin can cause nephro - and ototoxicity. adjuvant radiotherapy does not increase the survival when patients are treated with effective surgery and chemotherapy, and increase the risk for secondary tumors. however, radiation should be considered in the setting of an unresectable or incompletely resected primary tumor. in this case, the tumor was completely resected, but the resection margins were close due to the orbital localization of the tumor, making wide resection impossible. the first case was an 11-year - old boy who developed an eos of the orbit following radiotherapy for retinoblastoma in infancy. the second patient was a 22-year - old otherwise healthy male with no predisposing factor. our case is the oldest patient ever detected with an eos in the orbit with no history of previous radiotherapy or trauma. one could suggest a possible association between eos and patients known hd, but this has never been reported in the literature before. | primary extraskeletal osteosarcoma (eos) is an extremely rare malignancy. in this report, the clinical course of a 32-year - old man presenting with proptoses is described. medical history included hirschsprung disease (hd), horseshoe kidney, azoospermia, and vertebral anomalies. imaging of the orbit showed an oval, well - defined heterogeneous mass adjacent to the lateral wall of the orbit. the patient underwent a lateral orbitotomy and complete excision of the mass. the mass was not attached to the bone. histopathologic and immunohistochemical examination confirmed the diagnosis of an eos. the patient received chemotherapy and radiotherapy and is free of the disease 3 years after the diagnosis. genetic screening showed no mutations for both the ret proto - oncogene for hd and the p53 tumor suppressor gene for osteosarcoma. |
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